IMMUNOASSAY - ELISA

PHUBETH YA-UMPHAN National Institute of Health, Department of Medical Sciences 0bjective

After this presentation, participants will be able to

Explain how an ELISA test determines if a person has certain antigens or antibodies .

Explain the process of conducting an ELISA test.

Explain interactions that take place at the molecular level (inside the microtiter well) during an ELISA test.

Outline

- Principal of immunoassay

- Classification of immunoassay

Type of ELISA - ELISA ELISA Reagents

General - Applications Principal of ELISA ELISA workflow What is immunoassay?

Immunoassays are bioanalytical methods that use the specificity of an antigen-antibody reaction to detect and quantify target molecules in biological samples.

Specific antigen-antibody recognition Principal of immunoassay

• Immunoassays rely on the inherent ability of an antibody to bind to the specific structure of a molecule.

• In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding.

Classification of Immunoassays

Immunoassays can be classified in various ways.

Unlabeled Labeled

Competitive Homogeneous

Noncompetitive

Competitive Heterogeneous

Noncompetitive

https://www.sciencedirect.com/science/article/pii/S0075753508705618 Classification of Immunoassays

• Unlabeled - Immunoprecipitation

• Labeled Precipitation of large cross-linked Ag-Ab complexes can be visible to the naked eyes. - Fluoroimmnoassay (FIA) - Radioimmunoassay (RIA) - Enzyme Immunoassays (EIA) - Chemiluminescenceimmunoassay(CLIA) - Colloidal Gold Immunochromatographic Assay (ICA)

https://www.creative-diagnostics.com/Immunoassay.htm Classification of Immunoassays

• Homogeneous immunoassays Immunoassays that do not require separation of the bound Ag-Ab complex. (Does not require wash steps to separate reactants.)

Example: Home pregnancy test.

• Heterogeneous immunoassays Immunoassays that require separation of the bound Ag-Ab complex. (Incorporates wash steps to separate reactants.) Classification of Immunoassays

• Competitive (limited reagent assay) Patient serum and conjugated enzymes are added to the microwell, often at the same time, and compete for binding sites.

• Noncompetitive (excess reagent assay, sandwich assay) Only specific antibodies in the patient sample bind directly with the target antigen coated on the solid phase microwell. Enzyme ELISA is a plate based assay technique which is used for detecting and Linked quantifying substances such as peptides, Immuno proteins, antibodies and hormones. Sorbent

A ssays

An enzyme conjugated with an antibody reacts with colorless substrate to generate a colored product. Principal of ELISA

When a suitable substrate is added, the enzyme reacts with it to produce a color. The intensity of the color gives an indication of the amount of Ag or Ab.

A secondary antibodies that are attached to an enzyme are added to each well.

Serum is incubated in well. Ab (or Ag) present in serum are captured by corresponding Ag (or Ab) coated on to the solid surface.

Ag (or Ab) coated on to the solid surface.

https://www.nordicbiosite.com/blog/elisa-principles-101 Type of ELISA

Depending on the antigen-antibody combination, the assay is called a direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA etc.

https://www.bosterbio.com/newsletter-archive/20170728-which-elisa Direct ELISA Type of ELISA

In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then added, producing a signal that is proportional to the amount of analyte in the sample.

In the figure : Well coated with : Antigen (in the sample)

https://www.rndsystems.com/resources/what-is-an-elisa-and-elisa-types#:~:text=The%20four%20main%20types%20of,its%20own%20advantages%20and%20disadvantages. Indirect ELISA Type of ELISA

First, an unconjugated primary detection antibody is added and binds to the specific antigen. A conjugated secondary antibody directed against the host species of the primary antibody is then added. Substrate then produces a signal proportional to the amount of antigen bound in the well.

In the figure : Well coated with : Antigen Detects in serum : Antibody

https://www.rndsystems.com/resources/what-is-an-elisa-and-elisa-types#:~:text=The%20four%20main%20types%20of,its%20own%20advantages%20and%20disadvantages. Sandwich ELISA Type of ELISA

Two specific antibodies are used to sandwich the antigen (matched antibody pairs). Capture antibody is coated on a microplate, sample is added, and the protein of interest binds and is immobilized on the plate. A conjugated-detection antibody is then added and binds to an additional epitope on the target protein. Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample. Sandwich ELISAs are highly specific, since two antibodies are required to bind to the protein of interest.

In the figure : Well coated with : Antibody Detects in serum : Antigen

https://www.rndsystems.com/resources/what-is-an-elisa-and-elisa-types#:~:text=The%20four%20main%20types%20of,its%20own%20advantages%20and%20disadvantages. Competitive ELISA Type of ELISA

A reference antigen is bound to the bottom of microplate wells. Sample plus specific enzyme conjugated antibody (primary Ab conjugate) to the antigen are added to the wells. If there is an antigen in the sample (inhibitor antigen in the picture), it competes with the antigen bound to the well for binding the specific antibody. Unbound material is washed away. The more antigen is in the sample, the less specific antibody ends up bound to the coated antigen, and the lower the final signal.

In the figure : Well coated with : Antigen Ag in the Detects in serum : Antigen sample

https://www.moleculardevices.com/applications/enzyme-linked-immunosorbent-assay-elisa#gref Comparison of the 4 types of ELISA

STEPS DIRECT INDIRECT SANDWICH COMPETITIVE Capture Ab Coating O O P O Antigen Coating P P O P Blocking P P P P Sample (Ag) Incubation O O P P Primary Ab Incubation P P P P Secondary Ab Incubation O P P P Substrate Prep P P P P Signal Detection P P P P Data Analysis P P P P General ELISA Workflow

Standard Preparation Lyophilized Recombinant Standard , Sample Diluent Buffer

Sample Dilution Sample Diluent Buffer

Sample (Antigen) Incubation 96-Well Plate Pre-Coated with Capture Antibody

Washing PBS Buffer

Biotinylated Antibody Preparation & Incubation Biotinylated Antibody , Antibody Diluent Buffer

Washing PBS Buffer

ABC Preparation & Incubation Avidin-Biotin-Peroxidase Complex , ABC Diluent Buffer

Washing PBS Buffer

Signal Detection TMB Color Developing Agent , TMB Stop Solution

https://www.bosterbio.com/protocol-and-troubleshooting/elisa-principle# ELISA Reagents

Specimen Biotinylated Antibody - Contain many proteins,Ab,Ag and other substances. - Blood protein that responds to the presence of specific - The specimen must be diluted. antigens that can be used as probes in cell research.

Calibrators Standards and Controls Conjugated Enzyme - Standard curve - Bind to the analyte. - Cut-off point - Reacting with the chromogenic. - Quality control - Horseradish peroxidase (HRP)/Alkaline phosphatase (ALP)

Blocking buffer Chromogenic Substrate - Bind to all potential sites of nonspecific interaction. - Create a color change when it reacts with the conjugated - Eliminating background. enzymes, forming a colored end product. - Bovine serum albumin / non-fat dry milk or casein. - Light can induce reactions in the substrate. - TMB (3,3' 5,5' tetramethylbenzidine)

Wash Buffer Stop Solution - Stabilize the high affinity Ag-Ab reactions. - Stop solution is typically an acid. - Removing excess reagents,unbound proteins, - To denature the enzyme and stop its activity. analyte, or interfering substances. - The effect of changing the substrate color from blue to - Keep the bound proteins from denaturing. yellow, or from yellow to orange (depending on the - Preserving the enzyme's activity. enzyme and substrate used.) ELISA Data Interpretation

Qualitative: ELISA can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.

Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.

Applications Salivary Biomarkers of Human Exposure to Mosquito Vector Bites

The physiological interaction between the human and the vector

(1) The vector injects its salivary proteins in the human skin during the probing.

(2) Once in the skin, the salivary proteins take the control of the human hemostatic system by inhibiting the platelet activation, clotting mechanism, and inflammatory system.

(3) The salivary proteins modulate the human immune response and promote the production of anti-saliva antibodies.

(4) If ever the vector carries a pathogen, the salivary proteins contribute to its transmission into the human. The use of specific biomarkers of human exposure to Anopheles bites to assess the risk of malaria transmission.

Antigen : Salivary gland extracts/ Peptide Antibody : Human blood samples

+ + - + + - + + - + + - High IgG Intensity of human antibody response to responder Anopheles bites.

Low IgG responder

Blank

Positive relationship between the IgG antibody level and the Human Biting Rate level. The use of specific biomarkers of human exposure to Anopheles bites to assess the risk of malaria transmission.

Map of Village Hot spot

Demographic, Social, and Environmental factors Spatial clusters of high IgG responders. associated with Human vector contact.

Selecting the appropriate vector control measures. Thank you