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Specimen(redacted)
Serotype 4 Serotype 6B Serotype 9V Serotype 143 Serotype 19F
Target ELISA Values1
Median Luminex Values2
Target ELISA Values1
Median Luminex Values2
Target ELISA Values1
Median Luminex Values2
Target ELISA Values1
Median Luminex Values2
Target ELISA Values1
Median Luminex Values2
1 4.3-10.1
11.1 2.9-6.9 4.9 0.9-2.1 3 39.7-92.5 26.1 5.8-13.6 9.7
2 5.4-12.6
4.5 1.3-3.1 3.7 1.9-4.5 3.2 193.3-450.9
322.1 6.8-15.8 11.3
3 1.4-3.2 2.3 7.7-18.1
12.9 4.6-10.8
7.7 11-25.8 18.4 1.5-3.5 6.9
4 3.7-8.7 6.2 5.4-13.9
9.9 1.3-2.9 4.8 4.4-10.4 7.4 6.9-16.1 11.5
5 1.1-2.5 1.8 14-32.6 23.3 6-14 10 2.2-5.2 3.7 5.7-13.3 9.5
6 1.4-3.2 2.3 0.6-1.4 4.3 2.5-5.9 4.2 6.3-14.7 10.5 9.9-23.1 16.5
7 5.8-13.6
9.7 6.1-14.3
1.02 10.2-23.8
17 16.7-38.9 51 38.4-89.7 35.3
8 8.8-20.4
14.6 2-4.6 3.3 9.5-22.1
15.8 96.5-225.1
160.8 8.5-19.7 14.1
9 1.4-3.2 2.3 1.3-2.9 2.1 1.1-2.52
1.8 11.5-26.9 19.2 4.1-9.5 6.8
10 2.4-5.6 4 11.3-35.1
23.2 5-11.6 8.3 10.3-24.1 17.2 13-30.4 21.7
11 0.4-1.0 0.7 1.5-3.5 2.5 2.8-6.4 4.6 8.4-19.6 14 2.1-4.9 3.5
12 1.2-2.8 2 5-11.6 8.3 2.5-5.9 4.2 69.4-161.8
115.6 4.4-10.2 7.3
A 5 Serotype Comparison between the Luminex and ELISA Methods for Estimating Pneumococcal IgG Antibody
ConcentrationD.L. Klein;1 M. H. Hickey;1 M. Steinhoff;2
1Flow Applications, Inc., Atlanta, GA; 2Johns Hopkins School of Public Health, Baltimore, MD
ABSTRACT• TITLE: Correlation between Luminex and ELISA methods for
estimating pneumococcal IgG antibody concentration
• BACKGROUND: The standard measurement of pneumococcal (Pn) antibody is by ELISA. The assay has several limitations that result in substantial laboratory efforts at a relatively high cost. We compared the standard ELISA with a new multiplex anti-Pn IgG Luminex-based assay that relies on the conjugation of Pn polysaccharide to an array of color-coded microspheres and a flow cytometric detection system. In situations where only small quantities of blood or large numbers of samples are involved, and immunoassay that measures multiple IgG specificities to multiple antigens simultaneously from a single sample is highly advantageous.
• METHODS: Using pre- and post-vaccination sera collected from 100 pregnant Bangladesh subjects immunized during their third trimester with a 23-valent Pn polysaccharide vaccine, we compared the ELISA data with the Luminex-based multiplex assay using a WHO calibrated Pn reference serum. All sera were assessed for anti-Pn IgG specific for Pn serotypes 4, 6B, 9V, 14, and 19F. We have evaluated the statistical correlation between the ELISA and multiplex data for each serotype.
• PRELIMINARY DATA: 11 serum specimens were evaluated in both assays, and the titers compared with a Mann Whitney test and regression analysis. For the serotypes 4, 9V, 14, and 19F, the median titers were statistically similar (p=0.7 to 0.5), and the correlation coefficients ranged from 0.84 to 0.99, with >90% of the values meeting the criterion of +/- 40% of the consensus value. The multiplex assay was also very reproducible and robust with coefficients of variation under 10% and in some cases under 2%.
• DISCUSSION: These data suggest that the Luminex-based multiplex assay has a high degree of correlation with the standard traditional ELISA. The use of the multiplex assay should reduce the cost and time to obtain Pn antibody data and help promote the licensure of future Pn vaccines and other encapsulated bacterial pathogens.
BACKGROUND
• It has been proposed by the World Health Organization (WHO) that vaccination of the mother and transfer of maternal antibody across the placenta to the newborn infant should provide protection against pneumococcal (Pn) infection. This approach is the current strategy for prevention of neonatal and newborn tetanus in developing countries. Based on previous studies, use of the 23-valent polysaccharide (Ps) vaccine during pregnancy is safe and results in maternal transfer of high levels of specific IgG antibody to the newborn and protects them from Streptococcus pneumoniae in early infancy. Thus, a strong case can be made for the use of maternal vaccination as a mechanism for preventing serious illness or death in both young infants and pregnant women.
• To control high morbidity and mortality rates in newborns due to Pn disease, 340 healthy pregnant women in Bangladesh were vaccinated with a 23-valent Pn vaccine during their third trimester as part of a randomized, double-blinded trial. The effect of maternal vaccination on anti-Pn IgG to capsular Ps was measured by both the traditional enzyme-linked immunosorbent assay (ELISA) and a modified multiplexed anti-Pn IgG Luminex-based quantitation assay (LEXA) that relies on novel chemistry for conjugating PnPs to an array of color-coded microspheres and a flow cytometric detection system. The latter assay was developed by Flow Applications, Inc. to avoid many of the limitations associated with the traditional antibody measurement of ELISA. A set of 12 WHO reference sera and clinical samples from the above trial was used to demonstrate accuracy and precision of the LEXA.
Quantitative IgG Luminex Assay
A human, bead-based, multiplex Luminex quantitative assay or LEXA was optimized and validated for the measurement of antibodies to PnPs in large numbers of patient sera associated with vaccine clinical trials and epidemiology studies. The assay relies on a rapid particle-based flow cytometric system that allows a single sample to be tested simultaneously for a number of different biomolecules/analytes. The Luminex 100 platform was used to acquire data from a multiplexed assay using a capture bead system developed by the Luminex Corporation (Fig. 1). Purified capsular PnPs were obtained from the ATCC. The labeled beads serve as a solid support for the antigen and permit the simultaneous analysis of antibodies with specificities for up to 100 different antigens in a single reaction and a high through-put screening of up to 1000 sera per day. The bead-serum reaction mixture was then read in a dual laser (two-color analysis) flow cytometer. Prior to use in the assay, sera were adsorbed with cell wall Ps and type 22 Ps to improve specificity by removing antibody to cell wall Ps, cross-reacting epitopes, and contaminating cell wall protein. This procedure was compared to the WHO standard ELISA to establish correlation values for each of 5 different Pn serotypes.
DESIGN•Population: A prospective, randomized, double-blinded, control trial was evaluated in 4 groups of mothers and their infants (Fig. 2). During the trial, a 23-valent PnPs vaccine or a licensed inactivated influenza vaccine was administered to 340 healthy women in Bangladesh 18 to 40 years of age who were in their third trimester of gestation. Mothers were recruited in private practices after signing consent forms.
•Primary outcome measures: 1) evaluate immunogenicity and safety characteristics of a conjugate Pn vaccine administered to infants of mothers who had been immunized with PnPs vaccine during their third trimester of pregnancy; 2) evaluate the effect of high levels of maternal antibody on the infant’s response to 3 doses of the Pn conjugate vaccine given in the routine infant schedule; 3) continue surveillance of infants from 6 months to 15 months of age and assess for illness and nasopharyngeal carriage; 4) determine duration of mothers’ Pn antibody response; and 5) determine whether immune infants following maternal immunization demonstrate an enhanced antibody response to exposure to PnPs at 12 months of age.
•Design Summary: Blood specimens were collected in the mothers before and after immunization and at the time of birth. For infants, cord blood, along with venous and nasal swab specimens, was collected at various times followed by a single dose of PnPs vaccine at 12 months and a subsequent dose of either Hib conjugate or Pn conjugate vaccine (Table 1). With regard to intervention, mothers received a single immunization during their third trimester of pregnancy while infants, born to these mothers, received the normal 3 doses of licensed Pn conjugate or Haemophilus influenzae type b (Hib) conjugate vaccine as well as all the other routine vaccines administered in childhood.
Discussion
• The LEXA has a greater dynamic range than the ELISA and, thus, significantly reduces the need for multiple dilutions of each serum sample in addition to increasing the accuracy and reproducibility of the assay.
• The LEXA showed a positive correlation with a conventional ELISA procedure for serotypes 4, 6B, 9V, 14, and 19F in mothers’ sera. In addition, both the LEXA and ELISA demonstrated strong post-vaccine responses in mothers’ sera that were in general agreement.
• On a routine basis, the incubation times and total assay times of the LEXA were much less than ELISA.
• The simultaneous quantitation of different antigens in a single sample from vaccinated or naturally infected subjects using the LEXA means that greatly reduced sample sizes are needed as well as greatly reduced amounts of time, labor, and reagent costs when measuring immunological activity of sera.
• Results from the LEXA suggest that it has a greater sensitivity and specificity than that of the standard ELISA.
• The LEXA provides the ability to measure a broad range of antibody titers within diverse clinical populations.
• Due to its increased sensitivity and dynamic range, the LEXA was able to more effectively measure anti-Pn IgG in adults compared to ELISA. This suggests that the LEXA, may be able to determine the protective role of a specific antibody even in adults, who generally have been exposed to massive amounts of different antigens over a lifetime that cross-react with the antigen in question.
Conclusion
Flow Application’s modified use of the Luminex platform provides a comprehensive assay system that can be applied to virtually any application that requires analysis of anti-Pn antibody, basic research, clinical diagnostic testing, environmental testing, and agricultural testing. The Luminex bead technology, as adapted by Flow Application, is unique in its ability to provide multiplexed, high-throughput analysis coupled with real-time data analysis. The assay system offers excellent sensitivity, precision, speed, and economy for the measurement of Pn antibody.
RESULTSVaccination of pregnant women during their third
trimester was safe and immunogenic.
The LEXA was compared to the standard ELISA for quantitation of IgG antibodies to each of the 5 Pn serotypes using sera collected from mothers pre and post vaccination.
Antibody concentrations for each of the tested sera were subjected to linear regression analysis with the results shown in Figure 3.
The correlation coefficients (r-value) showed a range of 0.53 to 0.96 for mothers’ sera run against the 5 Pn serotypes.
ELISA values never demonstrated 0 antibody levels due to background noise.
The lower limit of detection for the LEXA has not been established at this time. This will be described in later work. As a result, technical limitations exist for the assay on the lower end.
A quality WHO panel of 12 Goldblatt Pn sera was used to evaluate the LEXA for 5 Pn serotpes (Table 2). Correlation coefficient values were considered good for each of the serotypes tested and suggest the two assays (ELISA vs. LEXA) result in very similar IgG concentrations.
Total IgG antibodies to pneumococcal serotypes 4, 6B, 9V, 14, 19F were successfully measured in mothers in both the standard ELISA and LEXA before and 4 weeks after vaccination with the 23-valent PS vaccine (Table 3).
Although the vaccine response varied considerably among individuals to each of the 5 serotypes, vaccination with PnPs during pregnancy resulted in higher post-vaccination concentrations of pneumococcal IgG antibodies for all 5 vaccine serotypes in both assays, demonstrating the sensitivity of the assay (Table 3).
Fold-increase responses among the LEXA post-vaccination values compared to pre-values were consistently higher than that observed for ELISA (Table 3).
Correlation values are presented for the WHO reference sera and the clinical trial samples (Table 3). The 5 Pn serotypes, when tested with the standardized WHO sera set, yielded results that achieved 83% compliance the FDA?WHO criteria.
Measurements for limits of detection, sensitivity, and robustness are being evaluated for LEXA. Linearity and specificity have been established through previous published work.
Mean ug of IgG/ml
Serotype Pre-vaccination Post-vaccination Fold Increase –Pre vs. Post
ELISA LEXA ELISA LEXA ELISA LEXA
4 6B 9V 14 19F
0.807 3.151 1.459 4.406 3.052
0.231 1.531 0.131 8.746 2.615
1.928 3.342
2.038 48.414
5.452
1.112 3.950 1.438
103.338 8.988
2.39 1.06
1.40 10.99 1.79
4.81 2.58
10.98 11.82 3.44
Blood samples were obtained 4 weeks post-vaccination with 23 valent PnPs vaccine
Contact information:David L. Klein [email protected]
AcknowledgementsSpecial thanks to the Respiratory Disease Branch, CDC
Pneumococcal IgG Antibody ConcentrationLuminex Comparison to ELISA
Expected Concentration
0 2 4 6 8 10 12 14 16
Lum
inex
Con
cent
ratio
n
0
10
20
30
40
Serotype 4 Values
Plot 1 Regr
N = 8 runsr = 0.99
Expected Concentrations
0 5 10 15 20 25
Lum
inex
Con
cent
ratio
ns
0
20
40
60
80
100
120
Serotype 6B
Plot 1 Regr
n = 5 runsr = 0.77
Expected Concentrations
0 20 40 60 80 100 120 140 160 180
Lum
inex
Con
cent
ratio
ns
0
50
100
150
200
Serotype 14
Plot 1 Regr
n = 8 runsr = 0.95
Expected Data
0 2 4 6 8 10 12 14 16 18 20
Lum
inex
Con
cent
ratio
ns
0
5
10
15
20
25
30
Serotype 18C
Plot 1 Regr
n = 5 runsr = 0.61
Expected Data
0 10 20 30 40 50 60 70
Lum
inex
Con
cent
ratio
ns
0
20
40
60
80
100
120
Serotype 19F
Plot 1 Regr
n = 5 runsr = 0.99
Expected Concentrations
0 2 4 6 8 10 12 14 16 18 20
Lum
inex
Con
cent
ratio
ns
0
20
40
60
80
Serotype 23F
Plot 1 Regr
n = 8 runsr = 0.76
METHODS
Gestation Delivery/Birth
After Birth (mother and infant)
Groups 32 Wks 36 Wks 0 weeks 6 weeks 10 weeks 14 weeks 18 weeks
22 weeks
1 M M M, B, NP
B, BM, NP
B, BM, NP
B, BM, NP
BM, NP
BM, NP
2 M M M, B, NP
B, BM, NP
B, BM, NP
B, BM, NP
BM, NP
BM, NP
3 M M M, B, NP
B, BM, NP
B, BM, NP
B, BM, NP
BM, NP
BM, MP
4 M M M, B, NP
B, BM, NP
B, BM, NP
B, BM, NP
BM, NP
BM, NP
M – Maternal Blood SpecimensB – Infant Blood SpecimensBM – Breast MilkNP – Nasopharyngeal Cultures for S. pneumoniae Isolation and Serotyping
Figure 1. Luminex assay
Figure 2.
Table 2
Table 3.
Figure 3.
Table 1. Vaccination and Specimen Collection Schedule by Study Group
