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Development of new methodologies for biomolecules determination based on the use of immunoassays and inductively coupled plasma mass spectrometry detection
Emma Pérez Hernández
Departamento de Química Analítica, Nutrición y Bromatología
Facultad de Ciencias
Development of new methodologies for biomolecules
determination based on the use of immunoassays and
inductively coupled plasma mass spectrometry
detection
Emma Pérez Hernández
Tesis presentada para aspirar al grado de
DOCTOR/DOCTORA POR LA UNIVERSIDAD DE ALICANTE
MENCIÓN DE DOCTOR/DOCTORA INTERNACIONAL
DOCTORADO en CIENCIAS EXPERIMENTALES Y BIOSANITARIAS
Dirigida por:
Juan Mora Pastor Guillermo Grindlay Lledó
Catedrático de Universidad Profesor Contratado Doctor
Departamento de Química Analítica,
Nutrición y Bromatología
Departamento de Química Analítica,
Nutrición y Bromatología
JUAN MORA PASTOR, Catedrático de Universidad del Departamento de
Química Analítica, Nutrición y Bromatología y GUILLERMO GRINDLAY
LLEDÓ, Profesor contratado doctor del Departamento de Química Analítica,
Nutrición y Bromatología, ambos de la Universidad de Alicante.
CERTIFICAN QUE:
Dña. Emma Pérez Hernández, Licenciada en Química, ha realizado en
el grupo de investigación de Espectrometría Atómica Analítica perteneciente al
Departamento de Química Analítica, Nutrición y Bromatología de la Universidad
de Alicante, bajo nuestra dirección, el trabajo que lleva por título:
“Development of new methodologies for biomolecules determination
based on the use of immunoassays and inductively coupled plasma mass
spectrometry detection”, que constituye su memoria para aspirar al grado de
doctora, reuniendo, a nuestro juicio, las condiciones necesarias para ser
presentada y defendida ante el tribunal correspondiente.
Y para que conste a los efectos oportunos, en cumplimiento de la
legislación vigente, firmamos el presente certificado en Alicante a 11 de enero
de 2018.
Juan Mora Pastor Guillermo Grindlay Lledó
Catedrático de Universidad Profesor Contratado Doctor
Departamento de Química Analítica,
Nutrición y Bromatología
Departamento de Química Analítica,
Nutrición y Bromatología
To my family
Índices
III
ÍNDICE GENERAL
RESUMEN/SUMMARY
Resumen ............................................................................................................ 1
Summary ............................................................................................................ 3
OBJETIVOS Y ESTRUCTURA GENERAL DE LA TESIS DOCTORAL.
1. Objetivo de la Tesis Doctoral. ..................................................................... 9
2. Estructura de la Tesis Doctoral. .................................................................. 9
CAPÍTULO 1. Introducción general.
1. Introducción. .............................................................................................. 17
2. Conceptos básicos de inmunoensayos. .................................................... 19
2.1. Anticuerpos. ......................................................................................................... 19
2.2. Tipos de inmunoensayos. ................................................................................. 22
2.2.1. Inmunoensayos no-competitivos. .............................................................. 22
2.2.2. Inmunoensayos competitivos. ................................................................... 23
2.2.3. Immunoensayos en fase heterogénea y homogénea. ................................ 25
3. La Espectrometría de Masas de Plasma de Acoplamiento Inductivo como
herramienta de cuantificación de biomoléculas. ............................................... 26
3.1. Conceptos básicos de ICP-MS. ........................................................................ 29
3.2. Estrategias analíticas para la cuantificación de biomoléculas mediante ICP-
MS………….. ............................................................................................................ 31
3.3. Limitaciones de los inmunoensayos con detección mediante ICP-MS. .......... 37
4. Referencias. ................................................................................................. 38
CAPÍTULO 2. Determination of aflatoxin M1 in milk samples by means of
an inductively coupled plasma mass spectrometry-based immunoassay.
1. Introduction. .................................................................................................. 49
2. Experimental. ............................................................................................... 51
IV
2.1. Reagents and materials. ...................................................................................... 51
2.2. Buffers and solutions. .......................................................................................... 52
2.3. Immunoassay procedure. ..................................................................................... 53
2.4. ICP-MS instrumentation. ..................................................................................... 55
2.5. Calibration. .......................................................................................................... 57
2.6. Samples. ............................................................................................................... 58
2.7. Sample preparation. ............................................................................................. 58
3. Results and discussion. ................................................................................ 59
3.1. Immunoassay optimization. ................................................................................. 59
3.2. Method validation. ............................................................................................... 64
3.3. Comparison with other methodologies. ............................................................... 66
4. Conclusions. ................................................................................................. 69
5. References. .................................................................................................. 70
Supplementary data. ........................................................................................ 77
CAPÍTULO 3. Evaluation of different competitive immunoassay for
aflatoxin M1 determination in milk samples by means of inductively
coupled plasma mass spectrometry.
1. Introduction. .............................................................................................. 81
2. Experimental. ............................................................................................... 83
2.1. Reagents and materials. ....................................................................................... 83
2.2. Buffers and solutions. .......................................................................................... 84
2.3. Immunoassay procedures .................................................................................... 85
2.3.1 Antibody binding inhibition assay (ABIA). .................................................... 87
2.3.2 Capture inhibition assay (CIA). .................................................................... 88
2.3.3. Capture bridge inhibition assay (CBIA). ...................................................... 88
2.4. Instrumentation. ................................................................................................... 89
2.5. Calibration. .......................................................................................................... 91
2.6. Samples. ............................................................................................................... 91
2.7. Sample preparation. ............................................................................................. 92
3. Results and discussion. ................................................................................ 93
V
3.1. Optimization of the immunoassay procedures. ................................................... 93
3.1.1. Antibody Binding Inhibition Assay (ABIA). .................................................. 94
3.1.2. Capture Inhibition Assay. ............................................................................. 98
3.2. Aflatoxin M1 analysis in milk samples. ............................................................. 102
3.3. Comparison of different competitive immunoassay formats for AFM1
determination. ........................................................................................................... 105
4. Conclusions. ............................................................................................... 107
5. References. ................................................................................................ 109
CAPÍTULO 4. A sensitive size-exclusion inductively coupled plasma mass
spectrometry multiplexed assay for cancer biomarkers using antibodies
conjugated with a lanthanide-labelled polymer.
1. Introduction. ............................................................................................ 117
2. Experimental. .......................................................................................... 119
2.1. Reagents and materials. ................................................................................. 119
2.2. Buffers. .......................................................................................................... 121
2.3. Serum samples. .............................................................................................. 121
2.4. Instrumentation. ............................................................................................. 121
2.4.1. Size Exclusion Chromatography. ........................................................... 121
2.4.2. Inductively Coupled Plasma Mass Spectrometry. .................................. 122
2.5. Antibody labelling procedure. ....................................................................... 123
2.5.1. Partial reduction of the antibody. .............................................................. 124
2.5.2. Antibody labelling via the polymer labelling kit. ................................... 124
2.5.3. Antibody labelling via DOTA-chelate complexes. .................................. 125
2.6. Determination of the antibody labelling degree. .............................................. 125
2.6.1. Protein quantification. ................................................................................ 125
2.6.2. ICP-MS analysis of metal content. ............................................................. 125
2.7. Immunoassay procedure. ................................................................................... 126
3. Results and discussion. .............................................................................. 126
3.1. Preliminary studies with lanthanide-labelled polymer in SEC-ICPMS. ........... 126
VI
3.2. Analysis of cancer biomarkers in human serum by m eans o f S EC-ICPMS a nd
polymer-labelled antibodies. .................................................................................... 131
3.2.1. Optimization of polymer-labelled antibodies synthesis. ............................. 131
3.2.2. Influence of the incubation medium on immunocomplex formation........... 134
3.3.3. Optimization of the concentration of the polymer-labelled antibody. ........ 137
3.3.4. Figures of merit. ......................................................................................... 142
3.3.5. Comparison with other methodologies. ...................................................... 144
4. Conclusions. ............................................................................................... 144
5. References. ................................................................................................ 147
CAPÍTULO 5. Conclusiones generales.
Conclusiones………………………………………………………………………..155
CAPÍTULO 6. Futuros estudios
Futuros estudios…………………………………………………………………….159
VII
ÍNDICE DE FIGURAS
Figura 1.1. Esquema de la inmunoglobulina G (IgG). Las cadenas ligera (azul
claro) y pesada (azul oscuro) están conectadas por enlaces disulfuro. Las
partes con secuencia de aminoácidos variable representan los sitios de
unión al antígeno. VH: parte variable de la cadena pesada; VL: parte
variable de la cadena ligera. Tras la digestión con papaína, el anticuerpo se
disocia en tres partes: dos fragmentos Fab (unión al antígeno) y un
fragmento Fc (región cristalizable)…………………………………………….21
Figura 1.2. Inmunoensayo no- competitivo……………………………………….23
Figura 1.3. Inmunoensayo no-competitivo para la detección de anticuerpos...24
Figura 1.4. Inmunoensayo competitivo……………………………………………25
Figura 1.5. Inmunoensayo homogéneo…………………………………………...26
Figura 1.6. Procesos que sufre la muestra al ser introducida en el seno del
plasma…………………………………………………………………………….31
Figura 1.7. Número de publicaciones en la bibliografía que emplean ICP-MS
como detector de inmunoensayos para el análisis de biomoléculas. Motor
de búsqueda: Scopus. Palabras clave: ICP-MS y proteins/elemental
tags/immunoassays (enero 2018)…………………………………….……….35
Figura 1.8. Metodología de análisis en los inmunoensayos que utilizan ICP-MS
como sistema de detección………………………………………………….…35
Figura 1.9. Ligando bifuncional empleado para marcar anticuerpos. El quelato
se une a un grupo reactivo, lo que permite la conjugación con el anticuerpo.
Se introduce un espaciador entre el quelato y el grupo reactivo para
mejorar la reactividad. M3+ es típicamente un ion lantánido…………….….37
VIII
Figure 2.1. Scheme of the competitive ICPMS-based immunoassay for AFM1
analysis in milk samples..............................................................................54
Figure 2.2. Aflatoxin M1 calibration curve using different pAb concentrations. (-
�-) 0.5 µg mL-1 pAb concentration/incubation time 1h; (-�-) 0.25 µg mL-1
pAb concentration/incubation time 2.5 h. Aflatoxin M1-BSA concentration:
0.35 ng mL-1; secondary Ab dilution factor: 1:2000; streptavidin-Au
nanoparticles conjugate concentration: 0.08 µg mL-1..................................62
Figure S2.1. Influence of the streptavidin-Au nanoparticles conjugate
concentration on the 197Au+ normalized signal and signal standard deviation
in ICP-MS. Aflatoxin M1-BSA concentration: 0.35 ng mL-1; pAb
concentration: 0.5 µg mL-1; secondary antibody dilution factor: 1:2000;
incubation time in the microtiterplate of AFM1 standards and pAb solution: 1
h…………………………………………………………………………………...78
Figure 3.1. Scheme of the different competitive immunoassays formats tested
in this work……………………………………………………………………….87
Figure 3.2. Influence of the α-AFM1 pAb concentration on the limits of detection
of AFM1 operating with different streptavidin-Au nanoparticles conjugates.
(�) 40 nm (�) 80 nm. Aflatoxin M1-BSA concentration: 0.35 ng L-1;
secondary Ab concentration: 500 ng mL-1; streptavidin-nanoparticle
conjugate concentration: 0.08 µg mL-1.………………………………...……..96
Figure 3.3. Aflatoxin M1 calibration curve using different streptavidin-Au
nanoparticles conjugate for the CBIA procedure. (-�-) 40 nm; (-�-) 80 nm.
Aflatoxin M1-BSA concentration: 1.0 ng mL-1; streptavidin-Au nanoparticles
conjugate concentration: 0.08 µg mL-1………………………..……………..102
IX
Figure 4.1. SEC-ICPMS chromatograms of a goat polyclonal antimouse IgG
antibody (pAb) labelled with 165Ho polymer reagents (black line) and 165Ho
DOTA chelate complex (red line). pAb nominal concentration: 10 µg mL-1,
column: Superose 6 Increase 10/300 GL……………………………………127
Figure 4.2. SEC-ICPMS chromatograms obtained after incubation of a mouse
IgG1 antibody solution with a goat polyclonal antimouse IgG antibody (pAb)
labelled with (A) 165Ho polymer reagents and (B) 165Ho DOTA chelate
complex. (1) High molecular weight immunocomplex; (2) low molecular
weight immunocomplex; (3) unreacted labelled pAb; (4) free lanthanide
label. pAb nominal concentration: 10 µg mL-1; antigen concentration: 10 µg
mL-1; incubation medium: 100 mM ammonium acetate; column: Superose 6
Increase 10/300 GL………………………………………………………..…..129
Figure 4.3. SEC-ICPMS chromatograms obtained after incubation of a human
serum sample spiked with 50 ng mL-1 CEA and with its corresponding 165Ho
polymer labelled mAb at a nominal concentration of: (A) 6 ng mL-1 or (B) 2
µg mL-1. Column: Superose 6 Increase 10/300 GL………………………...138
Figure 4.4. SEC-ICPMS chromatograms obtained after incubation of a human
serum sample spiked with 50 ng mL-1 of sErbB2, CA 15.3 or CA 125
antigen with its corresponding polymer-labelled antibody at a nominal
concentration of 2 µg mL-1. Column: Superose 6 Increase 10/300
GL………………….....................................................................................141
XI
ÍNDICE DE TABLAS
Tabla 1.1. Comparativa entre los diferentes tipos de inmunoensayos……...…30
Table 2.1. Operating conditions employed in ICP-MS…………………………...55
Table 2.2. Aflatoxin M1 recovery assay for different kinds of milk
samples……………………………………………………………………….….65
Table 2.3. Comparison of diverse analytical methodologies proposed in the
literature for AFM1 determination in milk samples.......................................68
Table S2.1. Results of the checkboard titration experiments to optimize AFM1-
BSA and pAb concentration. Secondary Ab dilution factor: 1:2000;
streptavidin-Au nanoparticles conjugate concentration: 0.16 µg mL-1;
incubation time in the microtiterplate of AFM1 standards and pAb solution: 1
h…………………………………………………………………………………...77
Table 3.1. Operating conditions employed in ICP-MS………………………..….90
Table 3.2. Influence of the streptavidin-nanoparticles conjugate on ABIA
optimum experimental conditions, LoDs and dynamic range………………95
Table 3.3. Optimum experimental conditions, limits of detection and dynamic
range for CIA and CBIA formats…………………………………..…………...99
Table 3.4. Recovery analysis of AFM1 by ABIA and CBIA methodologies.
Sample pretratment: ABIA: dilution + acetonitrile extraction; CBIA:
immunocolumns…………………………………………………..……………104
Table 3.5. Comparison of diverse analytical methodologies propose in the
literature for AFM1 determination in milk samples…………………….……108
Table 4.1. Operating conditions of SEC-ICPMS………………………………..123
XII
Table 4.2. Labelling degree of the different mAbs using polymer-reagents and
DOTA-chelate complexes………………………………………………..……133
Table 4.3. Influence of the incubation medium on the HMW and LMW
immunocomplexes integrated signals obtained for CEA. Antibody nominal
concentration: 1 µg mL-1; CEA concentration: 50 ng mL-1 (mean ± t·s·n1/2, n
= 3, P = 95%).…………………………………………………………………..136
Table 4.4. Influence of the CEA concentration on the immunocomplexes
integrated signals after incubation with 165Ho polymer-labelled mAb at
nominal concentrations of 6 ng mL-1 or 2 µg mL-1. Incubation medium:
human serum. (mean ± t·s·n1/2, n = 3, P = 95%)……………...……………139
Table 4.5. Recovery values for the CEA, sErbB2, CA 15.3 and CA 125
biomakers of interest using polymer labelling kit (mean ± t·s·n1/2, n = 3, P =
95%)……………………………………………………………………………..142
Table 4.6. Sensitivity, LoDs and linear dynamic range obtained for CEA,
sErbB2, CA 15.3 and CA 125 analysis using both polymer labelling kit and
DOTA-chelate complexes *(mean ± t·s·n1/2, n = 3, P = 95%)……………..145
Table 4.7. Comparison of different methods for CEA, sErbB2, CA 15.3 and CA
125 analysis…………………………………………………………………….146
XIII
GLOSARIO DE ACRÓNIMOS Y TÉRMINOS
Término Descripción
Ab Antibody
Ag-Ab Antigen-antibody
AFB1 Aflatoxin B1
AFM1 Aflatoxin M1
AFM1-BSA Aflatoxin M1-Bovine serum albumin conjugate
CA 15.3 Cancer antigen 15.3
CA 125 Cancer antigen 125
CEA Carcinoembryonic antigen
BSA Bovine serum albumin
DOTA 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''- tetraacetic acid
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTPA Diethylentriamine-N,N,N',N'',N''-pentaacetic acid
EDTA Ethylenediaminetetraacetic acid disodium salt
ELISA Enzyme linked immunosorbent assay
ESI Electrospray ionozation
FIA Flow injection analysis
HMW High molecular weight
HPLC High-performance liquid chromatography
ICP-MS Inductively coupled plasma mass spectrometry
IgG Immunoglobulin G
IgG1 Immunoglobulin G subclass 1
XIV
LoD Limit of detection
lLoQ Lower quantification limit
LMW Low molecular weight
mAb Monoclonal antibody
MALDI Matrix-Assisted Laser Desorption/Ionization
mRNA Messenger ribonucleic acid
MS Mass spectrometry
pAb Polyclonal antibody
PBS Phosphate buffer solution
PEEK Polyether ether ketone
RIA Radioimmunoassays
RNA Ribonucleic acid
SEC Size-exclusion chromatography
sErbB2 Soluble form of human epidermal growth factor receptor 2
TCEP Tris (2-carboxyethyl) - phosphine hydrochloride
Tris Tris(hydroxymethyl)aminomethane
Tween 20 Polyethylene glycol sorbitan monolaurate
uLoQ Upper quantification limit
UV-Vis Ultraviolet–visible
Resumen/Summary
1
RESUMEN
La Espectrometría de Masas mediante ionización en Plasma Acoplado
Inductivamente (ICP-MS) es una técnica de análisis elemental que permite
cuantificar la mayor parte de elementos de la tabla periódica de forma rápida y
a niveles de ultratraza. A lo largo de la última década, y debido a sus
características analíticas, ICP-MS se ha utilizado como detector de
inmunoensayos para la determinación de bioméculas. A pesar de los avances
que se han producido, este tipo de estrategias todavía no están exentas de
inconvenientes. Entre los retos que aún están por resolver, se encuentran: (i)
desarrollo de metodologías para la determinación de haptenos (biomoléculas
de bajo peso molecular < 10 kDa), (ii) falta de criterio para seleccionar el tipo
de inmunoensayo más adecuado para una aplicación bioanalítica dada, sobre
todo cuando se trabaja con haptenos; y (iii) escaso aprovechamiento del
potencial del ICP-MS (sensibilidad, límites de detección, capacidad de análisis
multielemental, etc.). En la presente Tesis Doctoral se han desarrollado nuevos
métodos de análisis de biomoléculas con detección mediante ICP-MS tratando
de salvar los inconvenientes citados con anterioridad. Los métodos se han
aplicado al análisis de tóxinas en alimentos y de biomarcadores oncológicos en
fluidos biológicos (suero).
En primer lugar, debido a la gran preocupación existente por la seguridad
alimentaria y el efecto de la alimentación sobre la salud humana, se desarrolló
una nueva metodología para la cuantificación de la aflatoxina M1 (hapteno) en
muestras de leche que cumpla los niveles de seguridad requeridos por las
actuales políticas internacionales. Por otro lado, se compararó diferentes
Resumen/Summary
2
formatos de inmunoensayos competitivos para la determinación de esta misma
aflatoxina en leche. Además se estudió el efecto de la naturaleza y del tamaño
de las nanopartículas (es decir, Ag / Au, 80 nm / 40 nm) sobre los parámetros
analíticos. Finalmente, se presentó un nuevo enfoque con el objeto de
aumentar la sensibilidad de los inmunoensayos homogéneos con detección
mediante ICP-MS, con la introducción de un alto número de etiquetas por Ab
monoclonal mediante el empleo de polímeros como portadores de múltiples
iones lantánidos. Esta metodología se aplicó a la determinación simultánea de
4 biomarcadores de cáncer (CEA, sErbB2, CA 15.3 and CA 125) en suero
humano.
Resumen/Summary
3
SUMMARY
Inductively coupled plasma mass spectrometry (ICP-MS) is an elemental
analytical technique that permits quantify most of the elements of the Periodic
Table at ultratrace levels. Over the last decade, and due to its features, ICP-MS
has been used as a detector in immunoassays for the analysis of biomolecules.
Despite the fact that these strategies are in good state of development, there is
still a long way from exploiting the full potential of this technique. Among the
challenges which have still to be solved: (i) development of methodologies for
hapten analysis (low molecular weight biomolecules < 10 kDa); (ii) lack of
criteria to select the most suitable immunoassay format for a given bioanalytical
application; especially for hapten determination and (iii) under-utilisation of the
potential of ICP-MS (sensibility, limit of detection, multi-element capability, etc.).
In the following Ph.D. work, the line of research dealing with the developed of
new analytical methods for biomolecules analysis that allow to solve the
limitations previously reported that currently have the immunoassays with ICP-
MS detection is presented. The methods have been applied to the analysis of
toxins in food and to the analysis of oncological biomarkers in biological fluids
(serum).
Firstly, due to the great concern about food safety and its influence on
human health, a new methodology for aflatoxin M1 quantification in milk
samples at the security levels required by the current international policies with
accuracy and precision has been developed. On the other hand, different
competitive immunoassay formats have been evaluated. Again, aflatoxin M1
has been selected as model analyte to evaluate the benefits and drawbacks of
Resumen/Summary
4
the different approaches. In addition, the effect of the nanoparticle metal label
(i.e. Ag/Au; 80 nm/40 nm) on the analytical figures of merit has been studied.
Finally, for the first time an approach aiming to the increase in sensitivity of the
homogeneous immunoassay with ICP-MS detection by the introduction of a
high number of tags into a monoclonal Ab has been presented, with the use of
polymer-based lanthanide group as useful carriers of multiple lanthanide ions.
This methodology has been applied for the simultaneous determination of four
cancer biomarkers (CEA, sErbB2, CA 15.3 and CA 125) in human serum.
Objetivos y Estructura general
de la Tesis Doctoral
Objetivos y Estructura general de la Tesis Doctoral
9
1. Objetivo de la Tesis Doctoral.
La presente Tesis Doctoral tiene como objetivo general el desarrollo de
nuevas metodologías basadas en el empleo de inmunoensayos y detección
mediante espectrometría de masas mediante ionización en plasma acoplado
inductivamente para la determinación de biomoléculas. Con ello se pretende
resolver algunas de las limitaciones que presentan este tipo de estrategias
analíticas, entre otras: (i) desarrollo de nuevas metodologías para el análisis de
haptenos (i.e., compuestos de bajo peso molecular, < 10 kDa); (ii) falta de
criterio para seleccionar el inmunoensayo más adecuado para una aplicación
bioanalítica dada; y (iii) escaso aprovechamiento del potencial de la técnica
(sensibilidad, límites de detección, capacidad de análisis multielemental, etc.).
Para ello, se plantean los siguientes objetivos específicos:
- Desarrollo de nuevas metodologías para la determinación de la
aflatoxina M1 (hapteno) en leche a niveles de ultra-traza.
- Desarrollo y evaluación de diferentes formatos de inmunoensayo
competitivo para el análisis de haptenos mediante ICP-MS.
- Desarrollo de una nueva metodología basada en inmunoensayos en
fase homogénea y detección mediante ICP-MS para el análisis
multicomponente de biomoléculas usando anticuerpos conjugados con
polímeros de lantánidos.
2. Estructura de la Tesis Doctoral.
La Presente Tesis Doctoral ha sido realizada en el grupo de investigación
de Espectrometría Atómica Analítica perteneciente al Departamento de
Objetivos y Estructura general de la Tesis Doctoral
10
Química Analítica, Nutrición y Bromatología de la Universidad de Alicante.
Además, parte de la investigación ha sido desarrollada en colaboración con el
Laboratory of Bioinorganic Analytical and Environmental Chemistry (University
of Pau and Pays de l’Adour) dirigido por el Doctor Ryszard Lobinski.
Dado que la presente Tesis Doctoral opta a la Mención Internacional, los
capítulos de la misma correspondientes a los resultados obtenidos han sido
redactados en inglés, para así, cumplir con la normativa establecida.
Esta Tesis Doctoral se encuentra estructurada en un total de seis capítulos:
Capítulo 1. Introducción general.
Este capítulo presenta los conocimientos básicos que han hecho posible el
desarrollo de la presente Tesis Doctoral. Así, en primer lugar, se describen los
principios básicos de los inmunoensayos para seguir con la exposición de las
ventajas que ofrece el empleo de ICP-MS como sistema de detección frente a
los que se vienen empleando de forma tradicional en inmunoensayos. Además,
se exponen diferentes estrategias analíticas para la cuantificación de
biomoléculas mediante inmunoensayos con detección mediante ICP-MS.
Finalmente, se describen las limitaciones actuales de los inmunoensayos con
detección mediante ICP-MS, haciendo especial hincapié en aquellas que se
van a investigar en la presente Tesis doctoral.
Capítulo 2. Determination of aflatoxin M1 in milk samples by means of an
inductively coupled plasma mass spectrometry-based immunoassay.
Este trabajo describe un nuevo método para la determinación de la
aflatoxina M1 (AFM1) en niveles de ultra-traza en leche. La detección de la
Objetivos y Estructura general de la Tesis Doctoral
11
AFM1 se lleva a cabo mediante un inmunoensayo competitivo que utiliza
anticuerpos secundarios biotinilados y estreptavidina conjugada con
nanopartículas de Au. Después de la adición de ácido, las nanopartículas se
descomponen y la señal de Au es registrada por medio de ICP-MS. Los
resultados demuestran que, en condiciones óptimas, el límite de detección del
inmunoensayo (0.005 g kg-1) es lo suficientemente bajo como para cuantificar
la AFM1 según las normativas internacionales actuales (incluida la europea que
es la más restrictiva). Además, queda patente, que el uso del ICP-MS para el
análisis de micotoxinas en alimentos tiene un gran potencial debido a que
presenta un mayor rango dinámico, señales de fondo más bajas e
independencia de la respuesta analítica a los tiempos de incubación o
almacenamiento con respecto a los métodos de detección convencionales.
Los resultados de este capítulo han dado lugar a las siguientes
contribuciones científicas:
Publicaciones:
1. E. Pérez, P. Martínez-Peinado, F. Marco, L. Gras, J. M. Sempere, J.
Mora, G. Grindlay, Determination of aflatoxin M1 in milk samples by
means of an inductively coupled plasma mass spectrometry-based
immunoassay, Food Chemistry 230 (2017) 721-727.
Comunicaciones orales:
2. G. Grindlay, E. Pérez, F. Marco, P. Peinado, J. Mora. Analysis of
aflatoxin M1 in milk by means of ICP-MS. EWCPS 2015 – European
Winter Conference on Plasma Spectrochemistry 2015. Münster
(Germany), 22-26th February 2015.
Comunicaciones (Póster):
Objetivos y Estructura general de la Tesis Doctoral
12
3. E. Pérez, G. Grindlay, F. Marco, P. Martínez, J. Mora. Inductively
coupled plasma mass spectrometry as a tool for aflatoxin M1 detection in
milk samples. EUROANALYSIS 2015 Conference. Bordeaux (France),
6th-10th September 2015.
Capítulo 3. Evaluation of different competitive immunoassay for aflatoxin M1
determination in milk samples by means of inductively coupled plasma mass
spectrometry.
En este trabajo, se han comparado sistemáticamente diferentes formatos
de inmunoensayos competitivos para la determinación de haptenos mediante
ICP-MS utilizando nanopartículas de diferente naturaleza y tamaño. La
aflatoxina M1 ha sido seleccionada como analito modelo para evaluar las
ventajas e inconvenientes de los diferentes formatos estudiados, debido a su
alto riesgo para la salud y a los niveles de seguridad requeridos por las
actuales políticas internacionales. De acuerdo a los resultados obtenidos, el
inmunoensayo basado en el marcaje del anticuerpo, en lugar del antígeno,
parece ser el más adecuado para el análisis de haptenos mediante ICP-MS ya
que esta estrategia es la que mejor explota la gran capacidad de detección que
ofrece ICP-MS.
Los resultados de este capítulo han dado lugar a las siguientes
contribuciones científicas:
Publicaciones:
1. E. Pérez, F. Marco, P. Martínez-Peinado, J. Mora, G. Grindlay.
Evaluation of different inductively coupled plasma mass spectrometry-
Objetivos y Estructura general de la Tesis Doctoral
13
based immunoassays for the determination of aflatoxin M1 in milk. (En
redacción).
Comunicaciones orales:
2. E. Pérez, G. Grindlay, F. Marco, P. Martínez-Peinado, J. Mora.
Evaluation of different inductively coupled plasma mass spectrometry-
based immunoassays for the determination of aflatoxin M1 in milk.
EWCPS 2017 – European Winter Conference on Plasma
Spectrochemistry 2017. Sankt Anton (Austria), 19-24th February 2017.
Capítulo 4. A sensitive size-exclusion inductively coupled plasma mass
spectrometry multiplexed assay for cancer biomarkers using antibodies
conjugated with a lanthanide-labelled polymer.
Este trabajo muestra, por primera vez, que anticuerpos marcados con
polímeros de lantánido pueden emplearse para la determinación simultánea de
cuatro biomarcadores de cáncer (CEA, sErbB2, CA 15.3 y CA 125) en suero
humano mediante cromatografía de exclusión por tamaño con detección de
ICP-MS. Los polímeros de lantánido llevan 30 veces más átomos del lantánido
correspondiente que el complejo lantánido-DOTA, utilizado tradicionalmente
para este fin; lo que ha dado como resultado una disminución de 10 veces en
los límites de detección. Además, la metodología analítica desarrollada, mejora
los límites de detección e incrementa el número de muestras que pueden ser
analizadas en un período de tiempo, al mismo tiempo que reduce los costes
operativos con respecto a los kits ELISA comerciales.
Los resultados de este capítulo han dado lugar a las siguientes
contribuciones científicas:
Objetivos y Estructura general de la Tesis Doctoral
14
Publicaciones:
1. E. Pérez, K. Bierla, G. Grindlay, J. Szpunar, J. Mora, R. Lobinski. A
sensitive size-exclusion inductively coupled plasma mass spectrometry
multiplexed assay for cancer biomarkers using antibodies conjugated
with a lanthanide-labelled polymer. (Submitted Analytica Chimica Acta).
Capítulo 5. Conclusiones generales.
En este capítulo se presentan las conclusiones más relevantes de las
metodologías analíticas desarrolladas en la presente Tesis Doctoral.
Capítulo 6. Futuros estudios.
En este capítulo se presentan algunos de los posibles futuros estudios que
podrían derivar del trabajo desarrollado en la presente Tesis Doctoral.
Capítulo 1
Introducción general
Capítulo 1
17
Nothing should be done
if the consequences might not be serious.
George Bernard Shaw
1. Introducción.
La cuantificación de biomoléculas es de gran importancia en una amplia
rama de campos científicos (e.g. biología, medicina, toxicología, farmacología,
etc.) ya que las funciones específicas en una célula o en un organismo están
controladas por cambios en los niveles de expresión proteica bajo diferentes
condiciones fisiológicas [1-4]. Actualmente, la herramienta principal para
cuantificar biomoléculas son los inmunoensayos, donde el analito de interés se
cuantifica mediante la unión específica que se establece entre un anticuerpo y
su correspondiente antígeno. El uso de esta técnica se inició a finales de los
años 50 cuando S.A. Berson y R. Yalow utilizaron por primera vez anticuerpos
marcados con radioisótopos (radioinmunoensayos, RIA) para la cuantificación
de insulina [5]. Aunque los métodos de RIA son fiables, precisos y altamente
sensibles, sufren de los problemas relacionados con el uso de radioisótopos
(como son: riesgo para la salud, problemas de eliminación de residuos y
estabilidad limitada) que restringen su uso a laboratorios especializados. Como
consecuencia, se han propuesto inmunoensayos basados en una detección
alternativa [6] como los ensayos de inmunoabsorción ligado a enzimas [7,8],
inmunoensayos quimioluminiscentes [9,10], inmunoensayos
electroquimioluminiscentes [11,12,13], etc. En los últimos 50 años, se han
Introducción general
18
aplicado con éxito a un gran número de moléculas de diferente tamaño,
propiedades químicas y físicas, y actividad biológica. Debido a su alta
sensibilidad, alta especificidad y coste, los inmunoensayos se han convertido
en métodos rutinarios para la detección y cuantificación de cientos de
moléculas tanto endógenas de organismos vivos (e.g. enzimas, proteínas,
hormonas, etc.) como exógenas (e.g., toxinas, fármacos). Sin embargo, estas
técnicas convencionales de detección de inmunoensayos a menudo presentan
baja sensibilidad, intervalo dinámico limitado y no son siempre adecuadas para
realizar análisis multicomponente [14-17].
La espectrometría de masas de plasma acoplado inductivamente (ICP-MS)
es indudablemente el instrumento comercial más sensible y más utilizado para
la determinación de una amplia gama de metales y varios no metales [18-21].
Las ventajas del ICP-MS como detector elemental incluye: bajos límites de
detección (a niveles de pg mL-1 para la mayoría de los elementos), bajos
efectos de matriz, amplios intervalos dinámicos y alta resolución espectral para
elementos e isótopos [4,22-31]. Es por todo ello que actualmente el ICP-MS se
ha convertido en una de las técnicas con mayor potencial en inmunoanálisis
desde que fuera publicado por primera vez por Zhang et al. [32].
A continuación, se presentarán algunos conceptos básicos de
inmunoensayos para facilitar la comprensión de las investigaciones realizadas
en la presente Tesis Doctoral. Además, se describirán las ventajas del empleo
de ICP-MS como sistema de detección en inmunoensayos y las estrategias
analíticas que se han empleado hasta la fecha para cuantificar biomoléculas.
Finalmente, se expondrán los retos que presentan los inmunoensayos con
Capítulo 1
19
detección mediante ICP-MS en la actualidad, haciendo hincapié en los que se
van abordar en la Tesis Doctoral.
2. Conceptos básicos de inmunoensayos.
Un inmunoensayo es un tipo de prueba bioquímica que utiliza anticuerpos
contra un analito de interés como medio para detectar la presencia de este
último. El analito al que se unen los anticuerpos se llama antígeno; aunque
este término se refiere a aquella sustancia capaz de desencadenar la
formación de anticuerpos y puede causar una respuesta inmunitaria.
Generalmente, en un inmunoensayo, los anticuerpos se inmovilizan en una
superficie sólida y al ser muy selectivos, sólo se unen a su correspondiente
antígeno, incluso en matrices muy complejas. La detección del correspondiente
inmunocoplejo entre el anticuerpo y su analito siempre requiere del marcaje de
alguna de las especies con un heteroátomo que facilite la detección.
Los inmunoensayos destacan por su especificidad, sensibilidad y
flexibilidad únicas debido a tres importantes características de los anticuerpos:
- Su capacidad para unirse a una amplia gama de productos químicos, ya
sean naturales o artificiales; biomoléculas; células y virus.
- Su elevada especificidad.
- La fuerza de la unión entre un anticuerpo y su antígeno.
2.1. Anticuerpos.
La eficiencia de cualquier reacción inmune es altamente dependiente de la
especificidad y de la afinidad del anticuerpo por su antígeno. Es importante
tener en cuenta que la afinidad del anticuerpo es el factor limitante en una
Introducción general
20
reacción inmune. El tipo de anticuerpo utilizado en inmunoensayos es la
inmunoglobulina G (IgG), que representa el 75% de todas las inmunoglobulinas
séricas [33]. Consiste en dos cadenas pesadas idénticas (H) de 50 kDa y dos
cadenas ligeras idénticas (L) de 24 kDa [34]. Las cadenas ligera y pesada, así
como su homólogo idéntico, están unidas por fuerzas no covalentes y enlaces
disulfuro (ver Figura 1.1). Los extremos N-terminal de cada cadena ligera y
pesada son altamente variables y representan los dos sitios de unión al
antígeno. Los extremos C-terminal son constantes en su secuencia de
aminoácidos. Si el anticuerpo se hace reaccionar con la enzima papaína, éste
se divide en tres partes: dos fragmentos Fab (fragmento de unión al antígeno)
con actividad inmunogénica y un fragmento Fc (fragmento cristalizable).
La obtención de anticuerpos se puede realizar vacunando animales
huéspedes con el antígeno contra el que se quiere producir el anticuerpo
(inmunización) o mediante biotecnología. Los anticuerpos producidos pueden
ser de 3 tipos: policlonales, monoclonales y recombinantes. Los anticuerpos
policlonales se obtienen por inmunización de animales y son relativamente
baratos en grandes cantidades; sin embargo, pueden reaccionar de forma
cruzada con compuestos estructuralmente similares. Los anticuerpos
monoclonales se obtienen mediante biotecnología combinando un linfocito B
productor del anticuerpo específico de interés con una línea celular de
mieloma. Estos anticuerpos poseen características de unión específicas, ya
que no sólo reconocen a un mismo antígeno sino que también se unen al
mismo epítopo (porción de la molécula reconocida por el anticuerpo) del
antígeno, dando lugar a ensayos mucho más específicos. El principal
inconveniente de los anticuerpos monoclonales es que son caros de producir y
Capítulo 1
21
técnicamente exigente para generar y mantener una línea celular de hibridoma.
Por último, el desarrollo de la biología molecular ha permitido la producción de
anticuerpos en ausencia de inmunización, conocidos como anticuerpos
recombinantes. Los anticuerpos recombinantes ofrecen ventajas sobre los
anteriores en términos de facilidad de producción, mayor repertorio para
selección y versatilidad.
Figura 1.1. Esquema de la inmunoglobulina G (IgG). Las cadenas ligera (azul
claro) y pesada (azul oscuro) están conectadas por enlaces disulfuro. Las
partes con secuencia de aminoácidos variable representan los sitios de unión al
antígeno. VH: parte variable de la cadena pesada; VL: parte variable de la
cadena ligera. Tras la digestión con papaína, el anticuerpo se disocia en tres
partes: dos fragmentos Fab (unión al antígeno) y un fragmento Fc (región
cristalizable).
Introducción general
22
2.2. Tipos de inmunoensayos.
2.2.1. Inmunoensayos no-competitivos.
El inmunoensayo más sencillo (Figura 1.2) consiste en un anticuerpo,
inmovilizado en una superficie de plástico (e.g. un pocillo de una placa de
inmunológica), que captura el antígeno presente en la muestra. A continuación,
se utiliza un anticuerpo diferente, específico de otro epítopo, como base del
sistema de detección. Este segundo anticuerpo debe estar marcado con alguna
especie que facilite la detección del inmunocomplejo formado (trazador). La
especie que permite monitorizar el inmunocomplejo suele ser una enzima. Al
igual que los anticuerpos, las enzimas son proteínas que se unen a analitos
específicos; sin embargo, las enzimas también son capaces de catalizar
reacciones específicas. La molécula sobre la que actúa una enzima se llama
sustrato. Las enzimas empleadas como marcadores, con el sustrato
apropiado, pueden usarse para generar color o crear productos finales
fluorescentes o luminiscentes, que pueden medirse fácilmente mediante
equipos ópticos y electrónicos. Además, cada molécula de enzima puede
unirse a varias moléculas de sustrato, amplificando la señal. Este formato de
ensayo a menudo se conoce como ensayo de inmunoabsorción ligado a
enzimas (ELISA). En cualquier caso, independientente del trazador empleado,
la señal que proporciona el marcador es proporcional a la concentración de
antígeno presente en la muestra. Una etapa crítica del ensayo es la eliminación
del trazador no unido durante una la etapa lavado. El material (normalmente de
plástico) al que el anticuerpo de captura se une irreversiblemente se conoce
como la fase sólida. Debido a que los anticuerpos forman un sándwich
Capítulo 1
23
alrededor del antígeno, estos ensayos inmunométricos también son conocidos
como inmunoensayos tipo sándwich.
Figura 1.2. Inmunoensayo no- competitivo.
Otro tipo de ensayo inmunométrico consiste en detectar anticuerpos en una
muestra fijando el antígeno en el soporte. Esta aplicación es útil, por ejemplo,
para detectar la exposición previa a una enfermedad infecciosa específica. Las
proteínas que se producen en la superficie de un virus se inmovilizan en la fase
sólida y son las encargadas de capturar los anticuerpos específicos, para ese
virus, presentes en la muestra. En esta estrategia, como trazador podría usarse
un anticuerpo marcado (anticuerpo secundario) contra la región constante del
primer anticuerpo (Figura 1.3).
2.2.2. Inmunoensayos competitivos.
Los ensayos inmunométricos, descritos hasta el momento, funcionan bien
cuando el antígeno es una molécula grande con suficiente área superficial para
+ + +
Fase sólida recubierta de anticuerpo
Analito Anticuerpo marcado
Separación del anticuerpo marcado libre
Concentración de analito
Se
ña
l
Respuesta lineal
Saturación
Introducción general
24
acomodar dos moléculas de anticuerpo. Sin embargo, muchos inmunoensayos
tienen como objetivo determinar la presencia de moléculas pequeñas (tóxicos,
hormonas, etc.) que sólo presentan un epítopo. Estas moléculas se las conoce
como haptenos y para desarrollar anticuerpos específicos contra ellas
requieren conjugarlas con alguna proteína de forma que se estimule una
respuesta inmunitaria.
Figura 1.3. Inmunoensayo no-competitivo para la detección de anticuerpos.
En este tipo de ensayos, se utiliza normalmente un anticuerpo presente en una
concentración limitada (Figura 1.4). El otro reactivo clave, el trazador, consiste
en el antígeno marcado con, por ejemplo, un radioisótopo o una enzima. La
cantidad de trazador que se une al anticuerpo es indirectamente proporcional a
la concentración de antígeno presente en la muestra. En este tipo de ensayo,
conocido como inmunoensayo de tipo competitivo, las concentraciones de
anticuerpo y trazador son críticas, a diferencia de los inmunoensayos de tipo
sándwich donde se usa un exceso de anticuerpo respecto del antígeno. Es
importante señalar que este tipo de inmunoensayo también se puede llevar a
+ + +
Fase sólida recubierta de antígeno
Anticuerpo Anticuerpo secundario
Separación del anticuerpo marcado libre
Concentración de anticuerpo
Se
ña
l
Respuesta lineal
Saturación
Capítulo 1
25
cabo fijando la molécula de analito modificada en el soporte y utilizando el
anticuerpo como trazador. En ocasiones, para evitar que el marcado del
anticuerpo altere la afinidad con el antígeno se utilizan sistemas auxiliares
como un anticuerpo secundario.
Figura 1.4. Inmunoensayo competitivo.
2.2.3. Immunoensayos en fase heterogénea y homogénea.
Todos los inmunoensayos descritos hasta el momento dependían de la
separación física del trazador no unido antes de medir la señal correspondiente
al trazador unido. Sin una etapa de separación, la intensidad de señal sería
siempre la misma, independientemente de la concentración de analito. Estos
formatos de ensayo son todos ejemplos de inmunoensayos de tipo
heterogéneo. Por el contrario, los inmunoensayos de tipo homogéneo se
basan en un cambio en la actividad del inmunorreactivo marcado que se
produce cuando el antígeno se une al anticuerpo para formar el
inmunocomplejo y no requieren ningún paso de separación (Figura 1.5).
+ + +
Fase sólida recubierta de anticuerpo
Analito Analitomarcado(trazador)
Separación del analitomarcado libre
Concentración de analito
Se
ña
l
Introducción general
26
Figura 1.5. Inmunoensayo homogéneo.
Aunque la etapa de separación complica el procedimiento, los inmunoensayos
de tipo heterogéneo a menudo proporcionan límites de detección superiores
respecto a los inmunoensayos de tipo homogéneo, convirtiéndolos en los
métodos más frecuentemente adoptados.
3. La Espectrometría de Masas de Plasma de Acoplamiento
Inductivo como herramienta de cuantificación de biomoléculas.
En los últimos años, el desarrollo de nuevas herramientas de bioanálisis ha
permitido avanzar enormemente en campos como la metalómica, proteómica y
metabolómica. Gracias a ello, los laboratorios de análisis clínico, por ejemplo,
pueden controlar los niveles de biomarcadores tanto endógenos (e.g.,
proteínas cuyo contenido en fluidos biológicos se pueda ver alterado por
procesos bioquímicos o enfermedades como cáncer o infecciones) como
exógenos (e.g., compuestos tóxicos o toxinas), lo que permite mejorar el
diagnóstico, tratamiento y evolución de diversas enfermedades. También
+ + +
Anticuerpo Antígeno Antígenomarcado
Generación de señal por launión del antígeno marcadoal anticuerpo (no se requiereseparación)
Concentración de analito
Se
ña
l
Capítulo 1
27
facilita los estudios farmacocinéticos de los tratamientos recibidos, lo que
permite su ajuste o el diseño de fármacos más seguros.
Muchas de las biomoléculas de interés en proteómica y metabolómica se
suelen determinar mediante métodos de inmunoensayos basados en detección
mediante absorción en UV-Vis (ELISA), fluorescencia, o quimioluminiscencia,
entre otros. Sin embargo, estas técnicas convencionales de detección de
inmunoensayos a menudo no poseen un amplio intervalo dinámico y presentan
baja sensibilidad. Además, se produce la superposición de señales, no
pudiendo ser utilizados para la cuantificación multianalítica simultánea. La
Tabla 1.1 resume una comparativa de los inmunoensayos de uso común.
El ICP-MS es indudablemente el instrumento comercial más sensible para
la determinación de una amplia gama de metales y varios no metales
[18,19,20,21]. El gran atractivo del ICP-MS viene determinado por su: (i)
elevada sensibilidad y bajos límites de detección (ng Kg-1); (ii) amplio intervalo
dinámico (iii) capacidad multielemental; (iv) información isotópica; (v) robustez;
y (vi) facilidad de acoplamiento a técnicas cromatográficas. Esto ha hecho que
actualmente se haya convertido en una de las técnicas con mayor potencial en
inmunoanálisis desde fuera publicado por primera vez por Zhang et al. [32].
Intro
du
cción
gen
eral
Tab
la 1.1. Co
mp
ara
tiva
entre
los d
ifere
nte
s tip
os d
e in
mu
no
en
sa
yos.
D
etección
del in
mu
no
ensayo
R
adio
isóto
pos
UV
-Vis
Q
uim
iolu
min
iscencia
F
luore
ccencia
E
lectro
qu
imio
lum
inis
cencia
IC
P-M
S
Año d
e
inve
nció
n
1959
[5]
1971
[7]
1976
[9]
1979
[35
] 1991
[11
] 2001
[32
]
Sensib
ilidad
A
lta
Med
ia
Alta
Alta
Alta
Alta
Cara
cte
rístic
as
del tra
zador
Radio
activo
P
rodu
zca s
eñal
colo
rimétric
a
Pro
du
zca s
eñal
quim
iolu
min
iscente
Pro
du
zca s
eñal
fluore
scente
Pro
du
zca s
eñal
ele
ctro
quim
iolu
min
iscente
Sin
requerim
iento
especia
l
Vid
a ú
til de lo
s
reactiv
os
Corta
M
ed
ia
Med
ia
Med
ia
Med
ia
Larg
a
Esta
bilid
ad
M
ed
ia
Alta
M
ed
ia
Med
ia
Med
ia
Alta
Pote
ncia
l para
multiá
na
lisis
B
ajo
B
ajo
Bajo
Bajo
Bajo
Excele
nte
Coste
de
intru
menta
ció
n y
opera
tivo
Med
io
Bajo
M
ed
io
Med
io
Med
io
Alto
Capítulo 1
29
3.1. Conceptos básicos de ICP-MS.
El ICP-MS se introdujo a principios de la década de 1980 [36], y hoy en día
se ha convertido en una poderosa técnica para la determinación de elementos
traza, minoritarios y mayoritarios en una variedad de muestras [37,38,39]. En
esta técnica, la muestra se introduce en forma de aerosol líquido en la base de
un plasma de argón; siendo este último la fuente de excitación y/o ionización
más común en Espectrometría Atómica. A diferencia de las fuentes de
ionización suave, como la ionización por electroespray (ESI) y la
desorción/ionización láser asistida por matriz (MALDI), el ICP opera a elevada
temperatura, entre 6.000 y 10.000 K [37]; esta energía es suficiente para la
desolvatización, volatilización de la muestra, atomización e ionización de los
átomos formados (Figura 1.6). Los iones generados en el plasma se introducen
en el analizador de masas, donde los iones se separan por su relación masa-
carga (m / z). Tras ello, son detectados y finalmente convertidos en una señal
eléctrica.
Figura 1.6. Procesos que sufre la muestra al ser introducida en el seno del
plasma.
Aerosol con la muestra
RecombinaciónM+ + 1e- MM+ + O MO+
DesolvataciónH2O (l) H2O (g) Vaporización
MX (s) MX (g)
AtomizaciónMX (g) M + X
Ionización M M+
Analizador de masas
Analitopresente como M+
Introducción general
30
En un primer momento, la aplicación de ICP-MS en bioanálisis estuvo
limitada al análisis elemental inorgánico y, dadas las limitaciones de la técnica
para la determinación de elementos como C, O, N, etc., al de aquellas especies
orgánicas que contenían en su estructura algún metal, metaloide y/o no metal.
Así, por ejemplo, se aplicó a la determinación de proteínas que contenían
cisteína o metionina a partir de la señal de S [40]. De igual forma, se aplicó a la
determinación de otras especies que contenían heteroátomos como: (i) P (e.g.
proteínas fosforiladas y ADN) [41]; (ii) Se (e.g. selenoproteinas) [42]; (iii) As
(e.g. arsenoazúcares) [43]; y (v) metales (e.g. enzimas y metaloproteínas)
[44,45]. El gran atractivo de este tipo de aplicaciones radica en que, al conocer
de antemano el número de heteroátomos por molécula, se pueden emplear las
estrategias de calibración habituales en análisis inorgánico (i.e. patrones
externos, adición de estándar y dilución isotópica). No obstante, este tipo de
análisis presenta diversas limitaciones que afectan a la exactitud de los
resultados y a los límites detección. Así, la mayor parte de los heteroátomos
presentes en las biomoléculas (e.g., P, S, Se, As, etc.) son muy poco sensibles
en ICP-MS por su elevado potencial de ionización (> 9 eV). Por otro lado, la
determinación de este tipo de elementos en matrices biológicas está afectada
por interferencias espectrales y no espectrales [40,41,42,46] que limitan sus
aplicaciones. En comparación, un analizador de sector magnético posee una
alta capacidad de resolución para distinguir los analitos de las inferencias,
incluso en muestras de matrices complejas. Sin embargo, el uso de ICP-MS de
alta resolución es limitado debido a su alto coste. Para superar las
interferencias en el cuadrupolo del ICP-MS, se desarrolló el uso de una célula
de colisión / reacción combinado con el ICP-MS [47]. Los detalles sobre su
Capítulo 1
31
desarrollo y su aplicación pueden encontrarse en la bibliografía y no se entrará
en más detalle dado que no es objeto de este estudio [37,47,48]. Finalmente es
importante indicar que el ICP y otras fuentes de ionización blandas (por
ejemplo, ESI o MALDI) son realmente técnicas complementarias [25]. La
información estructural se obtiene preferiblemente por medio de ESI o MALDI-
MS, mientras que el ICP-MS es ideal para detectar y cuantificar metales en
proteínas, incluso cuando están presentes en niveles muy bajos de
concentración.
3.2. Estrategias analíticas para la cuantificación de biomoléculas
mediante ICP-MS.
Como se mencionó anteriormente, los métodos basados en detección
mediante ICP-MS ofrecen conceptos simples de cuantificación, efectos de
matriz bajos en comparación con las técnicas bioanalíticas convencionales, y
límites de detección biológicamente relevantes (LoD) en el intervalo bajo pg g-1.
Sin embargo, la mayor parte de analitos de interés en bioanálisis no contienen
heteroátomos detectables mediante ICP-MS. Es por ello que, para tratar de
aprovechar las ventajas de la técnica en este campo se han investigado
diferentes metodologías de marcaje de biomoléculas con heteroátomos [30]. La
clave para una cuantificación fiable es que se produzca una reacción de
marcaje [28] bajo las siguientes condiciones generales:
- formación de un enlace estable entre la etiqueta y la proteína (o
escindible a propósito),
- reacción completa (cerca del 100%),
Introducción general
32
- reacción específica (reacción sólo con el grupo funcional objetivo, sin
reacciones laterales),
- reproducibilidad, y
- las condiciones de reacción no deberían interferir en las siguientes
etapas analíticas.
Además, las reacciones rápidas y evitar el uso de un exceso de reactivo,
debido a que implica menores señales de fondo y coste, son favorables. Así, se
han empleado métodos de derivatización utilizando: (i) I (las proteínas que
contienen histidina y tirosina pueden ser marcadas en un anillo bencénico con
este no-metal [49]); (ii) compuestos organometálicos [28] (los grupos tiol de la
cisteína pueden ser marcados con compuestos organomercúricos [50]); y (iii)
quelatos de lantánidos (DOTA/DTPA) [51] (complejos altamente estables que
pueden añadirse a las proteínas a través de los residuos tiol y amino de los
aminoácidos). El principal inconveniente de este tipo de estrategias es que, al
basarse en reacciones químicas, es difícil controlar la selectividad y el grado de
marcaje del analito en mezclas complejas. Una estrategia cuyo interés y
aplicaciones se ha extendido en los últimos años en el campo del bioanálisis
(Figura 1.7) es el marcaje indirecto de biomoléculas mediante reacciones
antígeno-anticuerpo en las que el anticuerpo se funcionaliza previamente con
un heteroátomo detectable mediante ICP-MS [52,53].
Capítulo 1
33
Figura 1.7. Número de publicaciones en la bibliografía que emplean ICP-MS
como detector de inmunoensayos para el análisis de biomoléculas. Motor de
búsqueda: Scopus. Palabras clave: ICP-MS y proteins/elemental
tags/immunoassays (enero 2018).
La Figura 1.8 muestra un esquema del modo de trabajo empleado
habitualmente para cuantificar biomoléculas mediante el uso combinado de
inmunoensayos e ICP-MS.
Figura 1.8. Metodología de análisis en los inmunoensayos que utilizan ICP-MS
como sistema de detección.
0
10
20
30
40
50
Núm
ero
de p
ublic
acio
nes
Año
BIOMOLÉCULA ICP-MS
Tipo
FormatoAnticuerpo
Heteroátomo
Nanopartículas Lantánidos
1. FUNCIONALIZAR ANTICUERPO
2. INMUNOENSAYO 3. DETECCIÓN
CompetitivoNo competitivo
Nebulizador
Señal
Ablación laser
Pulsos
Single Particle
F. HeterogéneaF. Homogénea
Otros
Introducción general
34
En primer lugar, se debe funcionalizar el anticuerpo (o el trazador) con un
heteroátomo a través de los enlaces disulfuro de los residuos de cisteína o los
grupos amino de los aminoácidos. Aunque sobre el papel se puede utilizar casi
cualquier elemento de la tabla periódica para funcionalizar anticuerpos, la gran
parte de los estudios realizados hasta el momento se centran en el uso de
nanopartículas de Au y/o Ag así como en quelatos de lantánidos. Estos últimos
consisten en un grupo reactivo, un enlazador o espaciador y un quelato, tal y
como se muestra esquemáticamente en la Figura 1.9. Estos ligandos
bifuncionales a menudo se unen covalentemente a grupos -amino de residuos
de lisina y a extremos N-terminales de grupos -amino a pH alcalinos. Los
residuos de isotiocianatobencilo (SCN) y éster de N-hidroxisuccinimida (NHS)
son frecuentemente los grupos reactivos utilizados para este tipo de marcaje
químico. Alternativamente, los residuos de maleimidoetilacetamida (maleimido)
se conjugan a los residuos de sulfhidrilo después de una reducción selectiva de
los puentes disulfuro de los residuos de cisteína del anticuerpo. Esta etapa de
reducción es crítica, ya que puede afectar la eficacia de unión del anticuerpo.
Con respecto al grupo quelante, diferentes compuestos lineales o macrocíclicos
basados en el ácido poliaminocarboxílico se suelen utilizar para acomplejar el
metal. Estos grupos quelantes se seleccionan en función de la carga de este
último. Además, deben tener una alta constante de equilibrio para resistir al
intercambio metálico. Los compuestos más usados son: ácido dietilen-triamino-
tetraacético (DTTA), ácido dietilentriaminapentaacético (DTPA) y ácido
1,4,7,10-tetraazaciclododecano-1,4,7,10- tetraacético (DOTA). Los lantánidos
poseen una respuesta altamente sensible en ICP-MS debido a: (i) su baja
abundancia en la naturaleza y, en consecuencia, la señal de fondo en las
Capítulo 1
35
matrices de muestras biológicas es baja; (ii) las interferencias poliatómicas rara
vez son significativas; y, (iii) la eficacia de ionización de los lantánidos es alta
debido a que el primer potencial de ionización es bajo. Además, los lantánidos
tienen propiedades químicas similares y, por lo tanto, son muy adecuados para
desarrollar ensayos multicomponente basados en detección mediante ICP-MS.
Figura 1.9. Ligando bifuncional empleado para marcar anticuerpos. El quelato
se une a un grupo reactivo, lo que permite la conjugación con el anticuerpo. Se
introduce un espaciador entre el quelato y el grupo reactivo para mejorar la
reactividad. M3+ es típicamente un ion lantánido.
Las nanopartículas se suelen emplear en ensayos monocomponente por su
capacidad para amplificar la señal analítica, debido a que incluyen miles de
átomos por conjugado. El tamaño de partícula puede variar entre el más
pequeño de 2nm (clúster de oro) a más de 100nm (oro coloidal), dependiendo
de la aplicación. Especialmente las nanopartículas de Au son a menudo
empleadas como marcadores de anticuerpos y enzimas debido a su robustez y
fácil manejo. Las nanopartículas están conjugadas con grupos reactivos como
residuos de maleimido o NHS para el marcaje, así como también ligandos
Quelato
Espaciador
Ligando bifuncional
Grupo reactivo
Introducción general
36
bifuncionales. Pero hay algunos puntos críticos a tener en cuenta durante el
uso de nanopartículas de Au: el oro tiene una gran afinidad por las superficies
y, por lo tanto, las etapas de bloqueo y lavado son críticas. Además, es difícil
sintetizar nanopartículas de tamaño uniforme, las cuales son necesarias
cuando la finalidad es la cuantificación. No obstante, existen estrategias
prometedoras con lantánidos que permiten mejorar significativamente la
sensibilidad y los límites de detección (polímeros [54] y nanopartículas de
lantánidos [55]).
Una vez funcionalizado el anticuerpo con el heteroátomo, se procede a
llevar a cabo el inmunoensayo con la biomolécula de interés. Finalmente, la
última etapa del análisis es la detección del analito a través del heteroátomo
mediante ICP-MS. Normalmente, y dado que el volumen de muestra disponible
en un inmunoensayo no es muy elevado (100-200 µL), la muestra se analiza en
forma líquida con un sistema de inyección en flujo acoplado a un nebulizador y
a una cámara de nebulización [52,53]. No obstante, cuando se trabaja con
electroforesis en gel o directamente en tejidos se puede utilizar un sistema de
ablación laser. En este caso, además de estudiar la distribución de una
determinada especie, se mejora la velocidad de análisis y se evita la dilución
del analito (mayor sensibilidad). Finalmente, cabe indicar, que algunos autores
han sugerido el empleo del modo single particle cuando se trabaja con
nanopartículas [56]. En este caso, la concentración de analito está relacionada
con la frecuencia con la que se detectan los pulsos de señal de las
nanopartículas al atomizarse e ionizarse. Sin embargo, a pesar de las
potenciales ventajas que puede presentar (e.g. menores límites de detección),
este modo de medida está muy poco estudiado hasta el momento.
Capítulo 1
37
3.3. Limitaciones de los inmunoensayos con detección mediante
ICP-MS.
Tal y como acabamos de revisar, los estudios realizados hasta el momento
nos permiten afirmar que las metodologías de bioanálisis basadas en el empleo
de ICP-MS se encuentran en un buen estado de desarrollo. Sin embargo,
todavía se está muy lejos de conseguir explotar todo el potencial de la técnica,
ya que existen importantes retos aún por resolver [52,53]. Así, en lo que se
refiere al tipo de analito, la mayor parte de aplicaciones desarrolladas hasta el
momento se han centrado en la cuantificación de biomoléculas de elevado
peso molecular (proteínas, marcadores tumorales, enzimas, ADN, etc). Las
aplicaciones a biomoléculas sencillas de bajo peso molecular (haptenos; masa
molecular <10000 Da) han sido mucho más escasas ya que, al no provocar
respuesta inmunológica, es más complicado obtener anticuerpos para este tipo
de analito. Además, hace necesario el uso de inmunoensayos de tipo
competitivo [57,58] que, desde el punto de vista analítico, son claramente
menos atractivos que los ensayos tipo sándwich. De hecho, para este tipo de
analito se suelen utilizar técnicas cromatográficas acopladas a Espectrometría
de Masas en lugar de inmunoensayos. Además, en la bibliografía tampoco es
posible encontrar un estudio sistemático que compare las ventajas e
inconvenientes de los distintos formatos de inmunoensayos competitivos
comúnmente empleados para el análisis de haptenos. En cuanto al tipo de
heteroátomo elegido, las especies más empleadas por su disponibilidad
comercial y prestaciones analíticas son los quelatos de lantánidos y las
nanopartículas metálicas de Au/Ag [53]. Sin embargo, el efecto de la naturaleza
y del tamaño de las nanopartículas sobre los parámetros analíticos de ensayos
Introducción general
38
competitivos con detección mediante ICP-MS no ha sido estudiado en
profundidad. Otro aspecto a tener en cuenta es que la gran parte de
inmunoensayos se suele realizar en fase heterogénea ya que proporciona una
mayor capacidad de detección que los ensayos en fase homogénea. No
obstante, estos últimos presentan el gran atractivo que se pueden realizar en
una sola etapa con la consiguiente mejora en la velocidad de análisis, coste y
reproducibilidad al no utilizar placas u otro tipo de soporte. En este tipo de
ensayos, se requiere de una separación cromatográfica previa para separar el
inmunocomplejo del anticuerpo que no ha reaccionado [59]. Hasta la fecha, se
han realizado estudios en fase homogénea utilizando quelatos de lantánidos o
In [59,60,61]. Si bien los resultados son prometedores, sería interesante
evaluar el uso de polímeros de lantánidos ya que permitiría aumentar la
sensibilidad debido a la presencia de más de un lantánido por cada etiqueta en
el anticuerpo marcado. Además, a pesar de que la mayor parte de los estudios
realizados resaltan las capacidades de análisis multicomponente de ICP-MS,
en muy contadas ocasiones se aprovecha dicho potencial.
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[56] G. Han, Z. Xing, Y. Dong, S. Zhang & X. Zhang. One-step homogeneous
DNA assay with single-nanoparticle detection. Angew Chem Int Ed 50
(2011) 3462-3465.
[57] A.R. Montoro Bustos, L. Trapiella-Alfonso, J. Ruiz Encina, J.M. Costa-
Ferández, R. Pereiro & A. Sanz-Medel. Elemental and molecular detection
for quantum dots-based immunoassays: a critical appraisal. Biosensors and
bioelectronics 33 (2012) 165-171.
Introducción general
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[58] P. Jarujamrus, R. Chawengkirttikul, J. Shiowatana & A. Siripyanoud.
Towards chloramphenicol detection by inductively coupled plasma mass
spectrometry (ICP-MS) linked immunoassay using gold nanoparticles
(AuNPs) as element tags. J. Anal. At. Spectrom. 27 (2012) 884-890.
[59] M. Terenghi, L. Elviri, M. Careri, A. Mangia & R. Lobinski. Multiplexed
determination of protein biomarkers using metal-tagged antibodies and size
exclusion chromatography−inductively coupled plasma mass spectrometry.
Anal. Chem. 81 (2009) 9440-9448.
[60] L. López-Fernández, E. Blanco-González & J. Bettmer. Determination of
specific DNA sequences and their hybridisation processes by elemental
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(2010) 861-866.
Capítulo 2
Determination of aflatoxin M1 in milk
samples by means of an inductively coupled
plasma mass spectrometry-based
immunoassay
Chapter 2
49
1. Introduction.
Aflatoxins are secondary metabolites produced by different filamentous
fungi (mainly Aspergillus species) and they are known to represent a high risk
for human health due to their mutagenic and teratogenic effects. These
substances could be found in different kinds of food and animal feeds (e.g.
cereals, cocoa, coffee, etc.) that have been in contact with fungi through the
food chain under high temperature and humidity conditions [1].
Aflatoxin M1 (AFM1) is the most significant aflatoxin in milk and dairy
products. This compound is the hydroxylated form of the aflatoxin B1 (AFB1) and
it is usually present in milk when animals have been fed with feedstuffs
containing AFB1 [2]. Aflatoxin M1 has been classified as Group 2 human
carcinogen by the International Agency of Research on Cancer [3]. For this
reason, and taking into account the significance of milk and milk products in
human diet (especially for children), the maximum allowed levels of AFM1 are
strictly regulated worldwide [2,4]. Food and Drug Administration from USA limits
the concentration of AFM1 in milk and processed milk products at 0.50 µg kg-1
[5]. However, European Community Legislation is even more restrictive and
does not allow AFM1 levels in milk and infant formula above 0.050 and 0.025 µg
kg-1, respectively [6,7].
Aflatoxin M1 determination is usually carried out by means of high-
performance liquid chromatography (HPLC) or immunoassays after an
extraction treatment to reduce matrix effects and pre-concentrate the analyte
[8,9]. HPLC is considered the reference method for AFM1 analysis
[10,11,12,13]. The detection of AFM1 is generally achieved by means of both
fluorescence and mass spectrometry. However, HPLC analysis requires
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
50
laborious sample preparation treatments to reduce matrix effects and improve
analytical figures of merit. On the other hand, immunoassays (mainly Enzyme
Linked Immunosorbent Assay, i.e., ELISA) are widely used for screening
purposes due to their high sample throughput, simplicity and low budget
[14,15,16,17].
Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful
technique for inorganic analysis due to its: (i) low limits of detection (LoD)
(usually in the µg kg-1-ng kg-1 range), (ii) good precision; (iii) multi-element
capability, (iv) high dynamic range; and (v) the possibility to obtain analyte
isotopic information [18]. Traditionally, the analysis of organic molecules by ICP-
MS has been limited to those analytes containing metals, metalloids and some
non-metals (e.g. P or S) because of the difficulty to utilize C, H, N and O for
quantification purposes at ultra-trace levels. However, it has been demonstrated
that ICP-MS can be used as a detector for all kinds of organic molecules (e.g.
proteins, mRNA, DNA, etc.) after a derivatization procedure with a heteroatom
or a compound containing a heteroatom [19,20]. In this context, ICP-MS has
been employed as a detector of proteins and biomolecules in immunoassays in
view of it is quite straightforward to functionalized antibodies with elements
detectable by this technique [21,22]. In general, antibodies (or any other
species present in the immunoassay) are conjugated with elements presumably
not present in biological samples, such as lanthanide based chelates or Au
nanoparticles. The latter approach is especially advantageous to amplify the
analytical response because of the significant number of quantities of Au atoms
in each nanoparticle. The use of ICP-MS as a detector in immunoassays affords
several attractive features such as: (i) specificity to heteroatom detection; (ii)
Chapter 2
51
compound-independent detection sensitivity; (iii) high elemental sensitivity and
dynamic range; (iv) robustness (complex sample pre-treatments are not
required to diminish matrix effects); and (vii) multielement capabilities, since the
antibodies can be conjugated with different heteroatoms and detected in a
single run. In spite of the above mentioned features, the use of ICPMS-based
immunoassays in food analysis has been limited so far. Nonetheless, these
methods have been successfully applied to quantify peanut allergens [23],
ochratoxine A in wine [24] and progesterone in milk [25].
The goal of this work is to develop a new procedure to quantify AFM1 in milk
samples by ICP-MS at the security levels required by the current international
policies with accuracy and precision. The proposed methodology is based on a
competitive immunoassay using secondary biotinylated antibodies and
streptavidin-Au nanoparticles conjugate followed by Au detection by ICP-MS.
2. Experimental.
2.1. Reagents and materials.
All solutions were prepared using ultrapure water (Milli-Q water purification
system, Millipore Inc., Paris, France).
Sodium carbonate, sodium hydrogen carbonate, monosodium phosphate,
disodium phosphate, sodium chloride, biotinylated goat α-rabbit immunoglubulin
G (IgG) secondary antibody (secondary Ab), aflatoxin M1 (AFM1) from
Aspergillus flavus, AFM1-Bovine serum albumin conjugate (AFM1-BSA),
streptavidin-40 nm Au nanoparticles conjugate from Streptomyces avidinii,
polyethylene glycol sorbitan monolaurate (Tween 20) and HPLC-grade
acetonitrile were purchased from Sigma-Aldrich (Steinheim, Germany). Bovine
serum albumin (BSA) was obtained from Biowest (Nuaillé, France) whereas α-
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
52
AFM1 primary rabbit polyclonal antibody (pAb) was obtained from Agrisera
(Vännas, Sweeden). Iridium 1000 mg L-1 stock solution was provided by Merck
(Darmstadt, Germany). Thiourea, 69% w w-1 nitric acid and 35% w w-1
hydrochloric acid were purchased from Panreac (Barcelona, Spain).
F16 maxisorp polystyrene microtiter plates were obtained from Thermo-
Scientific (Roskilde, Denmark).
2.2. Buffers and solutions.
Standard stock solution of AFM1 (10 µg mL-1) was prepared in pure
acetonitrile in an amber vial. AFM1-BSA was dissolved in 2 mL of phosphate
buffer solution (PBS, 10 mol L-1 monosodium phosphate, 2 mmol L-1 disodium
phosphate, 154 mmol L-1 sodium chloride, pH 7.6) for a final concentration of
500 µg mL-1. Both solutions were kept at -200C. Primary rabbit polyclonal
antibody was dissolved in 500 µL ultrapure water and kept at 40C.
The following solutions were employed in the ICPMS-based immunoassay:
(a) carbonate/bicarbonate buffer solution (15 mmol L-1 sodium carbonate and
35 mmol L-1 sodium hydrogen carbonate, pH 9.6); (b) 1% w V-1 BSA in a PBS
solution for plate blocking; (c) 1% w V-1 BSA and 0.05% V V-1 Tween 20 in PBS
as incubation medium; (d) 0.05% V V-1 Tween 20 in PBS for washing microtiter
plate wells medium and (d) 4% V V-1 nitric acid and 12% V V-1 hydrochloric acid
for Au-nanoparticles digestion.
Chapter 2
53
2.3. Immunoassay procedure.
The analysis of AFM1 by ICP-MS is based on a competitive immunoassay
[24] in which varying amounts of free AFM1 inhibit the binding of specific pAb to
the solid phase coated with AFM1-BSA conjugate using secondary biotinylated
antibodies and streptavidin- Au nanoparticles conjugate for ICP-MS detection
(see Figure 2.1). First of all, the polystyrene microtiter plate wells were coated
with 100 µL of the appropriate AFM1-BSA concentration in carbonate-
bicarbonate buffer (step 1a). After 1 h incubation at room temperature, wells
were washed three times and blocked with 1% w V-1 BSA in PBS for 1 h at
room temperature. Simultaneously, samples or AFM1 standards were mixed
with the pAb solution (step 1b). The mixture was incubated 1 h at room
temperature and then 100 µL of it were transferred to the plate wells for another
incubation step of 2.5 h at room temperature (step 2). After washing -three
times in order to eliminate the antigen-antibody complexes present in the
solution as well as the free pAb, the microwell plates were sequentially
incubated with 100 µL of a secondary Ab solution (step 3) and then with 100 µL
of the streptavidin-Au nanoparticles conjugate solution (step 4). The incubation
time for the previous steps was 1 h at room temperature followed by three
washing steps. Finally, before ICP-MS analysis, Au nanoparticles were digested
(step 5) with 150 µL of the digestion acid mixture and spiked with 50 µL of a
1.0% w V-1 thiourea solution containing 2.5 µg L-1 Ir. All the AFM1 standards
were analyzed in triplicate wells whereas samples containing unknown AFM1
amounts in quintuplicate wells.
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
54
Figure 2.1. Scheme of the competitive ICPMS-based immunoassay for AFM1
analysis in milk samples.
+
Anti -AFM1 antibody
Y
Y
AFM1
Microtiterplate
YY
Microtiterplate
Y
YY
Free Anti-AFM1 IgG interacts withfree AFM1-BSA in the microtiterplate
Y YY Y
Y YY Y
Streptavidine-Au addition
MicrotiterplateY Y
Y
Biotinilated secondaryantibody addition
Y
Y
ICP-MS
Microtiterplate
Microtiterplate
Microtiterplate
AFM1-BSA
+
Step 2
Step 1a Step 1b
+
Step 3
Step 4
Step 5Au nanoparticles digestion
Incubation at room temperature (1 h)
Y
Acid mixtureInternal standard (Ir)
Incubation at room temperature (1h)
Incubation at room temperature (2.5 h)
Incubation at room temperature (1h)
Incubation at at room temperature (1h)
Chapter 2
55
2.4. ICP-MS instrumentation.
According to the immunoassay procedure described above, AFM1 is
quantified by means of ICP-MS using the 197Au+ signal. Despite the poor Au
ionization in the plasma because of its high ionization potential (9.23 eV) [26],
the use of Au nanoparticles is especially advantageous to amplify the analytical
response due to the high number of atoms present in each nanoparticle. ICP-
MS measurements were performed by means of a 7700x quadrupole-ICP-MS
system (Agilent, Santa Clara, USA). Operating conditions were daily optimized
to maximize 197Au+ following the instrument user’s guide (Table 2.1).
Table 2.1. Operating conditions employed in ICP-MS.
Agilent 7700x ICP-MS
Plasma forward power (W) 1550
Argon flow rate (L min-1):
Plasma 15
Auxiliary 0.9
Nebulizer 1.01
Sample introduction system
Nebulizer OneNeb micronebulizer
Spray chamber Double pass
Carrier flow rate (mL min−1) 0.6
Dwell time (µs) 0.5
Number of sweeps 100
Replicates 90
Signal nature Area
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
56
On account of the limited volume of sample available in the immunoassay
(200 µL), a micronebulizer (OneNeb, Ingeniatrics, Sevilla, Spain) coupled to a
double pass quartz spray chamber (Agilent, Santa Clara, USA) was selected as
the sample introduction system. Using this configuration, 197Au+ sensitivity for a
sample uptake rate of 0.6 mL min-1 was 3-fold higher than the one obtained
using the standard nebulizer provided with the instrument (i.e., Micromist, Glass
Expansion, Australia). These results were the expected taking into account the
higher aerosol generation efficiency of the former nebulizer operating at sample
uptake rates in the order of µL min-1 [27]. Initially, samples were tried to be
introduced into the spectrometer using self-aspirating conditions (i.e., without
using a peristaltic-pump) as suggested by Giesen et al. [24]. However, signal
reproducibility was poor (relative standard deviation, RSD, in the 5-8% range)
and strong Au memory effects were registered. In fact, wash-out times (defined
as the time required for reaching the 1% of the stable signal after blank
introduction) of around 300 s for 1 µg Au L-1 were obtained. This behavior could
be attributed to the Au surface sticky nature [21] and the low sample uptake rate
used [28]. In order to improve the reproducibility and the sample throughput, a
flow injection analysis (FIA) procedure was employed. In this operating mode,
samples were introduced into a carrier solution controlled by a peristaltic pump
(Model Minipulse 3, Gilson, France) with the aid of a V-451 flow injection
manifold (Upchurch Scientific, Silsden, United Kingdom) equipped with a 75 µL
loop valve and a syringe. Carrier flow rate was set at 0.6 mL min-1 for high
throughput analysis. To minimize Au wash-out times, different carrier solutions
(i.e. water, diluted nitric acid, etc.) were tested. Finally, a 1% V V-1 hydrochloric
acid and 1% w V-1 thiourea mixture was chosen since it provided the lowest
Chapter 2
57
wash-out times (i.e., lower than 40 s) [29]. Nonetheless, further improvement
was feasible spiking the digestion acid mixture for Au nanoparticles with 1% w
V-1 thiourea. Operating this way, no differences on wash-out times between Au
and other elements (e.g. Mn, Ir, etc.) were observed (25 s). Finally, because of
the discontinuous sample introduction mode of the FIA device, a peak-shape
signal is obtained. Microsoft Excel software was employed to integrate 197Au+
signals manually.
2.5. Calibration.
Aflatoxin M1 determination was performed by means of a calibration curve
built with the 197Au+ ICP-MS signal response of AFM1 standards of
concentrations ranging from 0.001 to 5 µg kg-1. To improve accuracy and
precision, Ir signal (193Ir+) was employed as internal standard for Au
measurements. Ionization potential and m/z values for Ir are closed to the Au
ones and, hence, matrix and drifts effects are expected to be similar for both
elements [30]. Therefore, the 197Au+ and 193Ir+ signal ratio was really employed
to build the calibration curve and quantify AFM1. Iridium was added to the
standards and the unknown samples with the acid mixture employed to digest
Au nanoparticles after the immunoassay procedure (section 2.3.) for a final
concentration of 2.5 µg L-1.
Finally, it is important to remark that, unlike the conventional ICP-MS
analysis, the use of a competitive immunoassay makes that a high ICP-MS
signal is related to a low AFM1 concentration. Thus, for instance, when no AFM1
is present in the sample, all the pAb is retained in the microtiter plate and, as a
consequence, the Au signal is maximum. Because of the sigmoidal curve
response of the competitive immunoassay procedure, analyte determination
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
58
was circumscribed to the curve section where there was a lineal relationship
between the analyte signal and AFM1 concentration logarithm (i.e. 0.010-2.5 µg
kg-1 under optimum assay conditions).
2.6. Samples.
The AFM1 certified reference material of whole milk powder, ERM-BD284
(certified at 0.44±0.06 µg kg-1), was purchased from the Institute for Reference
Materials and Measurements (European Commission Joint Research Centre,
Geel, Belgium). In addition, five cow milk samples (i.e. raw, pasteurized and
ultrahigh temperature pasteurized) were obtained from retail markets and
supermarkets, stored at 40C and analyzed before their respective expiration
dates.
2.7. Sample preparation.
The ERM-BD284 milk powder (10.0 g) was suspended in 50 mL of ultrapure
water previously heated up to 500C using a stirring rod until a homogeneous
mixture was obtained. After that, the solution was cooled and then diluted to
100 mL using ultrapure water. So, AFM1 concentration in the reconstituted milk
was of 0.044 µg L-1.
Both the certified and the commercial milk samples were pre-treated before
the immunoassay using the procedure described by Huang et al. [31] with some
minor modifications. Thus, 200 µL of milk were mixed with 800 µL of acetonitrile
in an Eppendorf tube to extract AFM1 and remove matrix components. The
extraction was performed using a vortex mixer for 2 min and sonicating the
mixture for 30 min (Vibramix, J.P. Selecta S.A., Barcelona, Spain). Then, the
extracts were centrifuged at 12100 x g for 10 min at 40C (A 5804R Centrifuge,
Chapter 2
59
Eppendorf, Hamburg, Germany). After that, 800 µL of the supernatant were
collected and evaporated up to dryness (miVac Quattro concentrator, Genevac
Ltd, Suffolk, UK). The residue was reconstituted with 100 µL of PBS and then
analyzed.
3. Results and discussion.
3.1. Immunoassay optimization.
The competitive immunoassay described in the present paper has been
developed with a polyclonal antibody produced in rabbits by immunization with
BSA haptenized with AFM1. Considering that not only may this IgG preparation
contain antibodies which react with BSA but also bovine milk contains a
significant amount of this compound (≈1.2 % w V-1), an excess of BSA (1% w V-
1) was included in all the incubation media (see section 2.2) in order to
neutralize any antibody activity specific to BSA in the assay. According to the
supplier, the pAb is highly specific for AFM1 determination with low cross-
reactivity against other aflatoxins (AFB1 2%; aflatoxin B2 0.4%; aflatoxin G1
0.4% and aflatoxin G2 0.1%).
Variables selected for the immunoassay optimization were the
concentration of: (i) AFM1-BSA conjugate; (ii) pAb; and (iii) streptavidin-Au
nanoparticles conjugate. The concentration of the secondary biotinylated
antibody was not optimized and the dilution factor recommended by the
manufacturer (1:2000) was employed. The optimal conditions for the inhibition
assay were chosen by checkerboard titration experiments as described for
ELISA [32,33]. Briefly, decreasing amounts of AFM1 antigen (AFM1-BSA) were
bound to microtiter wells and then incubated with serial concentrations of the
pAb. Inductively coupled plasma mass spectrometry readouts were evaluated to
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
60
identify the optimal amount of AFM1-BSA per well as well as the corresponding
concentration of pAb. Primary polyclonal antibody was prepared by serial
concentrations from 0.125 to 2 µg mL-1 whereas AFM1-BSA concentration was
modified between 0.07 and 10 ng mL-1. For these experiments, streptavidin-Au
nanoparticles conjugate concentration was kept constant at 0.08 µg mL-1. In
titration experiments, optimal conditions were defined as the amount of
reagents (both AFM1-BSA and pAb) producing signal-to-background ratio in
ICP-MS close to the 80% of the maximal signal at plateau, characteristic of an
antibody excess conditions [33]. The rationale for this criterion was that an
assay dependent on inhibition of antibody binding, sensitivity would be maximal
at the lowest concentration of antibody producing a consistent readout (i.e.
minimal variability in replicates and an adequate dose-response relationship,
when tested with the lowest possible amount of AFM1-BSA bound to solid
phase). Experimental results showed that optimum response was obtained for
0.35 ng L-1 of AFM1-BSA and a pAb concentration of 0.5 µg mL-1 (see Table
S2.1, supplementary material). Once the optimum AFM1-BSA and pAb
concentration were selected, streptavidin-Au nanoparticles conjugate
concentration was optimized following a similar procedure to ensure the
maximum 197Au+ response by the mass spectrometer. To this end, this
parameter was modified between 0.02 and 0.32 µg mL-1. As expected, when
the streptavidin-Au nanoparticles conjugate concentration was decreased, the
Au response in ICP-MS improved. A 6-fold signal improvement was registered
when the concentration was varied from 0.02 to 0.32 µg mL-1 (see Figure S2.1,
supplementary material). On the other hand, the precision in ICP-MS was also
observed to be dependent on the streptavidin- Au nanoparticles conjugate
Chapter 2
61
concentration employed. Gold signal RSD was improved up to 2-3% when
streptavidin-Au nanoparticles conjugate concentration was decreased down
0.08 µg mL-1. No further improvement of the RSD was obtained when higher
concentrations were used. Though higher streptavidin-Au nanoparticles
conjugate concentrations provide higher Au signals in ICP-MS, no real
improvement on analytical figures of merit was obtained (this topic will be
discussed in detail below). For this reason, 0.08 µg mL-1 streptavidin- Au
nanoparticles conjugate concentration was selected for further studies as a
compromise between analytical performance and cost.
For calibration purposes, AFM1 standards were treated under the optimum
competitive immunoassay experimental conditions (Figure 2.2). As it has been
described in the experimental section, the sample containing the AFM1 and the
pAb solution were incubated together (Figure 2.1, step 1.b) and then the
mixture was transferred to the microtiter plate for a second incubation step
(Figure 2.1, step 2). Initially, the incubation time of each step was 1 h. Analytical
response in Figure 2.2 is expressed as the inhibition factor, defined as (S0-
S)·100/S0 where S0 is the maximal signal obtained in wells with no inhibition
(i.e. no AFM1 was added) and S is the signal observed for each sample or
standard preparation. Figure 2.2 shows that the concentration of AFM1 giving
rise to a 50% inhibition factor (i.e., half maximal inhibitory concentration, IC50)
for the ICPMS-based immunoassay was 6.4 µg kg-1 whereas the limit of
detection (LoD) (calculated as three times the standard deviation of the signal of
15 blank replicates [16]) was around 0.070 µg kg-1, low enough to analyze
AFM1 according to USA legislation (AFM1max level: 0.500 µg kg-1). However,
unless a pre-concentration step was implemented, the immunoassay could not
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
62
be applied to the analysis of milk samples (AFM1max level: 0.050 µg kg-1) and
infant formula (AFM1max level: 0.025 µg kg-1) under the more restrictive EU policy.
In order to reduce the minimum AFM1 concentration level detectable and avoid
costly and long sample pretreatments based on immunocolums for AFM1
preconcentration [10,13], the immunoassay optimization procedure was revised.
Figure 2.2. Aflatoxin M1 calibration curve using different pAb concentrations. (-
�-) 0.5 µg mL-1 pAb concentration/incubation time 1h; (-�-) 0.25 µg mL-1 pAb
concentration/incubation time 2.5 h. Aflatoxin M1-BSA concentration: 0.35 ng
mL-1; secondary Ab dilution factor: 1:2000; streptavidin-Au nanoparticles
conjugate concentration: 0.08 µg mL-1.
Competitive immunoassay LoD is strongly linked to both primary antibody
concentration and detector capability to recognize the primary antibody retained
in the microtiter plate after the incubation step with the analyte containing
sample. Thus, pAb concentration could be decreased to make the
0
20
40
60
80
100
120
0.0001 0.001 0.01 0.1 1 10 100 1000
Inh
ibti
on
(%
)
AFM1 concentration (µg kg-1)
EU limit USA limit
Chapter 2
63
immunoassay more sensitive to low AFM1 levels. However, this means that pAb
retained in the microtiter plate is less significant, thus making detection more
difficult or even impossible. Accordingly, LoDs for the ICPMS-based
immunoassay are expected to improve by increasing the efficiency of pAb
retention in the microtiter plate and/or detection by ICP-MS. First of all, the
incubation time of the AFM1 and the pAb in the microtiter plate coated with
AFM1-BSA (step 2) was increased from 1 to 2.5 h with the aim to favor pAb
retention on it. Operating in this way, it was feasible to reduce pAb
concentration from 0.5 to 0.25 µg mL-1, thus improving sensitivity and LoDs
without compromising robustness. As a consequence, the IC50 was
approximately 10 times lower (0.42 µg kg-1) and LoD decreased down to 0.005
µg kg-1 (Figure 2.2). The use of higher incubation times together lower pAb
concentration (< 0.25 µg mL-1) was not further explored since sample
throughput is negatively affected and LoD were low enough to quantify AFM1
according to the current international policies for this analyte. Alternatively, the
feasibility of using lower streptavidin-Au nanoparticles conjugate concentration
to improve ICP-MS detection was checked but no real improvement was
obtained on LoDs. It should be taking into account that secondary Ab has a
limited amount of biotin moieties and, as a consequence, signal amplification
with streptavidin-Au nanoparticles conjugate is limited and could not
compensate the lower amount of pAb retained in the microtiter plate. Probably,
LoD could be improved using a sector field instrument or with a heteroatom-tag
with a higher sensitivity in ICP-MS in order to use lower pAb concentrations.
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
64
3.2. Method validation.
The analytical methodology was evaluated according to European
Conformity guidelines for analytical methods of food contaminants and
mycotoxins [6,34]. First of all, method accuracy and precision was checked by
analyzing an AFM1 certified reference material (ERM-BD284 milk powder, 0.044
± 0.006 µg kg-1) and different milk samples spiked with known amounts of
AFM1. Recovery test was performed spiking milk samples with AFM1 standard
for a final concentration of 0.030 and 0.080 µg kg-1. All the samples were
analyzed following the optimized immunoassay after an extraction treatment
with acetonitrile to mitigate the effects of matrix components (e.g. proteins, fats
etc.) on the antibody reaction. In fact, direct analysis of milk samples produced
systematically AFM1 recovery values between 200-300%. Sample dilution to
mitigate matrix effects was not explored due to its negative impact on LoDs.
Table 2.2 shows the recovery values for AFM1 in the certified reference
material and in the spiked milk samples after the sample pre-treatment with
acetonitrile. There is observed to be a good agreement between experimental
and theoretical values. Aflatoxin M1 recovery values ranged between 80% and
102%. These values were within the limits established by the EU for analyte
concentrations below 1 µg kg-1 (-40%/+20%). The repeatability (intra-assay
precision) of the method was determined by analyzing five replicates of each
sample on the same day. The RSD of the AFM1 concentration levels was within
the 5%-15% range. These values are the typical for immunoassays. The
immunoassay reproducibility (inter-assay precision) of the proposed
methodology was evaluated as the RSD of the measurements obtained for five
independent immunoassays performed in five different days. The average of
Chapter 2
65
AFM1 concentration obtained for the certified reference material was 0.042 ±
0.008 µg kg-1 which highlights the reproducibility of the assay. Similar
conclusions were obtained for the spiked milk samples.
Table 2.2. Aflatoxin M1 recovery assay for different kinds of milk samples.
AFM1 concentration (µg kg-1) Recovery (%)*
Sample Certified/Spike Experimental
Certified milk powder 0.044 ± 0.006 0.038 ± 0.006 87 ± 10
Raw milk 0.030 0.027 ± 0.003 90 ± 10
0.080 0.078 ± 0.004 93 ± 5
Pasteurized milk 0.030 0.024 ± 0.005 80 ± 17
0.080 0.078 ± 0.002 98 ± 2
UHT whole milk 0.030 0.026 ± 0.003 87 ± 8
0.080 0.079 ± 0.010 99 ± 10
UHT whole milk (2) 0.030 0.028 ± 0.003 93 ± 7
0.080 0.082 ± 0.008 102 ± 10
Replicates = 5, * mean ± standard deviation
Finally, the lower and the upper quantification limits of the immunoassay (lLoQ
and uLoQ) were estimated [35]. The lLoQ was defined as the analyte
concentration that has a response at least 3 times that of a blank sample and
repeatability lower than 20%. Similarly, the uLoQ was defined as the highest
concentration standard that signal response has repeatability lower than 20%.
The lower and the upper quantification limits of the immunoassay experimental
values were 0.012 µg kg-1 and 2.5 µg kg-1, respectively. These results
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
66
demonstrate the suitability of the ICPMS-based immunoassay for AFM1
determination in milk samples according to the current security policies for this
analyte. In fact, this methodology was applied to the analysis of different milk
samples (i.e. raw, pasteurized or ultra-high temperature pasteurized) but no
AFM1 was detected (< 0.005 µg kg-1).
3.3. Comparison with other methodologies.
Analytical figures of merit of the ICPMS-based immunoassay have been
compared with those previously reported in the literature (Table 2.3). In general,
the methodology developed in this work affords similar results to those obtained
with chromatographic-based approaches or even other immunoassays.
Comparing to chromatographic methods, poorer precisions are obtained (RSD
nearly 2 to 3-fold higher), but no laborious sample pretreatments based on solid
phase extraction or immunocolumns are required to preconcentrate and purify
the AFM1. Sample pretreatment proposed in the present work is quite simple
and it is only focused to eliminate the most significant matrix components
without compromising sample throughput. Nevertheless, a 2-fold AFM1
preconcentration factor is obtained operating on this way. On the other hand,
due to immunoassay simplicity, reagents and solvent requirements (as well as
wastes) are minimized. As regards other immunoassay procedures, ICPMS
detection offers a wider linear range. Moreover, the proposed method not only
shows lower background and blank levels but also an independent analytical
response from incubation and storages times. In fact, standards and samples in
the microtiter plate can be stored 2-3 weeks after acid mixture addition without
significant effects on AFM1 results. However, sample throughput is partially
Chapter 2
67
degraded since microtiter plate wells are sequentially analyzed by the ICP-MS
(3-4 h to analyze a 96-well plate).
Up to date, the use of ICP-MS as a detector for mycotoxin analysis has
been limited. In fact, it has only previously used to quantify ochratoxin A levels
in wine by means of a competitive immunoassay methodology [24]. Though this
previous work is not focused on AFM1, it is worth to compare both
methodologies due to their similarities. Thus, Ochratoxin A detection was
accomplished using secondary antibodies functionalized with Au nanoparticles
and a sector field based ICP-MS. The limit of detection using this configuration
was stablished at 0.003 µg kg-1. A priori the high detection capabilities found
could be attributed (at least in part) to the high sensitivities afforded by sector
field mass spectrometers which makes feasible to use a low pAb concentration.
However, our work shows that AFM1 can also be quantified in the ng kg-1 range
using a quadrupole-based mass spectrometer with a two-step signal
amplification procedure based on a biotinylated secondary antibody and
streptavidin-Au nanoparticles conjugate. The possibility of using quadrupole-
based ICP-MS instruments to quantify toxin at ultra-trace levels is
advantageous since they are the most spread mass spectrometers worldwide.
Furthermore, precision for the AFM1 immunoassay was significantly better (5 -
15%) than that for the Ochratoxin A (5 - 40%). The origin of these differences is
not clear due to the different procedures implemented in each immunoassay.
Nonetheless, it points out that the precision achievable for the ICPMS-based
immunoassay is mainly limited by the immunological step since uncertainty
derived by the ICP-MS lays below 5%.
Determ
inatio
n o
f aflatoxin
M1 in
milk sam
ples b
y mean
s of an
ind
uctively co
up
led p
lasma m
ass spectro
metry-b
ased im
mu
no
assay
Tab
le 2.3. Co
mp
aris
on
of d
ive
rse
an
aly
tica
l me
thod
olo
gie
s p
rop
ose
d in
the
litera
ture
for th
e d
ete
rmin
atio
n o
f AF
M1 in
milk
sa
mp
les.
Meth
od
olo
gy
Reco
very
(%)
Precisio
n
(%)
LO
D
(µg kg
-1)
Lin
ear rang
e
(µg kg
-1) R
eference
Ind
irect co
mp
etitive
imm
uno
assa
y (ICP
-MS
) 8
0-1
02
5-1
5
0.0
05
0.0
10
-2.5
T
his
wo
rk
Liq
uid
-liqu
id e
xtractio
n H
PL
C-F
luo
resce
nce
7
3-9
9
2-7
0
.05
- [1
1]
IAC
-HP
LC
-FD
1
16
5 0
.010
0.0
1-0
.20
[36
]
MS
FE
-HP
LC
-FD
9
1-1
02
5 0
.005
0.0
15
-10
[37
]
SP
E-L
CM
S
78
-108
5-1
0 0
.010
0.0
20
-1 [3
8]
UP
HL
C-M
S/M
S
84
-97
13
0.0
10
- [1
2]
80
-110
<1
0 0
.005
0.0
25
-10
[13
]
Ind
irect co
mp
etitive
imm
uno
assa
y (EL
ISA
) 8
0-1
02
5-1
7
0.0
40
0.0
40
-0.5
00
[39
]
Dire
ct c
om
pe
titive E
LIS
A
90
-110
<1
0
0.0
03
- [1
5]
Dire
ct c
om
pe
titive E
LIS
A
93
-98
5-8
0
.008
0.0
04
-0.2
50
[40
]
Chapter 2
69
4. Conclusions.
This work demonstrates that ultra-trace AFM1 analysis in milk samples is
feasible using an ICPMS-based immunoassay. Analytical figures of merit of this
method fulfil the most restrictive current international policies for AFM1 analysis
in milk and dairy products. Despite the fact that sample throughput could be
deteriorated in relation to other immunoassay methodologies described in the
literature, the use of ICP-MS for mycotoxin analysis has a great potential in food
analysis due to a higher dynamic range, lower background levels and the
independence of analytical response from incubation or storage times. In this
regard, aflatoxin detection by means of ICP-MS could be still improved. Thus,
LoDs for the competitive immunoassay employed in this work strongly depend
on the primary antibody concentration and ICP-MS Au sensitivity to detect the
amount of primary antibody retained in the microtiter plater. Therefore, detection
capabilities could be probably improved (below ng L-1) using lower primary
antibody concentration with a sector field mass spectrometer and/or labelling
with higher sensitive hetereoatoms in ICP-MS. On the other hand, methodology
sample throughput could be significantly enhanced determining other
mycotoxins together AFM1 by means of antibodies functionalized with different
heteroatoms. Operating on this way ICP-MS multiplexing capabilities are
exploited and the use of a mass spectrometer could be more beneficial than
other immunoassay detection procedures (e.g. ELISA, etc.). These experiments
are currently being carried out in our laboratories.
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
70
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aflatoxin M1 levels in milk in Anakara, Turkey. Food Control 17 (2006) 1-4.
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Chapter 2
77
Supplementary data.
Table S2.1. Results of the checkboard titration experiments to optimize AFM1-
BSA and pAb concentration. Secondary Ab dilution factor: 1:2000; streptavidin-
Au nanoparticles conjugate concentration: 0.16 µg mL-1; incubation time in the
microtiterplate of AFM1 standards and pAb solution: 1 h.
pAb concentration (µµµµg mL-1)
AFM1-BSA concentration (µg mL-1) 2 1 0.5 0.25 0.125
10 1.0 0.9 0.8 0.6 0.4
5 1.0 0.9 0.8 0.6 0.5
2.5 0.9 0.9 0.8 0.7 0.5
0.75 0.9 0.9 0.8 0.7 0.5
0.35 0.9 0.9 0.8 0.6 0.5
0.15 0.9 0.9 0.7 0.6 0.5
0.07 0.9 0.8 0.7 0.5 0.4
Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay
78
Figure S2.1. Influence of the streptavidin-Au nanoparticles conjugate
concentration on the 197Au+ normalized signal and signal standard deviation in
ICP-MS. Aflatoxin M1-BSA concentration: 0.35 ng mL-1; pAb concentration: 0.5
µg mL-1; secondary antibody dilution factor: 1:2000; incubation time in the
microtiterplate of AFM1 standards and pAb solution: 1 h.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
0
1
2
3
4
5
6
7
2 1 0.5 0.25 0.125
Sign
al st
anda
rd d
evia
tion
(%)
Nor
mal
ized
Au
sign
al
Streptavidin-Au concentration (µµµµg mL-1)
Capítulo 3
Evaluation of different competitive
immunoassay for aflatoxin M1 determination
in milk samples by means of inductively
coupled plasma mass spectrometry
Chapter 3
81
1. Introduction.
The significance of inductively coupled plasma mass spectrometry (ICP-
MS) for biomolecule analysis has exponentially increased over the years [1].
The use of ICP-MS in this field offers several attractive features regarding other
stablished approaches: (i) sensitivity; (ii) specificity; (iii) compound-independent
detection sensitivity; (iv) multi-element capabilities; (v) robustness; and (vi)
versatility to be coupled to separation techniques. Initially, the analysis was
limited to those species containing a heteroatom detectable by ICP-MS.
However, analytical figures of merit were severely compromised since the
heteroatoms naturally present in biomolecules (e.g. P, S, Se, As, etc.) suffer
from low sensitivity and (spectral and non-spectral) interferences due to matrix
components in biological samples (e.g. carbon, chloride, etc.) [1,2]. To improve
the analytical figures of merit as well as to address with the determination of
non-containing heteroatoms biomolecules, different labelling strategies have
been proposed in the literature. First, the biomolecule can be derivatized
through a chemical reaction with a heteroatom or an organometallic compound
[3,4]. Alternatively, the analyte of interest can be labelled indirectly by means of
immunoreaction using a heteroatom-labelled antibody [5,6]. To this end, non-
competitive immunoassays (i.e. sandwich type) have been traditionally
employed for the analysis of high molecular weight biomolecules such as
proteins, enzymes, etc. Dealing with haptens (i.e., biomolecules which
molecular weight is lower than 10 kDa), quantification is only accomplished by
means of competitive immunoassays since, given the volume of these
molecules, they only react with a single antibody. Competitive immunoassays
have been successfully reported for several hormones (thyroxine [7],
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
82
progesterone [8]), herbicides (2,4-dichlorophenoxyacetic acid [9]), antibiotics
(chloramphenicol [10]); and toxins (ochratoxin A [11], aflatoxin M1 (AFM1) [12])
determination.
Two different competitive assay formats have been mainly reported for
hapten analysis with ICP-MS detection so far. In the first approach, the sample
is spiked with a given amount of the specific labelled antibody and the mixture is
incubated in a solid phase coated with an antigen (analyte)-protein conjugate. A
competitive reaction is stablished between the antigen bound to the solid phase
and the antigen present in solution for the limited number of the binding sites of
the antibody. The final stage of the assay involves the measurement of the
signal related to the amount of the antibody retained on the solid phase.
Alternatively, the competitive reaction can be also stablished by the competition
between the labelled and the unlabelled antigen for the limited number of the
binding sites of the antibody coating the solid phase. Up to date, both
immunoassay formats have been indistinctively employed in the literature but
the benefits (and drawbacks) of each approach have not been systematically
investigated under a similar set of experimental conditions. Regardless of the
competitive immunoassay format, different heteroatoms have been employed in
the literature for hapten analysis: (i) Eu [8]; (ii) Fe [11]; (iii) Au nanoparticles
[10,12,13]; and (iv) Quantum-Dots [9]. A priori, the use of Au nanoparticles or
Quantum-Dots seems to be more advantageous to improve analytical figures of
merit (e.g. sensitivity, LoDs, etc.) of competitive immunoassays due to the high
number of atoms present in each nanoparticle. Nevertheless, no previous
attempt has been made so far to systematically evaluate the influence of the
nanoparticle characteristics operating with competitive immunoassays.
Chapter 3
83
The goal of this work is to systematically compare diverse competitive
immunoassay formats for hapten determination by means of ICP-MS detection
using nanoparticles of different nature and size. Aflatoxin M1 has been selected
as a model of small chemical analyte to evaluate the benefits and drawbacks of
the different approaches, due to its high health risk and restrictive legal
requirements. Competitive immunoassays based on the use of either antibody
or antigen-protein conjugate as tracer species have been investigated since
they are the most frequently employed. Nanoparticles covering different
elements (Ag/Au) and sizes (40/80 nm) have been used through this work.
Finally, the methodologies developed have been applied to the AFM1 analysis in
milk samples.
2. Experimental.
2.1. Reagents and materials.
Anti-aflatoxin M1 primary rabbit polyclonal antibody (pAb) (1 mg mL-1) was
obtained from Agrisera (Vännas, Sweeden). According to the supplier, this
antibody is highly specific for AFM1 determination with low cross-reactivity
against other aflatoxins (aflatoxin B1 2%; aflatoxin B2 0.4%; aflatoxin G1 0.4%
and aflatoxin G2 0.1%).
Biotinylated goat α-rabbit IgG (whole molecule) secondary antibody
(secondary Ab), aflatoxin M1 (AFM1) from Aspergillus flavus (5 g mL-1), AFM1-
Bovine serum albumin conjugate (AFM1-BSA) (500 µg mL-1), sodium carbonate,
sodium hydrogen carbonate, monosodium phosphate, disodium phosphate,
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
84
sodium chloride, polyethylene glycol sorbitan monolaurate (Tween 20), HPLC-
grade acetonitrile were purchased from Sigma-Aldrich (Steinheim, Germany).
Bovine serum albumin (BSA) was purchased from Biowest (Nuaillé,
France). Streptavidin – (80/40 nm) Ag or (80/40 nm) Au nanoparticles conjugate
were obtained from Cytodiagnostics (Ontario, Canada).
Iridium and rhodium 1000 mg L-1 stock solution were provided by Merck
(Darmstadt, Germany). Thiourea, 69% w w-1 nitric acid and 35% w w-1
hydrochloric acid were purchased from Panreac (Barcelona, Spain).
All solutions were prepared using ultrapure water (Milli-Q water purification
system, Millipore Inc., Paris, France).
F16 maxisorp polystyrene microtiter plates were obtained from Thermo-
Scientific (Roskilde, Denmark). Immunoaffinity columns (IACs) Afla M1 (Vicam,
Watertown, MA) with a capacity of approximately 150 ng and based on
monoclonal antibodies were employed for AFM1 determination in milk samples.
Amicon Ultra-4 Centrifugal Filter Units with Ultracel-10 membrane (Merck
Millipore, Cork, Ireland) were used throughout the work for washing steps
during the biotin labelling of AFM1-BSA.
2.2. Buffers and solutions.
Standard stock solution of AFM1 (10 g L-1) was prepared in pure
acetonitrile. Aflatoxin M1-BSA was dissolved in 2 mL of phosphate buffer
solution (PBS; 10 mM NaH2PO4, 2 mM Na2HPO4, 154 mM NaCl, pH 7.6) for a
final concentration of 500 g mL-1. Both solutions were kept at -200C. Primary
rabbit polyclonal antibody was dissolved in 500 µL ultrapure water and kept at
40C.
Chapter 3
85
The following solutions were employed in the different immunoassays
tested: (a) as microtiter plate coating medium: carbonate buffer solution (15mM
Na2CO3 and 35 mM NaHCO3, pH 9.6); (b) as plate blocking medium: 1% w V-1
BSA in a PBS solution; (c) as incubation medium: 1% w V-1 BSA and 0.05% V
V-1 Tween 20 in PBS; (d) as washing medium: 0.05% V V-1 Tween 20 in PBS;
(e) as Au-nanoparticles digestion medium: 4% V V-1 nitric acid and 12% V V-1
hydrochloric acid; and (f) as Ag-nanoparticles digestion medium: 2 % V V-1 nitric
acid. It is important to remark that the use of BSA in the incubation medium is
critical to ensure immunoassay reproducibility. The pAb could react either with
the AFM1 or BSA. Therefore, an excess of BSA was included in the incubation
medium to eliminate pAbs without affinity to AFM1. Moreover, this strategy is
also useful to avoid potential matrix effects for AFM1 analysis in milk samples
since they contain significant amount of BSA.
2.3. Immunoassay procedures.
In the present work, different competitive immunoassay formats have been
tested for AFM1 determination with ICP-MS detection (Figure 3.1); namely: (i)
antibody binding inhibition assay (ABIA). This immunoassay is based on that
described in our previous work [12] in which pAb are employed as the tracer
specie. The competitive reaction is stablished between the AFM1 present in the
sample and the AFM1-BSA, coating the solid phase, for the limited number of
the binding sites of the pAb spiked in the sample solution. A biotinylated
secondary Ab and streptavidin-nanoparticles conjugate were employed to
detect the pAb retained on the solid phase; (ii) capture inhibition assay (CIA).
This procedure is based on that previously reported by Trapiella-Alfonso et al.
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
86
[13] in which a labelled antigen is used as the tracer specie. In this case, the
competitive reaction is stablished between the AFM1 present in the sample and
the biotinylated AFM1-BSA conjugates (spiked to the sample) for the limited
number of the binding sites of the pAb immobilized on the solid phase. The
biotinylated AFM1-BSA retained on the solid phase is quantified using
streptavidin-nanoparticles conjugates; and (iii) capture bridge inhibition assay
(CBIA). For the first time, this immunoassay procedure has been proposed for
hapten determination with ICP-MS detection. It is based on the CIA procedure
but includes some modifications to favor immunocomplex formation. A mixture
of the pAb and the biotinylated AFM1-BSA is spiked to the sample. A
competitive reaction is stablished between the biotinylated AFM1-BSA and the
AFM1 present in the sample for the limited number of the binding sites of the
pAb. Next, and given the bivalent nature of the antibodies, the AFM1-pAb and
the biotinylated AFM1-BSA-pAb complexes generated are retained by the
AFM1-BSA which coates the solid phase. Streptavidin-nanoparticles conjugates
are used again to detect the biotinylated AFM1-BSA-pAb complexes retained on
the solid phase.
For all strategies, the influence of the nanoparticle type (i.e. Ag and Au) and
size (i.e. 40 and 80 nm) on the analytical figures of merit have been
investigated.
The biotinylated AFM1-BSA required for CIA and CBIA procedures was
obtained using a commercial biotin labelling kit. Briefly, 2.3 mg of succinimidyl-
6-(biotinamido)hexanoate was dissolved in 500 µL of dimetylformamide and
then added to a 200 µL AFM1-BSA solution. The reaction was carried out on ice
for 2 hours in the dark and the excess of non-reactive biotin reagent was
Chapter 3
87
removed on a cutoff filter by centrifugation with PBS and recovered at 150 µg
mL-1. The biotinylated AFM1-BSA solution was divided into single working
aliquots and stored at -200C.
Figure 3.1. Scheme of the different competitive immunoassay formats tested in
this work.
2.3.1 Antibody binding inhibition assay (ABIA).
Plates were coated at room temperature with 100 µL/well of the appropriate
AFM1-BSA concentration in 0.05 M carbonate–bicarbonate buffer (pH 9.6). After
incubation for 1 h, plates were washed three times and then blocked (200
µL/well) for 1 h at room temperature. Simultaneously, samples or AFM1
A. Antibody binding inhibition assay (ABIA)
B. Capture inhibition assay (CIA)
C. Capture bridge inhibition assay (CIA)
++
Y
Step 1.1 Step 1.2 Step 1.3 Step 1.4 Step 1.5
Step 2.1 Step 2.2 Step 2.3 Step 2.4
+ Digestion+
ICP-MS
AFM1 Anti-AFM1 antibody (primary)
AFM1-BSA Biotynilated antibody (secundary) Biotynilated AFM1-BSAStreptavidin-nanoparticles
M
M M
+
YYYYYY
YYYYYYM
YYYYYY
M M
Step 3.1 Step 3.2 Step 3.3
+
+
+
Step 3.4Y Y
M M M
M
Y
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
88
standards were incubated with the appropriate concentration of the pAb solution
for 1 h at room temperature (Figure 3.1, step 1.1). After another washing step,
100 µL/well of the AFM1-pAb mixture was added and incubated for 2.5 h at
room temperature (Figure 3.1, step 1.2). Following a washing step (Figure 3.1,
step 1.3), the secondary Ab solution (100 µL/well) was added and incubated for
1 h at room temperature (Figure 3.1, step 1.4). The plates were washed again,
and 100 µL/well of streptavidin-nanoparticles conjugate solution was added and
incubated for 1 h at room temperature (Figure 3.1, step 1.5).
2.3.2 Capture inhibition assay (CIA).
Plates were coated at room temperature with 100 µL/well with the
appropriate pAb concentration in 0.05 M carbonate–bicarbonate buffer (pH 9.6).
After incubation for 1 h, plates were washed three times and then blocked (200
µL/well) for 1 h at room temperature. Next, after a washing step, samples or
AFM1 standards and the appropriate biotinylated AFM1-BSA concentration were
mixed (Figure 3.1, step 2.1) and transferred to the solid phase (Figure 3.1, step
2.2). The mixture was incubated for 1 h at room temperature and, after a
washing step (Figure 3.1, step 2.3), 100 µL/well of streptavidin-nanoparticles
conjugate solution was added and incubated for 1 h at room temperature
(Figure 3.1, step 2.4).
2.3.3. Capture bridge inhibition assay (CBIA).
Plates were coated at room temperature with 100 µL/well of 1 ng L-1 AFM1-
BSA in 0.05 M carbonate–bicarbonate buffer (pH 9.6). After incubation for 1 h,
plates were washed three times and then blocked (200 µL/well) for 1 h at room
Chapter 3
89
temperature. Then, after a washing step, samples or AFM1 standards, pAb and
biotynilated AFM1-BSA solutions were mixed (Figure 3.1, step 3.1) and
transferred to the solid phase (Figure 3.1, step 3.2). Next, the mixture was
incubated for 1 h at room temperature. Following a washing step (Figure 3.1,
step 3.3), 100 µL/well of streptavidin-nanoparticles conjugate solution was
added and incubated for 1 h at room temperature (Figure 3.1, step 3.4).
Finally, before ICP-MS analysis and with independence of the
immunoassay format employed, nanoparticles in the microtiterplate wells were
acid digested and spiked with 50 µL of a 1% w V-1 thiourea solution containing
2.5 µg L-1 of the corresponding internal standard (Rh/Ir) for a final volume of 200
µL. All AFM1 standards were analyzed in triplicate wells whereas samples
containing unknown AFM1 amounts in quintuplicate wells.
2.4. Instrumentation.
Aflatoxin M1 is indirectly quantified by means of ICP-MS using the signal of
the element present in the nanoparticle (107Ag+ and 197Au+). Experimental
measurements were performed by means of a 7700x quadrupole-ICP-MS
system (Agilent, Santa Clara, USA). The Table 3.1 shows the operating
conditions employed in this work. The performance of the system was checked
to respect the manufacturer indications for 1 g L-1 of 7Li+, 89Y+, and 205Tl+ in 2%
HNO3.
The sample was introduced in the equipment using a micronebulizer
(OneNeb, Ingeniatrics, Sevilla, Spain) coupled to a double pass quartz spray
chamber (Agilent, Santa Clara, USA). Because of the limited volume of sample
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
90
available in the immunoassay (200 µL), a V-451 flow injection manifold
(Upchurch Scientific, Silsden, United Kingdom) equipped with a 75 µL loop
valve was employed. The samples were introduced with the aid of a syringe into
a carrier solution controlled by a peristaltic pump (Model Minipulse 3, Gilson,
France). Carrier flow rate was set at 0.6 mL min-1 for high throughput analysis.
Two different carrier solutions were selected depending on the nanoparticle
type. A mixture of 1% V V-1 nitric acid and 1% w V-1 thiourea solution was
employed for Ag measurements whereas a 1% V V-1 hydrochloric acid and 1%
w V-1 thiourea mixture was employed as carrier for Au determination. Operating
in this way, no significant memory effects were registered in ICP-MS [14].
Table 3.1. Operating conditions employed in ICP-MS.
Agilent 7700x ICP-MS
Plasma forward power (W) 1550
Argon flow rate (L min-1):
Plasma 15
Auxiliary 0.9
Nebulizer 1.01
Sample introduction system
Nebulizer OneNeb micronebulizer
Spray chamber Double pass
Carrier flow rate (mL min−1) 0.6
Dwell time (µs) 0.5
Number of sweeps 100
Replicates 90
Signal nature Area
Chapter 3
91
To improve accuracy and precision, 103Rh+ and 193Ir+ were employed as internal
standards for Ag and Au measurements, respectively. These internal standards
match m/z values and ionization potentials and, hence, matrix and drifts effects
are expected to be similar [15]. Rhodium and Ir were added to the standards
and the unknown samples with the acid mixture employed to digest the
nanoparticles after the immunoassay procedure (section 2.3) for a final
concentration of 2.5 µg L-1. Microsoft Excel software was employed to integrate
peak signals manually, given the peak-shape signal obtained operating the FIA
device.
2.5. Calibration.
Irrespective of the immunoassay format employed, standard curves were
obtained by plotting the inhibition factor value against the logarithm of the
analyte concentration because of the sigmoidal curve response of the
competitive immunoassay procedure. The inhibition value is defined as (S0-
S)/S0 × 100, where S is the signal ratio between 107Ag+ or 197Au+ and its
corresponding internal standard for each sample or AFM1 standard solution and
S0 is the signal ratio when no AFM1 is added.
2.6. Samples.
A certified reference material of whole milk powder (ERM-BD283, European
Commission Joint Research Centre, Geel, Belgium) at 111 ± 18 ng kg-1 of AFM1
and five cow commercial milk samples from retail markets have been analyzed
through this work. All the samples were stored at 40C and analyzed before their
respective expiration dates.
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
92
2.7. Sample preparation.
The ERM-BD283 milk powder (10.0 g) was suspended in 50 mL of ultrapure
water previously heated up to 50°C using a stirring rod until a homogeneous
mixture was obtained. After that, the solution was cooled and then diluted to
100 mL using ultrapure water. So, AFM1 concentration in the reconstituted milk
was 11.1 ± 1.8 ng kg-1.
All the milk samples were pre-treated before the analysis to mitigate
interferences caused by matrix components (e.g. proteins, lipids, etc.) since,
otherwise, accuracy and precision is severely compromised. To this end, two
different approaches have been employed through this work depending on the
immunoassay methodology employed. On one hand, in the case of ABIA, the
samples were pretreated using the procedure described by Huang et al. [16]
with some minor modifications. Thus, 200 L of milk were mixed with 800 L of
acetonitrile in an Eppendorf tube to extract AFM1 and remove matrix
components. The extraction was performed using a vortex mixer for 2 min and
sonicating the mixture for 30 min (Vibramix, J.P. Selecta S.A., Barcelona,
Spain). Then, the extracts were centrifuged at 12100 x g for 10 min at 4°C (A
5804R Centrifuge, Hamburg, Germany). After that, 800 L of the supernatant
were collected and evaporated up to dryness (miVac Quattro concentrator,
Genevac Ltd, Suffolk, UK). The residue was reconstituted with 100 L of PBS
and then analyzed. On the other hand, in the case of CBIA, milk samples were
pretreated using immunocolumns following the procedure suggested by the
manufacturer with some minor modifications. Briefly, a 100 mL volume of liquid
milk was centrifuged at 1614 × g for 15 min, to separate the fat and thin upper
fat layer was discarded. Fifty mL of the milk were passed through
Chapter 3
93
immunoaffinity column at 1-2 drops/second by gravity. Then, the column was
washed with 10 mL water at a rate of 1-2 drops/second until air comes through
column. Aflatoxin M1 was eluted slowly from column with 4 mL pure acetonitrile
at 1 drop/2-3 seconds by gravity. Two hundred µL of the sample eluate were
evaporated up to dryness (miVac Quattro concentrator, Genevac Ltd, Suffolk,
UK). The residue was reconstituted with 100 L of PBS and then analyzed.
3. Results and discussion.
Competitive immunoassays described in the literature shows several
differences on the experimental setup according to the tracer specie monitored:
the specific antibody against the analyte [12,13] or the antigen-protein
conjugate [9,10]. For the first time, immunoassays based on both approaches
are compared under a similar set of experimental conditions for AFM1 analysis.
From now on, antibody inhibition immunoassay (ABIA) will denote the
procedure based on the antibody detection whereas capture inhibition assay
(CIA) will be used as the immunoassay format based on the antigen-protein
conjugate detection.
3.1. Optimization of the immunoassay procedures.
The optimization of the immunoassay formats tested were performed by
means of checkerboard titration experiments as previously described for
Enzyme Linked Immunosorbent Assay (ELISA) [17,18]. The optimal conditions
were defined as the amount of reagents producing signal-to-background ratio in
ICP-MS close to 80% of the maximal signal at plateau, characteristic of a tracer
excess conditions [18]. The rationale for this criterion was that an assay
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
94
dependent on inhibition of tracer binding, its sensitivity would be maximal at the
lowest concentration of the tracer producing a consistent readout.
3.1.1. Antibody Binding Inhibition Assay (ABIA).
In our previous work [12], ABIA experimental conditions were optimized
using streptavidin-40 nm Au nanoparticles conjugate for AFM1 determination in
milk samples. It was observed that the limit of detection (LoD) was strongly
related to the pAb concentration. As long as the heteroatom-label allows the
accurate detection of the pAb retained on the solid phase, pAb concentration
could be decreased to improve immunoassay detection capabilities to low AFM1
levels. In this context, high sensitive heteroatom labels could be required. Thus,
operating with nanoparticles, ICP-MS signal mainly depends on the
nanoparticle size and the properties of the element in the nanoparticle (e.g.
ionization energy). Therefore, for the first time, this works explores the influence
of nanoparticle characteristics on the analytical figures of merit for a competitive
immunoassay based on the use of antibodies as tracers. To this end,
nanoparticles of different elements (Au and Ag) and size (80 nm and 40/80 nm,
for Au and Ag, respectively) conjugated to streptavidin were investigated. For
each of them, the following concentrations were optimized: (i) pAb; (ii) AFM1-
BSA; and (iii) streptavidin-nanoparticles conjugates. The concentration of the
secondary Ab was not optimized, and the value recommended by the
manufacturer was employed (0.5 µg mL-1). For the sake of comparison,
previous data with streptavidin-40 nm Au nanoparticles conjugate was
employed.
Table 3.2 summarizes the optimum immunoassay conditions for each
streptavidin-nanoparticles conjugate.
Chapter 3
95
Table 3.2. Influence of the streptavidin-nanoparticles conjugate on ABIA
optimum experimental conditions, LoDs and dynamic range.
Ag Au
Parameter 40 nm 80 nm 40 nm 80 nm
AFM1-BSA (µg mL-1) 0.35 0.35 0.35 0.35
pAb (ng mL-1) 240 15 240 15
Biotinylated secondary-Ab (ng mL-1) 500
Streptavidin-Au nanoparticles (µµµµg mL-1) 0.08
LoD (ng L-1) 12 2 5 0.1
LLoQ -ULoQ (ng L-1) 30-5000 6-1250 10-2500 0.3-300
Firstly, the optimum AFM1-BSA and pAb concentration was investigated by
means of a checkerboard tritation experiments. The AFM1-BSA concentration
was modified between 0.07 and 10 µg mL-1 whereas the pAb concentration
ranged from 16 to 2000 ng mL-1. The streptavidin-nanoparticles conjugate
concentration was kept at 0.08 µg mL-1 for these experiments. The optimum
AFM1-BSA concentration was similar for all streptavidin-nanoparticles
conjugates (i.e. 0.35 µg mL-1), but significant differences were noted on the pAb
concentration. The optimum pAb concentration using both 80 nm Ag and Au
nanoparticles was 15 ng mL-1 whereas it was 250 ng mL-1 for 40 nm Ag
nanoparticles. Interestingly, the optimum pAb concentration using 40 nm Ag
nanoparticles was similar to that previously found for 40 nm Au counterparts
[12]. From these findings, it could be derived that for this assay format,
analytical figures of merit afforded were strongly dependent on the nanoparticle
size.
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
96
Figure 3.2 shows the influence of the pAb concentration on LoD operating
with streptavidin-80 and 40 nm Au nanoparticles conjugate.
Figure 3.2. Influence of the α-AFM1 pAb concentration on the limits of detection
of AFM1 operating with different streptavidin-Au nanoparticles conjugates. ()
40 nm () 80 nm. Aflatoxin M1-BSA concentration: 0.35 ng L-1; secondary Ab
concentration: 500 ng mL-1; streptavidin-nanoparticles conjugate concentration:
0.08 µg mL-1.
It is important to remark that the only difference between the experimental set
up of both Au nanoparticles was the pAb concentration. So, it was feasible to
directly evaluate the influence of the pAb concentration on the LoD. Limit of
detection was calculated as three times the standard deviation of the signal of
15 blank replicates [19]. Experimental data for 40 nm Au nanoparticles was
measured following previously reported experimental conditions [12]. As
expected, irrespective of the nanoparticle considered, LoD increased when the
pAb concentration was increased. Under optimum pAb concentration,
0.01
0.1
1
10
100
15 30 60 120 240 480 960
LoD
(ng
L-1)
Ab concentration (µg L-1)
Chapter 3
97
streptavidin-80 nm Au nanoparticles conjugate afforded a LoD of 0.1 ng L-1, i.e.
50-fold lower than that obtained with the 40 nm Au counterparts. Similar
findings were observed for Ag nanoparticles but the LoD obtained were
significantly higher than those obtained using Au nanoparticles (Table 3.2).
Thus, the LoDs for 80 nm and 40 nm Ag nanoparticles were 2 and 12 ng L-1,
respectively. The origin of these differences is not clear; since, for a given
nanoparticle size, optimum immunoassay conditions do not depend on the
element in the nanoparticle. Nevertheless, it was noted that the use of Ag
nanoparticles was tricky due to a build up of Ag metallic deposits into the
injector tube that could even lead to full blockage. This phenomenon was also
noticed for Au nanoparticles but the metallic deposit formation was significantly
lower. Finally, the influence of the streptavidin-nanoparticle conjugate
concentration on the analytical figures of merit was also investigated. This
parameter was modified between 0.02 µg mL-1 and 0.32 µg mL-1. It was
observed that the signal registered by the mass spectrometer increased with
the streptavidin-nanoparticle conjugate concentration but no changes were
produced either on the optimum pAb and AFM1-BSA concentration or the LoD
for the nanoparticles tested. These results were similar to those previously
reported for 40 nm Au nanoparticles [12]; thus, pointing out that this parameter
was not critical for the immunoassay performance. It should be considered that,
given the limited amount of biotin moieties in the secondary Ab, signal
amplification is not high enough to reduce the pAb concentration in the
immunoassay.
Finally, the lower and upper quantification limits (lLoQ and uLoQ,
respectively) obtained with each streptavidin-nanoparticle conjugate were
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
98
evaluated (Table 3.2). The lLoQ was defined as the analyte concentration that
has a response at least 3 times that of a blank sample and repeatability lower
than 20%. Similarly, the uLoQ was defined as the highest concentration
standard that signal response has repeatability lower than 20% [20]. As
expected by the changes on detection capabilities, lLoQ and uLoQ depends on
the streptativin-nanoparticle conjugate employed. For instance, lLoQ and uLoQ
experimental values for streptavidin-80 nm Au nanoparticle conjugates were 0.3
ng L-1 and 300 ng L-1, respectively. Nevertheless, the dynamic linear range for
the different nanoparticles tested was similar (3/2-order of magnitude).
3.1.2. Capture Inhibition Assay.
In this immunoassay format, unlike the previous one, the sample containing
AFM1 was spiked with biotinylated AFM1-BSA and both species compete for the
limited number of the binding sites of the pAb which coated the solid phase. The
variables selected for the optimization of this immunoassay were the
concentration of: (i) pAb; (ii) biotinylated AFM1-BSA; (iii) streptavidin-
nanoparticles conjugate; and (iv) the type and size of the nanoparticle in the
streptavidin-nanoparticles conjugate. Firstly, the pAb and the biotinylated AFM1-
BSA concentration was optimized by means of checkerboard titration
experiments. The pAb concentration was ranged from 0.6 µg mL-1 to 10 µg mL-1
whereas the biotinylated AFM1-BSA concentration was modified between 0.08
and 5 µg mL-1. For these experiments, the streptavidin-nanoparticles conjugate
concentration was kept constant at 0.08 µg mL-1. Experimental results showed
that the optimum response was obtained for 2.5 µg mL-1 pAb and 0.03 ng mL-1
biotinylated AFM1-BSA. Under these experimental conditions, a calibration
curve was built with AFM1 standards to evaluate the immunoassay detection
Chapter 3
99
capabilities and the dynamic range. It was observed that a LoD of 100 ng kg-1
was always obtained, regardless the size and the element of the nanoparticle
employed (Table 3.3).
Table 3.3. Optimum experimental conditions, limits of detection and dynamic
range for CIA and CBIA formats.
Parameter CIA CBIA
Nanoparticle Ag/Au (40/80 nm) Ag/Au (40/80 nm)
AFM1-BSA (µg mL-1) - 1
pAb (ng mL-1) 2500 2000
Biotinylated AFM1-BSA (µg mL-1) 0.03 0.04
Streptavidin-Au nanoparticles (µµµµg mL-1) 0.08 0.08
LoD (ng L-1) 100 5
LLoQ -ULoQ (ng L-1) 300-5000 15-2500
Though this value was similar to that previously reported for antigen-protein
conjugate employed as tracer for progesterone analysis [9], it was significantly
higher than the value obtained by means of ABIA. To improve the LoD, the
immunoassay optimization was repeated using higher streptavidin-
nanoparticles conjugate concentrations. The concentration of this reagent was
modified from 0.08 to 0.64 µg mL-1. As it was shown for ABIA, analyte signal
increased with streptavidin-nanoparticles conjugate concentration but no
significant changes were produced on the optimum pAb and biotinylated AFM1-
BSA concentration values previously obtained and, consequently, on the LoDs.
Therefore, the streptavidin-nanoparticles conjugate concentration was kept at
0.08 µg mL-1 to minimize operative cost. Alternatively, the influence of the
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
100
incubation time of the AFM1 and the biotinylated AFM1-BSA mixture on the
microtiter plate was studied since it favored tracer retention on the solid phase;
thus, making feasible to operate with lower tracer concentrations [12]. The
incubation time was increased from 1 to 10 hours but, again, no significant
improvement on the LoD was obtained. From these findings, it can be derived
that detection capabilities were limited by the immunoassay procedure itself. It
should be considered that, given the size of the BSA moieties (66 kDa), high
steric effects are expected when biotinylated AFM1-BSA molecules are
captured by the pAbs coating the microtiter plate. Obviously, steric effects are
more significant when decreasing the AFM1 content in the sample since more
biotinylated AFM1-BSA molecules are captured on the solid phase. To address
this issue, the use of antibodies containing a spacer was proposed. From a
practical point of view, the use of antibodies with a spacer would reduce sample
throughput due to an additional incubation step would be required and, hence,
this approach was discarded. Instead, some modifications were implemented in
this immunoassay format to favor the immunocomplex formation. To this end, a
mixture of the pAb and the biotinylated AFM1-BSA was initially spiked to the
sample containing the AFM1. A competitive immunoreaction was stablished
between the AFM1 and the biotinylated AFM1-BSA for the limited number of the
binding sites of the pAb. In this way, the immunocomplexes were generated in a
homogeneous phase rather than in a heteregoenus phase; thus, minimizing
steric effects. Finally, given that the antibodies are bivalent molecules, the
mixture was incubated in a microtiter plate coated with AFM1-BSA in order to
capture AFM1-BSA-pAb and AFM1-pAb immunocomplexes. According to this
scheme, AFM1-BSA coating the microtiter plate merely served as a capturing
Chapter 3
101
agent to fix the immunocomplexes formed on the solid phase. To our best
knowledge, this is the first time that this strategy is employed for hapten
analysis with ICP-MS detection. From now on, this immunoassay will be called
capture bridge immunoassay (CBIA).
The optimization of CBIA was carried out likewise the CIA procedure but
including the concentration of the AFM1-BSA coating the microtiter plate. After
some preliminary test, it was observed that this concentration was not critical for
the immunoassay. This reagent had to be in excess to maximize the
immunocomplex capture and, hence, this parameter was kept at 1 ng L-1. Next,
a checkerboard tritation procedure was employed to optimize the biotinylated
AFM1–BSA and the pAb concentration. To this end, the pAb concentration was
modified from 0.125 µg mL-1 to 2 µg mL-1 whereas the biotinylated AFM1 –BSA
concentration was tested from 0.01 µg mL-1 to 0.64 µg mL-1. A streptavidin-
nanoparticles conjugate concentration of 0.08 µg mL-1 was employed. A
satisfying compromise between an optimal analytical sensitivity with a
consistent readout was obtained using 2 µg mL-1 pAb and 0.04 µg mL-1
biotinylated AFM1-BSA for all streptavidin-nanoparticles conjugate. Under these
conditions, as it was previously noticed for the CIA procedure, LoDs for AFM1
determination were again independent on the streptavidin-nanoparticles
conjugate, both type and concentration. This phenomenon is clearly observed in
Figure 3.3 where the calibration curves obtained using different streptavidin-Au
nanoparticles conjugate are presented. Nevertheless, steric effects were less
significant with the new approach since the LoD for CBIA format (5 ng mL-1)
was improved 20-fold regarding CIA strategy. From these results, and given its
better analytical performance, CBIA strategy was selected for further studies.
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
102
Figure 3.3. Aflatoxin M1 calibration curve using different streptavidin-Au
nanoparticles conjugate for the CBIA procedure. (--) 40 nm; (--) 80 nm.
Aflatoxin M1-BSA concentration: 1.0 ng mL-1; streptavidin-Au nanoparticles
conjugate concentration: 0.08 µg mL-1.
3.2. Aflatoxin M1 analysis in milk samples.
Both ABIA and CBIA formats were validated for AFM1 analysis in milk
samples. The streptavidin-80 nm Au nanoparticles conjugate was used for ABIA
measurements due to its higher detection capabilities. As regards CBIA, though
the nanoparticle characteristics are not critical, 80 nm Au nanoparticles were
also employed for the sake of comparison. Method accuracy and precision were
checked by analyzing an AFM1 certified reference material (ERM-BD283 milk
powder, at 11.1 ± 1.8 ng kg-1) and different commercial milk samples spiked
with known amounts of AFM1. Recovery test was performed spiking milk
samples with AFM1 standard at two different levels: above (80 ng kg-1) and
below (30 ng kg-1), the limits stablished by the European Union policy.
0
20
40
60
80
100
120
1 10 100 1000 10000
Inhi
bitio
n (%
)
AFM1 concentration (ng Kg-1)
Chapter 3
103
Initially, all samples were analyzed after an extraction pretreatment with
acetonitrile to mitigate the effects of matrix components (e.g. proteins, fats, etc.)
on the immunoreaction following the optimized ABIA and CBIA procedures.
However, AFM1 recoveries were systematically higher than 100% for both
approaches. These results were totally unexpected since this approach was
previously employed for AFM1 analysis with the ABIA procedure using the
streptavidin-40 nm Au nanoparticles conjugate [12]. The higher matrix effects
registered for the ABIA format using streptavidin-80 nm Au nanoparticles
conjugate could be attributed to the lower pAb concentration employed since it
makes the immunoassay more sensitive to low AFM1 concentrations but also to
the matrix components. Given the LoD afforded by the ABIA procedure, sample
dilution was explored to mitigate matrix effects due to its simplicity and lower
cost regarding immunocolumns. To this end, before the extraction pretreatment,
milk samples were diluted with the appropriate amount of ultrapure water. Table
3.4 shows the recovery values for AFM1 determination in the certified reference
material and in the spiked milk samples by means of ABIA procedure using
streptavidin-80 nm Au nanoparticles conjugate after a 4-fold milk dilution. There
is observed to be a good agreement between the experimental and the
theoretical values. Aflatoxin M1 recovery values ranged between 97% and
107%. These values were within the limits established by the EU for analyte
concentrations below 1 µg kg-1 (-40%/+20%) [23,24]. It is important to remark
that the use of a lower dilution factor did not allow accurate AFM1
determinations. Taking into account the dilution factor applied, LoD for AFM1
determination in milk samples by means of ABIA format was 0.4 ng L-1. The
repeatability (intra-assay precision) was determined by analyzing five replicates
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
104
of each sample on the same day. The RSD of the AFM1 determination was
within the 5%-15%. The immunoassay reproducibility (inter-assay precision)
was evaluated as the RSD of the measurements obtained for five independent
immunoassays performed in five different days. The reproducibility was within
10-25% for the ABIA format.
Table 3.4. Recovery analysis of AFM1 by ABIA and CBIA methodologies.
Sample pretratment: ABIA: dilution + acetonitrile extraction; CBIA:
immunocolumns.
Immuoassay Sample
AFM1 concentration (ng kg-1)*
Recovery (%)* Certified
/expected Experimental
ABIA
Commercial whole milk 80 81 ± 9 101 ± 8
Commercial whole milk 30 29 ± 3 98 ± 8
Commercial fresh milk 80 81 ± 4 107 ± 7
Commercial fresh milk 30 32 ± 2 105 ± 4
ERM®-BD283 11.1 ± 1.8 11 ± 1 103 ± 7
CBIA
Commercial whole milk 80 71 ±2 0 89 ± 20
Commercial whole milk 32 34 ± 10 106 ± 30
Commercial fresh milk 80 75 ± 15 93 ± 20
Commercial fresh milk 32 31 ± 11 97 ± 30
ERM®-BD283 11.1 ± 1.8 11 ± 3 100 ± 28
* mean ± standard deviation, replicates = 5.
As regards the CBIA format, the poor recoveries obtained for milk analysis after
the extraction pretreatment pointed out that this immunoassay was quite
sensitive to matrix components. It should be taking into account that matrix
components affect both the biotinylated AFM1-BSA-pAb immunocomplex
Chapter 3
105
formation and its retention on the microtiter plate. Unlike ABIA, dilution strategy
could not be employed with this immunoassay since LoD did not allow to
quantify AFM1 according to European Union Policy. Consequently,
immunocolumns were required for AFM1 determination. This approach made
feasible AFM1 analysis but at the expense of a significant precision increase
(Table 3.4). The RSD for intra-assay and inter-assay determination of AFM1
was 30%. Due to the extraction-preconcentration pretreatment, LoD for CBIA
format was approximately improved 50-fold (i.e. 0.1 ng mL-1) and, consequently,
detection capabilities for CBIA strategy were similar to those shown by ABIA. It
is interesting to note that irrespective of the immunoassay format, LoD for AFM1
determination were significantly lower (30/45000-fold) than those previously
reported for other hapten analysis by means of competitive immunoassays with
ICP-MS detection.
Finally, both methodologies were applied to the analysis of commercial
products (i.e. raw, pasteurized and ultra-high temperature pasteurized milk)
obtained from retail markets and supermarkets. However, none of them showed
detectable levels of AFM1.
3.3. Comparison of different competitive immunoassay formats for
AFM1 determination.
Aflatoxin M1 has been classified as Group 2 human carcinogen by the
International Agency of Research on Cancer [21] and, consequently, the
maximum allowed levels of this substance are strictly regulated worldwide. For
instance, European Community legislation limits AFM1 levels in milk and infant
formula at 50 and 25 ng kg-1 [22,23] whereas Food and Drug Administration
from USA limits do allow AFM1 levels up to 500 ng kg-1 [24]. Therefore, the
Evaluation of different competitive immunoassay for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry
106
analytical parameters (i.e., LoD, dynamic range, sample throughput, matrix
effects, etc.) of the different immunoassay formats developed have been
compared for AFM1 analysis.
From previous data shown in Sections 3.1 and 3.2, it could be derived that
both ABIA and CBIA could be employed for AFM1 analysis according to the
international policies. Nevertheless, the use of pAb as tracer specie (ABIA)
seems to be more advantageous than the use of the antigen-protein conjugates
as the tracer one (CBIA) for AFM1 analysis. Immunocomplex formation in the
ABIA format is not impeded by steric effects since, given the volume occupied
by the BSA residues on the solid phase, pAbs have enough space to form the
immunocomplex without interacting with its neighborhoods (Figure 3.1). In this
case, the use of nanoparticles with bigger diameter is indeed advantageous to
detect lower amounts of pAb retained on the solid phase. As regard CIA and
CBIA, steric effects control the immunocomplex formation between the
biotinylated AFM1-BSA and the pAbs and, consequently, no direct advantages
are derived from using different kind of nanoparticles for tracer detection.
Probably, steric effects explain the higher matrix effects and lower dynamic
range shown by CBIA regarding ABIA. On the other hand, despite ABIA
immunoassay procedure takes longer than CBIA (7 vs 4 hours), no real
advantage on sample throughput is derived from the latter since sample
preparation is more complex, time consuming and costly due to the use of
immunocolumns. It is important to note that cost derived from the use of 100-
fold less amount of pAb is lower for ABIA in comparison to CBIA. Nevertheless,
this feature is partially counterbalanced due to the use of a secondary Ab.
Chapter 3
107
Finally, analytical figures of merit of ABIA and CBIA procedures have been
compared with those previously reported in the literature (Table 3.5).
Comparing to other competitive immunoassays and sensors, the LoD and the
dynamic range are clearly improved by the immunoassays with ICP-MS
detection. Nevertheless, sample throughput is partially deteriorated due to the
sequential nature of the mass spectrometer. As regard chromatographic
methods, the ABIA format affords similar LoDs without requiring complex
sample pretreatments based on solid phase extraction or immunocolumns to
preconcentrate and purify the AFM1. In this regard, CBIA format is clearly less
attractive since immunocolumns are mandatory for AFM1 analysis. The main
benefit of mass spectrometry is the feasibility of multi-compound analysis.
4. Conclusions.
This work demonstrates that the tracer specie used (i.e. antibody or
antigen-protein) in competitive immunoassays with ICP-MS detection is critical
for AFM1 analysis. Using the antibody as tracer specie, the heteroatom-label
exerts a great influence on the immunoassay experimental conditions and,
consequently, on the analytical figures of merit. This type of immunoassay
affords better analytical figures of merit and lower matrix effects than those
based on the use of antigen-protein conjugate as tracer. The immunocomplex
formation in the latter strategy is severely hampered by steric effects caused by
the protein moiety in the antigen-protein conjugate. According to these results,
competitive immunoassays based on the use of antibodies as tracer specie
seem to be more suitable for hapten analysis by means of ICP-MS detection
since this strategy exploits better the detection capabilities afforded by the
technique.
Evalu
ation
of d
ifferent co
mp
etitive imm
un
oassay fo
r aflatoxin
M1 d
etermin
ation
in m
ilk samp
les by m
eans o
f ind
uctively co
up
led p
lasma m
ass sp
ectrom
etry
Tab
le 3.5. Co
mp
aris
on
of d
ive
rse
an
aly
tica
l me
thod
olo
gie
s p
rop
ose
d in
the
litera
ture
for A
FM
1 de
term
ina
tion
in m
ilk s
am
ple
s.
M
etho
do
log
y R
ecovery
(%)
Precisio
n
(%)
LO
D
(ng
kg-1)
Lin
ear rang
e
(ng
kg-1)
Referen
ce
Immunoassays
AB
IA- IC
PM
S
98-1
07
< 1
0
0.4
1-3
00
This
work
CB
IA-IC
PM
S
89-1
06
< 3
0
0.1
15-2
500
This
work
Dire
ct c
om
petitive
ELIS
A (U
V-V
is d
ete
ction)
90-1
10
< 1
0
3
- [2
5]
Dire
ct c
om
petitive
ELIS
A
(chem
ilum
inis
cente
dete
ctio
n)
80-1
20
< 1
0
1
2-7
.5
[26]
Indire
ct c
om
petitive
ELIS
A (U
V-V
is d
ete
ctio
n)
80-1
02
5-1
7
40
40-5
00
[27]
Liquid chromatography
Hig
hly-s
ensitive
time-re
solve
d flu
ore
scent
imm
unochro
mato
gra
phic a
ssay
80-1
10
5-1
2
0.3
0.1
-200
[28]
UH
PLC
-MS
/MS
79
< 1
5
0.1
8
50-5
00000
[29]
SP
E-U
PLC
-MS
/MS
89-1
20
2-9
0.3
1-3
00
[30]
Liq
uid
-liquid
extra
ctio
n H
PLC
-Flu
ore
scence
73-9
9
2-7
50
- [3
1]
MS
PE
-HP
LC
-FD
91-1
02
5
5
15-1
0000
[32]
Sensors
Optic
al im
munosensor
- -
0.6
U
p to
1.8
× 1
03
[33]
Ele
ctro
chem
ical im
munosensor
- -
12
15-1
000
[34]
Flo
w in
jectio
n in
munoass
ay
80-1
20
8
11
20 - 5
00
[35]
Chapter 3
109
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[5] C. Giesen, L. Waentig, U. Panne & N. Jakubowski. History of inductively
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for Quantum Dots-based immunoassays: A critical appraisal. Biosens.
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[9] A.P. Deng, H.T. Liu, S.J. Jiang, H.J. Huang & C.W. Ong. Reaction cell
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[10] P. Jarujamrus R. Chawengkirttikul, J. Shiowatana & A. Siripinyanond.
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[13] L. Trapiella-Alfonso, J.M. Costa-Fernández, R. Pereiro & A. Sanz-Medel.
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progesterone determination in bovine milk. Biosens. Bioelectronics 26
(2011) 4753-4759.
[14] W. Chen, P. Wee & I.D. Brindle. Elimination of the memory effects of gold,
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[15] F. Vanhaecke, H. Vanhoe, R. Dams & C. Vandecasteele. The use of
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ochratoxin A, zearalenone and -zearalenol in milk by UHPLC–MS/MS.
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[17] J. R. Crowter (2001) The ELISA Guidebook. (2nd ed.) New Jersey,
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[18] S.J. Gee, T. Miyamoto, M.H. Goodrow, D. Buster & B.D. Hammock.
Development of an enzyme-linked immunosorbent assay for the analysis of
the thiocarbamate herbicide molinate. Journal of Agricultural and Food
Chemistry 36 (1988) 863–870.
[19] W. Jiang, Z. Wang, G. Nolke, J. Zhang, L. Niu & J. Shen. Simultaneous
determination of aflatoxin B1 and aflatoxin M1 in food matrices by enzyme-
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[21] International Agency for Research on Cancer (2002). Monographs on the
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[22] EC (2004). Commission Regulation, Regards Aflatoxins and Ochratoxin a
in Foods for Infants and Young Children, Official Journal of the European
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[23] EC (2006). Commission Regulation (EC) No. 1881/2006 of 19 December
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[24] FDA, Sec. 527.400 Whole milk, Low fat milk, skim milk – aflatoxin M1 (CPG
7106.10), FDA/ORA Compliancy Guides 2005.
[25] D. Guan, P. Li, W. Zhang, D. Zhang & J. Jiang. An ultra-sensitive
monoclonal antibody-based competitive enzyme immunoassay for aflatoxin
M1 in milk and infant milk products. Food Chemistry 125 (2011) 1359-1364.
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ultrasensitive direct chemiluminescent enzyme immunoassay for
determination of aflatoxin M1 in milk. Food Chemistry 158 (2014) 310-314.
[27] S.C. Pei, Y.Y. Zhang, S. A. Eremin & W.J. Lee. Detection of aflatoxin M1 in
milk products from China by ELISA using monoclonal antibodies. Food
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[28] X. Tang, Z. Zhang, P. Li, Q. Zhang, J. Jiang, D. Wang & J. Lei. Sample-
pretreatment-free based high sensitive determination of aflatoxin M1 in raw
milk using a time-resolved fluorescent competitive immunochromatographic
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utra-high-performance liquid chromatography/tandem mass spectrometry.
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[31] P. Diniz Andrade, J. Laine Gomez da Silva & E. Dutra Caldas.
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liquid-liquid extraction with low temperature purification (LLE-LTP). Journal
of Chromatography A 1304 (2013) 61-68.
[32] M. Hashemi & Z. Taherimaslak. Determination of aflatoxin M1 in liquid milk
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[33] Y. Wang, J. Dostálek & W. Knoll. Long range surface plasmon-enhanced
fluorescence spectroscopy for the detection of aflatoxin M1 in milk. Biosens.
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Characterization of the gold-catalyzed deposition of silver on graphite
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[35] M. Badea, L. Micheli, M.C. Messia, T. Candigliota, E. Marconi, T. Mottram,
M. Velasco-Garcia, D. Moscone & G. Palleschi. Aflatoxin M1 determination
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520 (2004) 141-148.
Chapter 4
A sensitive size-exclusion inductively
coupled plasma mass spectrometry
multiplexed assay for cancer biomarkers
using antibodies conjugated with a
lanthanide-labelled polymer
Chapter 4
117
1. Introduction.
Recently, a number of immunoassays based on the use of metal-labelled
antibodies and the determination of antibody-antigen complexes by inductively
coupled plasma mass spectrometry (ICP-MS) have been proposed for the
determination of biomolecules, and, in particular, proteins [1,2]. The ICP-MS
quantification offers several advantages over the conventional detection
techniques employed in immunoassays (colorimetry, fluorimetry, etc.), such as,
e.g., (i) specificity to heteroatom detection; (ii) compound-independent detection
sensitivity; (iii) high elemental sensitivity and dynamic range; (iv) limited sample
treatment; (v) stability of the reagents against time, temperature and light (the
isotopic masses do not change, bleach or degrade); (vi) reduction of non-
specific background; (vii) independence of analytical response from incubation
or storage times and (viii) multiplexed detection [2,3].
Antibodies have usually been labelled by either metal nanoparticles [4,5] or
lanthanides [6,7]. The advantage of elemental nanoparticles is the possibility of
the introduction of a significant number of atoms per conjugate which allows the
amplification of the analytical response. This advantage is set off by the high
affinity of nanoparticles to surface of labware and/or ICP-MS sample
introduction system, increasing wash-in and wash-out times, and by the
difficulty to synthetize nanoparticles of uniform size. Lanthanides are introduced
as DOTA or DTPA chelates due to its extraordinary thermodynamic stability [6].
The similar chemical properties make lanthanides well suited for multiplex
assays: different antibodies can be easily and specifically labelled with different
lanthanides in the same experimental workflow. Of particular interest, because
of their simplicity, are homogenous immunoassays in which a mixture of
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
118
antibodies, each labelled with a different lanthanide, is made react within a
liquid sample and the antigen-antibody (Ag-Ab) complexes formed are isolated
and specifically determined [8]. Size-exclusion chromatography (SEC) coupled
with ICP-MS first proposed by Terenghi et al. [9] is a convenient method to
isolate the Ag-Ab complex from the excess of reagent offering: (i) multiplexed
capability; (ii) low sample amount consumption; and (iii) virtually no sample
preparation. Hann et al. [10] reported a SEC-ICPMS determination of an In-
DOTA labelled peptide Bβ15–42-antibody complex in cellular extracts. López-
Fernández et al. [11] developed a methodology for monitoring and determining
oligonucleotide sequences by means of DOTA-lanthanide labelled DNA probes
followed by SEC-ICPMS detection. Though the above-mentioned works
demonstrated the benefits of using homogeneous-based immunoassays with
ICP-MS detection, signal amplification was limited since only a single lanthanide
atom was introduced per binding site of the antibody. The number of lanthanide
atoms per antibody can be increased by using metal-loaded polymers [12],
which leads to an increase in sensitivity. This labelling strategy has been
successfully employed for single-cell ICP MS analysis [13,14,15]. Waentig et al.
[16] compared polymer-based lanthanide labelling with other lanthanide-based
labelling strategies for protein quantification in solid phase immunoassays
(Western Blot, SDS-PAGE, etc.). These authors noted that this labelling
strategy improves significantly sensitivity which results in limits of detection in
the low fmol range. However, there has been no attempt so far to investigate the
potential of antibodies conjugated with metal-labelled polymers in homogenous
assays.
Chapter 4
119
The aim of this work was to increase the sensitivity, by at least a factor of
ten, of a direct homogenous multiplexed assay for four cancer biomarkers
(CEA, sErbB2, CA 15.3 and CA 125) in human serum by -labelling the relevant
monoclonal antibodies with a different polymer-based lanthanide group. Size-
exclusion chromatography was used to isolate the antigen-antibody complexes
whereas ICP-MS on-line detection was used for quantification. The method was
benchmarked against the ones using the labelling with DOTA-chelates [9].
2. Experimental.
2.1. Reagents and materials.
Carcinoembryonic antigen (CEA) was obtained from Sigma-Aldrich (St.
Quentin-Fallavier, France). The soluble form of human epidermal growth factor
receptor 2 (sErbB2) was purchased from antibodies-online (Aachen, Germany).
Cancer antigen 15.3 (CA 15.3) was obtained from MyBioSource (San Diego,
CA) and CA 125 was from Fitzgerald (MA). Goat polyclonal antimouse IgG
antibody (H&L)was purchased from Abcam (Cambridge, UK).
Mouse Immunoglobulin G subclass 1 (IgG1) antihuman monoclonal
antibody (mAb) for α-CEA (clone 1C11) and mouse IgG1 antihuman mAb for α-
CA 125 (clone X325) were purchased from Gene Tex (Irvine, CA). Mouse IgG1
antihuman mAb for α-sErbB2 (clone 5J297) was obtained from antibodies-
online (Aachen, Germany) and mouse IgG1 antihuman mAb for α-CA 15.3
(clone M002204) was from LifeSpan BioSciences (Seattle, WA). The antibody
(Ab) solutions should not contain additives, such as bovine serum albumin
(BSA) or gelatin, because the latter could be labelled as well and cause
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
120
interferences. Upon reception, mAb were divided into single working aliquots
and stored at -20ºC.
MAXPARTM- polymer -Ab labelling kits were obtained from Fluidigm (Les
Ulis, France). Human Albumin AlbuteinTM 20% was purchased from Grifols
Biologicals Inc. (Los Angeles, CA). 1,4,7,10 – Tetraazacyclododecane - 1,4,7 -
tris(aceticacid) - 10-maleimido - ethylacetamide (DOTA) was obtained from
Macrocyclics (Dallas, TX). Tris (2-carboxyethyl) - phosphine hydrochloride
(TCEP), TrizmaTM base, lanthanide chlorides (HoCl3, TbCl3, TmCl3, PrCl3) with
natural isotopic abundance, ammonium acetate ( 98%, for molecular biology),
monosodium phosphate, disodium phosphate, ethylenediaminetetraacetic acid
disodium salt (EDTA), dimethyl sulfoxide (DMSO), sodium chloride and
polyethylene glycol sorbitan monolaurate (Tween 20) were from Aldrich
(Schelldorf, Germany). Acetic acid glacial and 69% w w-1 nitric acid were
purchased from Panreac (Barcelona, Spain). Rare earth 100 µg mL-1 Complete
Standard was provided by Inorganic Ventures (Lakewood, Colorado) and DC™
Protein Assay Kit was from Bio-Rad (Marnes-la-Coquette, France).
Ultrapure water 18 M cm from a Milli-Q water purification system
(Millipore, Paris, France) was used throughout the work.
AmiconTM Ultra-0.5 mL centrifugal filters for DNA and protein purification
and concentration (Merck Millipore, Cork, Ireland) with different cutoff limits (3,
30 and 50 kDa) were used throughout the work for washing steps and buffers
exchange during labelling procedure of Abs using a EppenforfTM microcentrifuge
5415R (Eppendorf AG, Hamburg, Germany).
Chapter 4
121
2.2. Buffers.
The buffers used were: (a) ammonium acetate buffer (100 mM, pH 6.8 as
elution buffer and 20 mM, pH 6.0 for metal complexation), (b) phosphate buffer
(100 mM, pH 7.2, 2.5 mM EDTA) for the partial reduction of the antibody with
TCEP and (c) Tris buffer saline (20 mM Tris-HCl, 0,45% NaCl, pH 7.0 for
antibody storage media and 20 mM Tris-HCl, 0,45% NaCl, 10 mM EDTA, pH
7.0 for removing TCEP).
2.3. Serum samples.
Serum samples were provided from Hospital General Universitario of
Alicante (Alicante, Spain).
2.4. Instrumentation.
2.4.1. Size Exclusion Chromatography.
The chromatographic analyses were performed on an Agilent 1200 series
(Agilent Technologies, Santa Clara, CA) equipped with an autosampler.
Separations were carried out isocratically at 0.5 mL min−1 using 100 mM
ammonium acetate (pH 6.8) as mobile phase and sample injection volume of
100 L. Two size exclusion columns of different separation range (GE
Healthcare, Buckinghamshire, UK) were tested: a Superose 6 Increase 10/300
GL (cross-linked agarose composite stationary phase; 10 mm x 300 mm x 8.6
µm average beads size) with the approximate bed volume of 24 mL and an
optimum separation range of 5-5000 kDa for globular proteins and a Superdex
200 HR 10/300 (cross-linked agarose and dextran composite stationary phase;
10 mm x 300 mm x 8.6 µm average beads size) with the approximate bed
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
122
volume of 24 mL and an optimum separation range of 10-600 kDa for globular
proteins. The performance of the size exclusion column Superose 6 Increase
10/300 GL was verified with a mixture of blue dextran (Mr 2 000 kDa),
thyroglobulin (Mr 669 kDa), ferritin (Mr 440 kDa), aldolase (Mr 158 kDa),
ovalbumin (Mr 44 kDa), ribonuclease A (Mr 13.7 kDa) and ubiquitin from bovine
(Mr 8.6 kDa) using UV-Vis detection at 280 nm with baseline evaluation at 800
nm. Retention times (in minutes) plotted versus the logarithm of molecular mass
(in kDa) does not give a straight line (two straight lines were obtained: y = -
0.049x +4.104; r2 = 1 for proteins which Mr 440 kDa and y = -0.170x +7.754;
r2 = 0.998 for proteins which Mr 440 kDa). Concerning the size exclusion
column Superdex 200 HR 10/300, it was calibrated similarly to the Superose 6
Increase 10/300 GL but without using blue dextran. Retention times (in minutes)
plotted versus the logarithm of molecular mass (in kDa) gave a straight line (y =
-0.103x +4.881; r2 = 0.991).
2.4.2. Inductively Coupled Plasma Mass Spectrometry.
Detection was carried out by means ICPMS 7700x quadrupole – ICPMS
system (Agilent) equipped with a pneumatic concentric nebulizer and a double-
pass spray chamber. The connection between the exit of the column and the
nebulizer was performed directly by means of polyether ether ketone (PEEK)
tubing. The operating conditions and the nuclides measured are listed in Table
4.1. Instrumental conditions for ICP-MS were daily optimized according to the
protocol described in the user's manual. In order to evaluate the plasma
ionization conditions and the matrix load of the plasma, the 138Ba2+/138Ba+ and
156CeO+/140Ce+ signal ratios were also registered. Quantification was based on
peak areas using the Agilent ChemStation software.
Chapter 4
123
Table 4.1. Operating conditions of SEC-ICPMS.
Parameters
RF Power (W) 1500
Argon flow rate (L min-1):
Plasma gas 15
Auxiliary gas 0.9
Carrier gas 1.15
Carrier
Type 100 mM Ammonium acetate (pH 6.8)
Flow rate (mL min-1) 0.5
Sample introduction system
Injection volume (µL) 100
Nebulizer Pneumatic concentric
Spray chamber Double-pass Scott
Nuclides 141Pr, 159 Tb, 165Ho, 169Tm
2.5. Antibody labelling procedure.
Antibodies (Ab) have been labelled with either a lanthanide-labelled
polymer or a DOTA-lanthanide chelate complexe. Antibody labelling procedure
was based on a chemical reaction between a maleimide residue employed as a
linker of the different metal labels and free sulfhydryl groups obtained after a
partial reduction of the Ab’s cysteine-based disulfide bridges with TCEP. This
procedure was preferred over other approaches due to its lower complexity [6].
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
124
2.5.1. Partial reduction of the antibody.
The labelling method started with a pre-rinse of the ultrafiltration
membranes with phosphate buffer. Thereupon, a monoclonal antibody (mAb)
washing by centrifugation (1 x 500 µL, 10000 x g, 15 min, 4ºC) with phosphate
buffer and a partial reduction of the mAb using an excess of TCEP for 30 min at
370C were carried out. According to the polymer Ab labelling kit protocol, a
molar excess of 60 of TCEP relative to Ab molarity has to be used which has
been optimized for a multitude of IgG isotypes. However, it was observed that
the Ab do not show antigen selectivity in the immune reaction and, hence,
TCEP concentration was optimized. The reduction step for DOTA labelling was
carried out using a 6-fold molar excess [9]. It has to be noted that TCEP is not
particularly stable in phosphate buffers, especially at neutral pH; so the working
solutions have to be prepared immediately before use. EDTA was added to
prevent oxidation of the generated sulfhydryl groups by trace metals [16]. The
mAb was quickly washed (1 × 500 L) with Tris buffer saline to remove the
TCEP in solution by centrifugation and resuspended in the same buffer at 1 mg
mL-1. Then, the mAb was labelled following different procedures, namely: (i)
DOTA-chelate complexes or (ii) polymer labelling kit.
2.5.2. Antibody labelling via the polymer labelling kit.
The mAb was labelled following the protocol of the reagent supplied. Briefly,
the polymer was pre-loaded with a lanthanide for 30 - 40 min at 370C. Then, the
mAb was conjugated with the lanthanide - loaded polymer for 1 h at 370C. The
excess of the ligand was removed from the mAb solution by ultracentrifugation.
Chapter 4
125
2.5.3. Antibody labelling via DOTA-chelate complexes.
This labelling procedure was based on that described by Terenghi et al. [9].
Briefly, the mAb was reacted with a 50-fold molar excess of DOTA for 1 h at
370C. Then, the lanthanide (III) ion was made react with DOTA for 30 min at
370C. The excess of the ligand was removed from the mAb solution by
ultracentrifugation.
In both labelling strategies, it is important to avoid moisture condensation;
otherwise the maleimide moiety will hydrolyze and become non-reactive. The
four mAbs towards the four protein molecules chosen: CEA, sErbB2, CA 15.3,
CA 125, were labelled with the lanthanide ions: 165Ho, 159Tb, 169Tm and 141Pr,
respectively, following both labelling methods described above. Element-
labelled mAbs were stored at – 200C in Tris buffer saline until use.
2.6. Determination of the antibody labelling degree.
2.6.1. Protein quantification.
The concentrations of labelled mAbs were measured by a microplate
spectrophotometer (SPECTROstar Nano, BMG LabTech, Champigny s/Marne,
France) at 750nm using a DC™ Protein Assay Kit. The DC™ (detergent
compatible) protein assay is a colorimetric assay, similar to the well-
documented Lowry assay [17], for protein concentration following detergent
solubilization. Bovine serum albumin was used as calibration standard.
2.6.2. ICP-MS analysis of metal content.
A 0.15 µL volume of all mAbs conjugated with the labelled polymer and 3
µL of mAbs labelled with the DOTA-chelate complexes were diluted up to 5 mL
with 3.5% V/V nitric acid for the determination of the labelling degree of the
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
126
mAbs. An external calibration series from 1 ng mL-1 to 1 µg mL-1 was prepared
using a rare earth multielemental standard solution. Samples were analyzed by
an ICPMS 7700x quadrupole – ICPMS system using the operating conditions
listed in Table 4.1.
2.7. Immunoassay procedure.
A human serum aliquot (120 L) was incubated overnight at 40C with a
mixture of labelled mAbs 2 µg mL-1 or 10 µg mL-1, for the polymer and DOTA-
chelate labels, respectively, and subsequently, analyzed by SEC-ICPMS.
The incubation was performed at 40C in order to avoid protein degradation.
3. Results and discussion.
3.1. Preliminary studies with lanthanide-labelled polymer in SEC-
ICPMS.
Given that polymer-based lanthanide labels have not been tested for the
analysis of biomolecules in homogeneous-based immunoassays so far, a proof
of concept test was initially carried out to evaluate the potential benefits and
drawbacks of this labelling approach. First, following the procedure described in
the experimental section, a goat polyclonal antimouse IgG antibody (pAb) was
labelled with the 165Ho polymer reagents and analyzed by SEC-ICPMS.
Likewise, for the purpose of evaluating the results obtained, this assay was also
carried out using 165Ho DOTA chelate complexes.
Figure 4.1 shows the chromatograms obtained for a solution containing a
nominal concentration of 10 µg mL-1 pAb labelled with 165Ho polymer or 165Ho
DOTA using the Supereose 6 Increase 10/300 GL column. Irrespective of the
Chapter 4
127
labelling approach selected, two 165Ho-related peaks were approximately
obtained at 34 and 40 min. In agreement with the theoretical values expected
from the column calibration curve and UV-Vis measurements at 280 nm, the
first peak corresponds to the 165Ho-labelled pAb; whereas the second one was
identified as metal impurities from the Ab labelling procedure. In fact, the
retention time of the second peak was similar to that obtained from a solution
containing either free 165Ho polymer or free 165Ho DOTA chelate complexes. As
can be seen in Figure 4.1, in the case of using 165Ho polymer, the signal of the
labelled pAb (measured as peak height) was approximately two orders of
magnitude higher than that obtained for the 165Ho DOTA chelate complexes.
These results are totally expected taking into account that there is an average
of 30 chelators per polymer label [18]. Nevertheless, given the signal difference
between both labelling approaches, it could be concluded that the Ab labelling
efficiency achieved with the polymer reagents is at least three times higher than
that afforded by the DOTA chelate complexes.
Figure 4.1. SEC-ICPMS chromatograms of a goat polyclonal antimouse IgG
antibody (pAb) labelled with 165Ho polymer reagents (black line) and 165Ho
DOTA chelate complex (red line). pAb nominal concentration: 10 µg mL-1,
column: Superose 6 Increase 10/300 GL.
0
50
100
150
200
250
300
350
0 5 10 15 20 25 30 35 40 45
165 H
o+
inte
nsi
ty·1
0-4(c
ps)
Time (minutes)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
20 24 28 32 36 40 44
165 H
o+in
tens
ity·
10-4
(cps
)
Time (minutes)
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
128
Next, with the aim to verify the pAb activity and the immunocomplex
formation, a 10 µg mL-1 mouse IgG1 Ab (antigen) solution in ammonium acetate
was incubated overnight at 40C with pAb labelled with either 165Ho polymer or
165Ho DOTA chelate complex at nominal concentration of 10 µg mL-1 and, then,
the mixture obtained was analyzed by SEC-ICPMS (Figure 4.2). In the case of
using the polymer reagents (Figure 4.2 A), the elution profile shows, in addition
to those shown in Figure 4.1, two new peaks at 17 and 31 min, respectively.
Given that the separation in SEC is based on the size of the molecules as they
pass through the column, these results suggest that two different
immunocomplexes have been formed: the first peak corresponds to a high
molecular weight (HMW) immunocomplex whereas the second one to a low
molecular weight (LMW) immunocomplex. According to the retention time
observed for blue dextran (16.4 min) and thyroglobulin (26.2 min) during column
mass calibration, the size of the HMW immunocomplex might be ranged
between 2000 and 700 kDa. On the other hand, the LMW immunocomplex peak
might be related to small antigen-pAb complex given its proximity to the
unreacted pAb peak. The peak corresponding to the unreacted pAb was still
observed either because of the excess of the pAb used or because of its partial
deactivation during the labelling procedure. Interestingly, the chromatographic
profile registered for the mixture of the antigen with the 165Ho DOTA labelled
pAb (Figure 4.2 B) was different to that obtained using the 165Ho polymer-
labelled pAb. The elution profile just shows one new peak at 17 min that, in
agreement with the literature [9] and previous observations with the 165Ho
polymer-labelled pAb, should be related to a HMW immunocomplex. No peak
corresponding to other type of immunocomplexes was registered. Therefore, it
Chapter 4
129
could be concluded that the HMW immunocomplex formation is favored by the
use of 165Ho DOTA labelled pAb over the use of 165Ho polymer-labelled one.
Figure 4.2. SEC-ICPMS chromatograms obtained after incubation of a mouse
IgG1 antibody solution with a goat polyclonal antimouse IgG antibody (pAb)
labelled with (A) 165Ho polymer reagents and (B) 165Ho DOTA chelate complex.
(1) High molecular weight immunocomplex; (2) low molecular weight
immunocomplex; (3) unreacted labelled pAb; (4) free lanthanide label. pAb
nominal concentration: 10 µg mL-1; antigen concentration: 10 µg mL-1;
incubation medium: 100 mM ammonium acetate; column: Superose 6 Increase
10/300 GL.
0
50
100
150
200
250
300
0 5 10 15 20 25 30 35 40 45
165 H
o+
inte
nsi
ty·1
0-4(c
ps)
Time (minutes)
(1)
(2)
(3)
(4)
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30 35 40 45
165 H
o+
inte
nsi
ty·1
0-4(c
ps)
Time (minutes)
(1)
(3)
(4)
B
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
130
The origin of this behavior could be related to steric effects caused by the
polymer chains linked to the Ab which make difficult the formation of big
antigen-antibody (Ag-Ab) aggregates. In fact, both HMW and LMW
immunocomplex signals were observed to be strongly dependent on the Ag:Ab
ratio tested. Thus, for a given antigen amount, a reduction of the Ab
concentration favours the HMW immunocomplex formation at the expense of
the LMW immunocomplex. Conversely, the LMW immunocomplex formation
was improved increasing the Ab concentration. So, when the 165Ho polymer-
label is used, either HMW or LMW immunocomplex signals could be
theoretically employed for protein quantitation purposes. Nevertheless, given
the interdependence between both immunocomplexes, the analytical figures of
merit are expected to be strongly dependent on the Ab concentration employed
in the immunoassay. The above mentioned phenomenon was not observed for
the 165Ho DOTA labelled pAb and, hence, protein quantification could only be
performed using the signal of the HMW immunocomplex [9].
Previous works in SEC showed that unwanted interactions between the
sample components and the chromatographic stationary phase could occur,
thus negatively affecting quantitative analysis [9,19,20]. For this reason,
lanthanide content emerging from the Superose 6 Increase 10/300 GL column
for both labelling strategies was compared to that initially present in the sample
before the chromatographic run. Holmium recovery using the polymer Ab
labelling kit was quantitative (106 ± 3%) but not for the DOTA chelate
complexes (70 ± 5%). The origin of the low recoveries obtained with the latter
approach was unclear. The chromatographic recovery was therefore
determined using an alternative SEC column (Superdex 200 HR 10/300) to that
Chapter 4
131
initially employed in this work (Superose 6 Increase 10/300 GL). While the
lanthanide recovery for DOTA labelling with the alternative column was
quantitative and acceptable (113 ± 13%), the peak resolution between the LMW
immunocomplex and the unreacted Ab for lanthanide-labelled polymer was
compromised. No further differences were observed in the chromatograms
between both columns. Therefore, further studies for the mentioned labelling
strategies were carried out using different SEC columns: the Superose 6
Increase 10/300 GL column for lanthanide-labelled polymer and the Superdex
200 HR 10/300 column for DOTA chelate complexes.
3.2. Analysis of cancer biomarkers in human serum by means of
SEC-ICPMS and polymer-labelled antibodies.
Once the feasibility of using the lanthanide-labelled polymer for protein
analysis in homogeneous-based immunoassays was successfully proved, this
labelling approach was applied for the multiplex determination of cancer
biomarkers in human serum samples; namely: CEA, sErbB2, CA 15.3 and CA
125. To this end, mAbs against the above-mentioned biomarkers have been
labelled with 165Ho, 159Tb, 169Tm and 141Pr, respectively.
3.2.1. Optimization of polymer-labelled antibodies synthesis.
The labelling degree of the polymer-labelled mAbs depends on the number
of sulfhydryl groups obtained after reducing the Ab’s cysteine-based disulfide
bridges with TCEP. To achieve the highest labelling efficiency, it is necessary to
reduce as many disulfide bridges of the mAb as possible. However, the
experimental conditions should not be too harsh so the labelled Ab still shows
antigen selectivity in the immune reaction. In other words, the conditions have
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
132
to be as mild as possible, so that the Ab is not separated into its heavy and light
chain by the breaking of too many disulfide bridges. Preferably, the disulfide
bridges of the hinge region can be cleaved resulting in two identical and still
binding Ab fragments. To evaluate both labelling efficiency and mAb’s activity, it
has been proceeded as follows. First, aliquots of the different mAbs were
reduced with a given molar excess of TCEP, respectively. Next, after labelling
the different mAb with the polymer reagent, a solution containing a nominal
concentration of 1 µg mL-1 of labelled mAb is made to react with different
amounts of their corresponding antigen (0-50 ng mL-1) in human serum. Finally,
the mixture was analyzed by SEC-ICPMS.
Initially, a molar excess rate of 60 of TCEP (concentration recommended by
the reagent supplier) relative to mAb molarity was tested but no
immunocomplexes were registered for all the mAb tested. Similar findings were
observed for 20-fold molar excess, suggesting that the reduction of the disulfide
bridges was too harsh leading to a denaturation of the mAbs. These results
were totally unexpected taking into account polymer manufacturer
recommendations and previous data reported by Waentig et al. [16]. As in the
IgG1 subclass the 2 heavy chains are connected in the hinge region by 2
disulfide bonds [21] and each disulfide bridge needs at least to be reduced 2
protons from the TCEP, the molar excess of TCEP was further reduced ranging
from 2 to 8-fold. For a molar excess of TCEP lower than or equal to 8-fold, the
chromatographs obtained showed the four peaks previously mentioned (free
lanthanide-labelled polymer, unreacted labelled mAb, LMW and HMW
immunocomplexes) thus showing that the mAbs conserved their binding
properties. The elution time for all the peaks was similar to that previously has
Chapter 4
133
pointed out. In general, with a decreasing amount of TCEP, the intensities of the
different immunocomplexes also decreased. Because of signal differences
between 4-fold and 8-fold molar excess of TCEP were lower than 5% and the
reduction step is critical in keeping mAb binding properties, the former was
finally chosen for further studies.
Finally, the labelling degree of the labelled mAbs was evaluated as
described before (section 2.6.). To this end, the metal content of the labelled
mAbs was measured by ICP-MS. In advance, the total amount of the mAb after
labelling was measured with the DC™ Protein Assay Kit in a microtiter plate
because during sample preparation and in particular during the purification step
losses can occur. The labelling degrees (n(Ln):n(Ab)), which correspond to the
number of lanthanide atoms labelled to the mAb, are shown in Table 4.2.
Table 4.2. Labelling degree of the different mAbs using polymer-reagents and
DOTA-chelate complexes.
CEA (165Ho) sErbB2 (159Tb) CA 15.3 (169Tm) CA 125 (141Pr)
Polymer reagents 25 31 36 28
DOTA-chelate
complexes 0.2 0.11 0.3 0.09
On average, there were 29 lanthanides per mAb and, considering that the
lanthanide-labelled polymer contains an average of 30 chelators per label [18],
it points out that almost one polymer label is attached to each Ab. These values
are about 6 times lower than those reported elsewhere [16] but it should be
taking into account that the molar excess of TCEP employed for the partial
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
134
reduction of the mAb in this work was 15 times lower. For the sake of
comparison, the mAbs were also labelled with DOTA-chelate complexes. The
experimental conditions selected were those previously described by Terenghi
et al. [9] where a 6-fold molar excess of TCEP (with regard to the Ab) was used
for the partial reduction of the mAbs. Data gathered in Table 4.2 clearly show
that the use of DOTA-chelate complexes was a less efficient approach for mAb
labelling. In agreement with previous works [16], approximately every thirtieth
Ab was modified with SCN-DOTA which covalently bound to amino groups.
From these experiments, and considering the differences in the lanthanide
content, better analytical figures of merit should be expected for the lanthanide-
labelled polymer.
3.2.2. Influence of the incubation medium on immunocomplex
formation.
Thereupon different solution media were evaluated for incubating the
polymer-labelled mAbs with the biomarkers. Previous works [22] have shown
that nonspecific proteins may assist the formation and stabilization of Ag-Ab
complexes by maintaining the correct conformation of the Ab and antigen for
optimum binding. For this purpose, a solution containing a nominal
concentration of 1 µg mL-1 of the polymer-labelled mAbs was incubated
overnight at 40C with the maximum concentrations of wished-to be determined
antigens (namely: 50 ng mL-1 of CEA, 100 ng mL-1 of sErbB2, 100 IU mL-1 of
CA 15.3 and 100 IU mL-1 CA 125) in the pertinent incubation medium. The
resulting mixture was subsequently analyzed by SEC-ICPMS. The incubation
media tested were: (i) 100 mM ammonium acetate (SEC carrier); (ii) 0.1 % w w-
1 Tween 20; (iii) 6% w w-1 human serum albumin; and (iv) human serum. In this
Chapter 4
135
experiment, the antigen and the mAb concentration was modified regarding
previous sections. The antigen concentration was selected according to the
concentration range of interest in clinical sample analyses whereas the mAb
nominal concentration was decreased 10-fold due to the high signals afforded
by the polymer-labelled Abs and the low biomarker concentration tested.
As expected, regardless of the biomarker, HMW and LMW
immunocomplexes were observed using 0.1 % w w-1 Tween 20, 6% w w-1
human serum albumin or human serum as incubation medium. No detectable
immunocomplex signal was obtained for ammonium acetate despite this
medium was successfully employed in the preliminary studies. Table 4.3
summarizes the experimental data obtained for CEA with the different
incubation media tested. From these data, it was concluded that, given the low
levels of the biomarkers expected in human serum samples, the incubation
medium should contain surfactants and/or proteins to favor immunocomplex
formation [9,22]. In fact, the absence of both HMW and LMW
immunocomplexes signals with ammonium acetate could be probably attributed
to the low levels of the biomarkers tested and the incubation medium
inefficiency to stabilize the Ab and the Ag-Ab complexes.
Human serum from a healthy person contains significant levels of all the
biomarkers studied (CEA, sErbB2, CA 15.3 and CA 125) and, hence, the
concentration values obtained for unknown human serum samples have been
relative to their content in the control human serum employed in the incubation
step. While this situation is not the ideal from an analytical point of view, it
should not be especially troublesome for clinical sample analyses since its main
interest is focused on status changes from reference range concentrations.
A sen
sitive size-exclusio
n in
du
ctively cou
pled
plasm
a mass sp
ectrom
etry mu
ltiplexed
assay for can
cer bio
markers u
sing
antib
od
ies con
jug
ated
with
a lanth
anid
e-labelled
po
lymer
Tab
le 4.3. Influ
en
ce
of th
e in
cub
atio
n m
ed
ium
on
the H
MW
an
d L
MW
imm
un
oco
mp
lexe
s in
teg
rate
d s
ign
als
ob
tain
ed
for C
EA
.
An
tibod
y n
om
ina
l co
nce
ntra
tion
: 1 µ
g m
L-1; C
EA
con
ce
ntra
tion: 5
0 n
g m
L-1 (m
ea
n ±
t·s·n
1/2, n
= 3
, P =
95
%).
C
EA
con
centratio
n (n
g m
L-1)
Imm
un
oco
mp
lex A
mm
on
ium
acetate T
ween
20
(0.1% w
w-1)
Hu
man
serum
albu
min
(6% w
w-1)
Hu
man
serum
HM
W
No
t dete
cte
d
35
00
± 6
00
33
00
± 4
00
91
00
± 1
300
LM
W
No
t dete
cte
d
11
00
00
± 8
00
0
98
00
0 ±
900
0
24
50
00
± 1
00
00
Chapter 4
137
Obviously, this makes imperative to use a control human serum with a known
concentration of all the biomarkers. In this work, a pooled serum, prepared from
15 healthy patients with a declared amount of tumor biomarkers determined
with the conventional heterogeneous immunoassays usually employed in the
clinical analytical laboratories, was used. The concentration levels for all the
biomarkers studied in the control human serum were: 1.7 ng mL-1 CEA, 7 ng
mL-1 sErbB2, 13 IU mL-1 15 IU mL-1 CA 15.3 and CA 125.
3.3.3. Optimization of the concentration of the polymer-labelled
antibody.
As it has been pointed out (section 3.1. and elsewhere [23]), the Ag:Ab ratio
employed in the immune reaction determines which types of immunocomplexes
are formed. To investigate this effect in detail, two types of experiments were
carried out. First, a human serum sample containing a fixed amount of each
biomarker was incubated with variable amounts of the corresponding polymer-
labelled mAb. Alternatively, the concentration of the polymer-labelled mAb was
fixed and the biomarker concentration was modified.
Figure 4.3 shows the chromatograms obtained after incubation overnight at
40C of a human serum sample spiked with 50 ng mL-1 CEA and with the
corresponding 165Ho polymer-labelled mAb at a nominal concentration of 6 ng
mL-1 or 2 µg mL-1. As expected, the Ag:Ab ratio employed was critical on
immunocomplex formation. Thus, incubating the antigen with the polymer-
labelled mAb at a nominal concentration of 6 ng mL-1, just the HMW
immunocomplex was formed and no LMW immunocomplex signal was
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
138
detectable. The opposite behavior was observed for the polymer-labelled mAb
at a nominal concentration of 2 µg mL-1.
Figure 4.3. SEC-ICPMS chromatograms obtained after incubation of a human
serum sample spiked with 50 ng mL-1 CEA and with its corresponding 165Ho
polymer labelled mAb at a nominal concentration of: (A) 6 ng mL-1 or (B) 2 µg
mL-1. Column: Superose 6 Increase 10/300 GL.
Alternatively, human serum samples containing concentrations from 5 to 50
ng mL-1 of CEA were incubated with the corresponding 165Ho polymer-labelled
mAb at the nominal concentrations of 6 ng mL-1 or 2 µg mL-1 (Table 4.4).
0
0.5
1
1.5
2
2.5
0 10 20 30 40
165 H
o+
inte
nsi
ty·1
0-3(c
ps)
Time (minutes)
A
0
100
200
300
400
500
600
0 10 20 30 40
165 H
o+
inte
nsi
ty·1
0-3(c
ps)
Time (minutes)
B
Chapter 4
139
Table 4.4. Influence of the CEA concentration on the immunocomplexes
integrated signals after incubation with 165Ho polymer-labelled mAb at nominal
concentrations of 6 ng mL-1 or 2 µg mL-1. Incubation medium: human serum.
(mean ± t·s·n1/2, n = 3, P = 95%).
CEA concentration (ng mL-1)
Immunocomplex 5 15 30 50
HMW$ 34100±200 36000±600 34100±200 32000±700
LMW& 4391000±2000 4624000±5000 5089000±2000 5589000±3000
$ 6 ng mL
-1 polymer-labelled mAb;
& 2 µg mL
-1 polymer-labelled mAb.
Interestingly, the HMW immunocomplex signal did not increase at increasing
antigen concentration when the polymer-labelled mAb nominal concentration
was 6 ng mL-1. Nevertheless, the LMW immunocomplex signal did show a
linearly increased response for a polymer-labelled mAb nominal concentration
of 2 µg mL-1. The fact that, in the former case, the assay dose response had a
maximum is related to the Hook effect [24] and it is caused by excessively high
concentrations of antigen saturating all of the available binding sites of the Ab
without forming complexes. Consequently, the immunocomplex formation is not
favored and the SEC-ICPMS signal decreases instead of increasing. This
phenomenon is common in one-step immunometric assays, as the one
developed in this work, affecting negatively the dynamic linear range. The Hook
effect can be mitigated by either decreasing the amount of antigen or increasing
the concentration of the Ab. From a practical point of view, the only feasible
approach to deal with this problem is to modify the concentration of the
polymer-labelled mAb. However, as indicated above, when the concentration of
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
140
the polymer-labelled mAb was increased, the LMW immunocomplex was clearly
favored over the HMW one. As a result, the use of the HMW immunocomplex
signal for quantitative purposes must be discarded in favor of the LMW
immunocomplex signal. No Hook effect was observed when the LMW
immunocomplex signal was used for quantification since the Ag-Ab reaction did
not go into antigen excess. These findings were similar for all the biomarkers
studied (Figure 4.4) and, hence, the mAb nominal concentration was set at 2 µg
mL-1 for further studies.
At this point, it is interesting to compare the above-mentioned findings with
experimental data obtained for DOTA labelled mAbs. In agreement with
Terenghi et al. [9] observations, no Hook effect was observed for biomarker
quantification using the HMW immunocomplex signal. This behavior is
explained considering that optimum mAb nominal concentration used (10 µg
mL-1) was 5-fold higher than that using reagents due to the lower signal
amplification afforded by DOTA-chelate complexes.
Chapter 4
141
Figure 4.4. SEC-ICPMS chromatograms obtained after incubation of a human
serum sample spiked with 50 ng mL-1 of sErbB2, CA 15.3 or CA 125 antigen
with its corresponding polymer-labelled antibody at a nominal concentration of 2
µg mL-1. Column: Superose 6 Increase 10/300 GL.
0
100
200
300
400
500
600
0 10 20 30 40
141 T
b+
inte
nsi
ty·1
0-3(c
ps)
Time (minutes)
A
0
100
200
300
400
500
600
0 10 20 30 40
169 T
m+
inte
nsi
ty·1
0-3(c
ps)
Time (minutes)
B
0
100
200
300
400
500
600
0 10 20 30 40
141 P
r+in
ten
sity
·10-3
(cp
s)
Time (minutes)
C
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
142
3.3.4. Figures of merit.
Due to the lack of a certified biomarker reference material for CEA, sErbB2,
CA 15.3 and CA 125 antigen, method accuracy was evaluated by means of a
recovery test. To this end, a control human serum was spiked with the four
tumor biomarkers at three different known concentration levels. This assay was
performed using the optimum operating conditions described in previous
sections. Table 4.5 shows the average of the recovery values of the multiplexed
method developed for the 4 biomarkers.
Table 4.5. Recovery values for the CEA, sErbB2, CA 15.3 and CA 125
biomakers of interest using polymer labelling kit (mean ± t·s·n1/2, n = 3, P =
95%).
Antigen [Antigen]spiked [Antigen]calc Recovery (%)
CEA
3.5 ng mL-1 3.4 ± 0.4 ng mL-1 103 ± 11
16 ng mL-1 16.6 ± 0.3 ng mL-1 96 ± 2
32 ng mL-1 33 ± 2 ng mL-1 98 ± 5
sErbB2
4 ng mL-1 4.0 ± 0.2 ng mL-1 101 ± 6
20 ng mL-1 19 ± 1.2 ng mL-1 105 ± 7
60 ng mL-1 57 ± 3 ng mL-1 106 ± 6
CA 15.3
11 IU mL-1 11.6 ± 0.4 IU mL-1 95 ± 3
40 IU mL-1 41 ± 5 IU mL-1 98 ± 3
68 IU mL-1 69 ± 4 IU mL-1 98 ± 5
CA 125
10 IU mL-1 10.2 ± 0.8 IU mL-1 98 ± 8
35 IU mL-1 34.8 ± 0.5 IU mL-1 100 ± 2
65 IU mL-1 67 ± 2 IU mL-1 97 ± 3
For all the biomarkers studied, the recovery values were quantitative ranging
from 90% to 114%. The repeatability (intra-assay precision) of the method was
Chapter 4
143
determined by analyzing four replicates of each sample on the same day. The
RSD of the biomarkers concentration levels was in the 0.6 - 4% range.
Reproducibility (inter-assay precision) was also verified by analyzing the spiked
human serum samples in four different days with RSDs ranging from 4 to 8%.
The limit of detection (LoD), sensitivity (defined as the slope of the
calibration curve) and linear dynamic range are given in Table 4.6. Given that
the control human serum employed as incubation medium (blank solution) is not
antigen free, a theoretical LoD was roughly estimated dividing 3 times the
standard deviation of the instrument response for blank matrix by the slope of
the calibration curve, from the linear regression analysis [25]. For the sake of
comparison, these parameters were also calculated for the DOTA labelling
approach. In this case, since no LMW immunocomplex signal was observed,
the calibration was carried out using the signal of the HMW immunocomplex. In
general, the sensitivity and the LoDs obtained using polymer reagents were
improved 10-fold (on average) regarding the DOTA labelling. These results
were lower than expected according to the differences in the labelling degree
between both approaches. It should be considered that both unreacted labelled
mAb and LMW immunocomplex were not well-resolved in the chromatogram
and, hence, signal reproducibility for low biomarker concentrations was partially
compromised. As regards linear dynamic range, this parameter was also
improved by 10-fold using polymer reagents. From data shown in Table 4.6,
and despite the low chromatographic resolution, there is no doubt that polymer–
labelling significantly improves the analytical figures of merit of the previous
labelling approach employed in ICPMS homogeneous-based immunoassays for
biomolecules analysis. Moreover, it is worth to mention that the concentration of
A sensitive size-exclusion inductively coupled plasma mass spectrometry multiplexed assay for cancer biomarkers using antibodies conjugated with a lanthanide-labelled polymer
144
the polymer-labelled mAb required in the immunoreaction is decreased 5-fold to
that required using DOTA-labelled mAbs. As a consequence, a considerable
reduction of the cost per analysis is obtained using polymer reagents.
3.3.5. Comparison with other methodologies.
Analytical figures of merit of CEA, sErbB2, CA 15.3 and CA 125 analysis
using polymer labelled mAbs and SEC-ICPMS have been compared with those
previously reported in the literature for other immunoassay-based
methodologies (Table 4.7).
In general, the analysis of these biomarkers is usually carried out by
heterogeneous-based immunoassays which have wider dynamic range but are
time consuming and costly. The major advantage of the proposed methodology
is the possibility to analyze simultaneously 4 biomarkers. In fact, comparing to
commercial sandwich ELISA spectrophotometric kits, detection limits are
improved approximately 10-fold.
4. Conclusions.
This work shows that lanthanide-labelled polymers conjugated with
antibodies can be successfully employed for multiplexed biomarkers analysis
using a homogeneous-based immunoassay and SEC-ICPMS detection. This
new approach improves detection limits 10-fold regarding the lanthanide-DOTA
complex traditionally employed for antibody conjugation.
The established method affords lower LODs than those obtained by commercial
sandwich ELISA spectrophotometric kits for CEA, s ErbB2, CA 15.3 and CA
125 determination as well as higher sample throughput and lower operative
cost.
Ch
apte
r 4
Tab
le 4
.6.
Se
nsitiv
ity,
Lo
Ds a
nd
lin
ea
r d
yn
am
ic r
an
ge
of
CE
A,
sE
rbB
2,
CA
15
.3 a
nd
CA
12
5 a
na
lysis
usin
g b
oth
po
lym
er
lab
elli
ng
kit a
nd D
OT
A-c
he
late
co
mp
lexe
s *
(me
an
± t
·s·n
1/2
, n
= 3
, P
= 9
5%
).
P
oly
mer
lab
ellin
g k
it
DO
TA
-ch
elat
e co
mp
lexe
s
Bio
mar
kers
S
ensi
tivi
ty*
LO
D
Lin
ear
dyn
amic
ran
ge
Sen
siti
vity
* L
OD
L
inea
r d
ynam
ic r
ang
e
CE
A
(4.3
±0.3
)·1
05
0.1
2 n
g m
L-1
0
.4 - 5
0 n
g m
L-1
(4
.2 ±
0.6
)·1
04
3 n
g m
L-1
1
1 -
50
ng m
L-1
sErb
B2
(5.8
± 0
.4)·
10
4
0.5
ng m
L-1
2
- 1
00
ng m
L-1
(0
.8 ±
0.2
)·1
04
8 n
g m
L-1
2
7 -
100
ng m
L-1
CA
15.
3 (1
.0 ±
0.4
)·1
05
0.6
IU
mL
-1
2 -
10
0 IU
mL
-1
(0.3
2 ±
0.0
2)·
10
5
5 I
U m
L-1
1
8 -1
00 I
U m
L-1
CA
125
(1
.13
± 0
.08
)·1
06
0.5
IU
mL
-1
2 -
10
0 IU
mL
-1
(0.4
4 ±
0.0
6)·
10
6
7 I
U m
L-1
2
4 -
100
IU
mL
-1
A sen
sitive size-exclusio
n in
du
ctively cou
pled
plasm
a mass sp
ectrom
etry mu
ltiplexed
assay for can
cer bio
markers u
sing
antib
od
ies con
jug
ated
with
a lanth
anid
e-labelled
po
lymer
Tab
le 4.7. Co
mp
aris
on
of d
iffere
nt m
eth
od
s fo
r CE
A, s
Erb
B2
, CA
15
.3 a
nd
CA
12
5 a
na
lysis
.
An
alytical meth
od
T
arget p
rotein
C
on
centratio
n ran
ge
LO
D
Referen
ce
Com
mercial E
LISA
kit C
EA
0.343 - 250 ng m
L-1
0.2 ng mL
-1 A
bcam (C
ambridge, U
.K.)
Com
mercial E
LISA
kit sE
rbB2
0.819 - 200 ng mL
-1 0.8 ng m
L-1
Abcam
(Cam
bridge, U.K
.)
Com
mercial E
LISA
kit C
A 15.3
5 - 240 UI m
L-1
5 UI m
L-1
Abcam
(Cam
bridge, U.K
.)
Com
mercial E
LISA
kit C
A 125
5 - 400 UI m
L-1
5 UI m
L-1
Abcam
(Cam
bridge, U.K
.)
Chem
iluminescent im
munoassay
CE
A
0.5 - 100 ng mL
-1 0.12 ng m
L-1
[26]
ICP
-MS
based
magnetic
imm
unoassay C
EA
0.2 - 50 ng m
L−
1 0.05 ng m
L-1
[27]
Chip-based
magnetic
imm
unoassay-
ET
V-IC
P-M
S
CE
A
0.2 - 50 ng mL
-1 0.06 ng m
L-1
[28]
Am
perometric m
agnetoimm
unosensor sE
rbB2
0.1 - 32.0 ng mL
-1 0.03 ng m
L-1
[29]
Gold
nanorod-based plasm
onic
sensor C
A 15.3
0.0249 - 0.2387 UI m
L-1
- [30]
Optical m
icroresonators C
A 125
Limit of linearity of 10 U
I mL
-1 ~ 1.8 U
I mL
-1 [31]
Fluorescence spectroscopy
CA
125 Lim
it of linearity of 500 UI m
L-1
0.26 UI m
L-1
[32]
Chapter 4
147
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Capítulo 5
Conclusiones generales
Capítulo 5
155
Los resultados obtenidos en la presente Tesis Doctoral demuestran el
enorme potencial que presenta la Espectrometría de Masas mediante
ionización en Plasma Acoplado Inductivamente como técnica de análisis de
biomoléculas. La selección adecuada del formato de inmunoensayo y la
correcta optimización de las condiciones de trabajo son el punto crítico que
determinará los parámetros analíticos del método desarrollado. Así:
- Es posible determinar haptenos a niveles de ultratraza (sub-ppt)
empleando inmunoensayos competitivos.
- En general, para los haptenos, se obtienen mejores resultados analíticos
cuando el inmunoensayo competitivo emplea como especie trazadora
anticuerpos en lugar del analito conjugado a proteínas.
- El empleo de polímeros de lantánidos, como estrategia de marcaje en
inmunoensayos en fase homogénea, permite llevar a cabo (mediante la
adecuada separación cromatográfica) el análisis multiparamétrico de
biomoléculas mediante ICP-MS con elevada sensibilidad, precisión y
exactitud.
Capítulo 6
Futuros estudios
Capítulo 6
159
La gran mayoría de trabajos referentes a la determinación de biomoléculas
mediante inmunoensayos con detección por medio de ICP-MS, llevan a cabo la
cuantificación de las biomoléculas mediante el registro continuo de la señal que
proporciona el heteroátomo. Cuando se trabaja con nanopartículas, también se
puede utilizar el modo single particle para relacionar la concentración de analito
con la frecuencia de pulsos que provocan las nanopartículas. Los resultados
muestran que los límites de detección entre ambos métodos son similares
cuando se trabajan con inmunoensayos. No obstante, una revisión de la
(escasa) bibliografía en este campo pone de manifiesto que los límites de
detección mediante single particle podrían ser mejorados respecto al modo
convencional mediante la selección adecuada del inmunoensayo, el disolvente
con el que se introducen las nanopartículas en el ICP-MS y el sistema de
introducción de muestras. Otro aspecto a tener en cuenta es que, a pesar de la
capacidad de análisis multicomponente del ICP-MS, en muy contadas
ocasiones se aprovecha dicho potencial para el análisis de haptenos. Otro de
los problemas a resolver en cuanto a la cuantificación es la variabilidad en el
grado de funcionalización del anticuerpo con el heteroátomo. Ello dificulta la
cuantificación del analito de forma exacta y reproducible e impide el empleo de
algunas de las técnicas de calibración que se pueden emplear en ICP-MS (e.g.
dilución isotópica).