Immunosensor: Antibodies based affinity biosensors · Luminex technology: Consist of combining...

Immunosensor: Antibodies based affinity biosensors

2015-02-19

Valerio.beni@liu.se

Outline

� Nomenclature

� What is an Antibody

� Monoclonal and Polyclonal antibodies

� Enzyme-Linked ImmunoSorbent Assay ELONA

� Assay formats

� Some examples

� Further examples

� Mass based sensing assay

� Lateral flow assay

Of what we will talk? Nomenclature

Immunosensors: Use antibody as biorecognition element.

Affinity biosensors: Biosensors in which bio-recognition molecules bind analyte molecules leading to formation or dissociation of complex.

Possible receptor molecules: i) antibodies ii) DNA iii) receptors

Design parameters for affinity biosensors

Assay format strongly influenced by:• Nature of analyte• Transduction approach

What are Antibodies?

Antibodies are proteins produced by B cells and that are used by the immune system to identify foreign substances (e.g. bacteria, virus).

Antibodies are specifically binding to an unique part (epitope) of their specific target molecule (antigen).

Antibodies contain a region with high variability, the Fab region, that is involved in antigen recognition and a constant region (fiveisotypes: IgG, IgA, IgD, IgM, IgE are known) the Fc region, that modulate the cellular function of the antibodies.

Synthetic antibodies can exist in the monoclonal or polyclonal form; these differ in their ability of interacting with the antigen.

Basic Structure

1. Variable region (Fab)

2. Constant region (Fc)

3. Heavy chain: contain a

variable domain (VH) and a

constant domain (CH1), a

connection chain, and two more

constant domains (CH2 y CH3).

4. Light chain: with a variable

domain (VL) and a constant

domain (CL)

5. Recognition region

6. Connection region.

Cabohydrate

chain

Monoclonal Antibodies (Mab)

Antibodies produced by theprogeny (cell derived from) of asingle parent B cell.

The produced antibodies will allhave the same specificity(recognise a single epitope ofthe antigen).

Polyclonal Antibodies (Pam)

antigen

When different B-cells produceantibodies against the sameantigen (antigen inoculated inan animal), the producedantibodies will have slightlydifferent specificity and will beagainst different epitopes ofthe antigen.

Polyclonal antibodies (mixtureof monoclonal antibodies).

Antibodies production

Polyclonal Monoclonal

Enzyme-Linked ImmunoSorbent Assay (ELISA)

1. Immobilisation of capture antibodies 2. Addition of samples 3. Addition of detection antibodies (with an

enzyme label) 4. Addition of enzyme substrate for colour

development 5. Measure by optical transducers

How to read?

Multiple-wells spectrophotometer

ELISA: Direct assay

substrate

product

Concentracion

•Used to detect the presence of antigens

in a sample.

•Robust assay

•It can be performed using either MAbs

or Pabs

•Not suitable for small molecules

Primary antibody (biorecognition

element)

Secondary labeled antibody (reporter);

usually Pam against the antigen).

Analyte (antigen)

ELISA: Indirect assay

substrate

product

Concentracion

Primary antibody (target/recognition

element)

•Used for the detection of

antigenes/antibodies in serum

•Robust assay: all reagent are in excess

Secondary antibody (reporter); usually Pam

against the primary antibody.

Antigen (recognition element/target)

Procedures for direct affinity assay

Martin Mak

Interpretation of direct affinity assay

Martin Mak

ELISA: Competitive assay

Concentration

•Sample and marked antigenes are

incubated with the antibodies at the

same time

•Suitable for small molecules

•Low sensitive assay

•Require a fine optimisation of the

concentration of the reagents to be

used

Procedures for competitive affinity assay

Martin Mak

ELISA: Displacement assay

Concentration

•Single step assay

•Low sensitivity high speed

•Requires fine tuning of the reagents concentration to be used.

Interpretation of competitive affinity assay

Martin Mak

Sensitivity in affinity biosensors

Imagine each candle representing an antibody and you are an optical transducer, you can easily detect if one candle light up (direct affinity assay)

You cannot easily detect if one candle is being off (competitive affinity assay)

Beyond ELISA:Other possible assay formats

Membrane-based lateral flow affinity devices

Lateral flow format (1980’S till Today)

• Use capillary forces to allow movement of biomolecules across different

zones: conjugation zone, test zone and control zone.

• Colloidal gold nanoparticles are used as support for bio-recognition molecules

and as labels (give color)

• “One-step” test concept (Whole blood assay)

• Smart use of biochemical interaction for performing different functions.

Membrane-based lateral flow affinity devices: how do they work?

AuNPs work as support for bio-recognition molecule immobilisation and as label (give colour).

The design of the bio-label is crucial for detection to happen.

How can I get my information?

Clearblue Pregnancy tests.Detect the hormone hCG (Human chorionic gonadotropin)

using a combination of different antibodies. Semi-Qualitative evaluation:

Reflectance: The ratio of the total amount of radiation reflected by a surface to the total amount of incident radiation.

Imaging: Combination of digital camera and advance images processing tools.

www.clearblue.com/

Luminex technology: Consist of combining beads-based assay, flow-cytometry and fluorescence detection to perform fast

• The wells used of ELISA assay are replaced by beads• High throughput analysis (up to 100 targets at the time) can be performed in

homogeneous format.• Require lower sample volume.• It is fast and allow significant statistical analysis.

Luminex assay

Polymeric beads containing 2 different fluorophores are used as support.

Direct Assay

Use flow-cytometry for fat high throughput analysis.

Detection principle in Luminex

Flow-cytometry system

Lasers for beads identification

The ratio of the responses from the 2 fluorophores in the beads is used to identify the beads and a result the assay.

Detector for reporter

The fluorescence from the reporters is used to quantify the presence of the different antigens.

Detectors for beads

Electrochemical:

Acustic (QCM):

SPR:

Magnetic:

Other possible transduction approaches

Antibodies immobilisation

Optimal orientation of the antibodies results in:

• Greater affinity for the antigen

• Increased sensitivity of the immunosensor

• High reproducibility in the immobilisation

Ammino linking of antibodies

Early methods of immobilisation using the NH2 and COOH groups of

the antibody for covalent attachment. These groups, being distributed throughout

the molecule surfaces with antibodies generated randomly oriented and low

affinity.

This phenomenon is particularly critical if NH2 groups are used for the

immobilisation because they are preferentially located in the recognition site

NH2

NH2

COOH

NH2

CONH

Clever design of immobilisation

An alternative is to modify the carbohydrate groups of the antibody by oxidation

withperiodate and subsequent introduction of amino groups to achieve a more

effective orientation.

NH2

O O

NH2

1) NaIO42) RNH2/NaBH4

NH2

COOH CONH

Fab immobilisation

+enzymatic

digestion

antibody

(Fab)2 fragment

Fc fragments

(discarded)reducing

agent

Fab fragments-SH

-SH

Preparation of Fab fragment

Advantages

Higher sensitivity; due to the immobilisation of higher number

and better oriented recognition molecules.

Reduced background response: lower unspecific response

due to the absence of the Fc fragment (recognised by several

receptors).

Summary

• We saw how antibodies can be used in biosensing.

• We saw 3 different commercial formats of immunoassays: ELISA, lateral flow strip test and Luminex.

• We saw how different formats or ELISA can be used to detect different targets and how results can be interpreted.

• We saw how optimal orientation of antibodies on surfaces can be achieved.