ANSWER 3- Procedure of ELISA:

ELISA involves a series of steps designed to detect and quantify specific molecules, typically proteins
or antibodies. The basic procedure of ELISA can be summarized as follows:

1. Coating :

- A microtiter plate is coated with an antigen of interest, which could be a protein, peptide, or
other molecules. This is done by adding the antigen to each well and incubating to allow it to adhere
to the surface.

2. Blocking :

- Uncoated sites on the plate are blocked with a protein such as bovine serum albumin (BSA) or
milk powder to prevent non-specific binding of antibodies in subsequent steps.

3. Primary Antibody Binding :

- The sample containing the target antibody is added to the wells. If the target antibody is present,
it will bind specifically to the coated antigen.

4. Washing :

- The plate is washed to remove any unbound proteins or antibodies, leaving only the specific
antibodies bound to the coated antigen.

5. Secondary Antibody Binding :

- A secondary antibody, conjugated with an enzyme (e.g., horseradish peroxidase or alkaline

phosphatase), is added. This secondary antibody recognizes the primary antibody and binds to it.

6. Washing :

- The plate is washed again to remove unbound secondary antibodies.

7. Enzyme Reaction :

- A substrate solution containing a chromogenic or fluorogenic substrate for the enzyme is added.
If the secondary antibody is bound to the primary antibody (indicating the presence of the target
antibody), an enzymatic reaction occurs, producing a detectable signal.
8. Signal Detection :

- The intensity of the signal is measured using a spectrophotometer or fluorometer. The signal's
intensity is directly proportional to the amount of target antibody present in the sample.

Significance of ELISA:

ELISA has several significant advantages and applications in immunology:

1. High Sensitivity : ELISA can detect antigens or antibodies at very low concentrations, making it a
highly sensitive assay.

2. Quantification : ELISA allows for the quantification of specific molecules, providing researchers
with precise concentration measurements.

3. Specificity : ELISA is highly specific, as it relies on antigen-antibody interactions. This specificity is

crucial in distinguishing between closely related molecules.

4. High Throughput : ELISA can be easily adapted to analyze multiple samples simultaneously,
making it suitable for screening large numbers of samples.

5. Versatility : ELISA can be used to detect a wide range of molecules, including antibodies, antigens,
cytokines, hormones, and more, making it a versatile tool in immunology research.

Types of ELISA:

There are several types of ELISA, each with its specific applications:

1. Direct ELISA :

- In a direct ELISA, a labeled primary antibody is used to directly bind to the antigen. It is a simple
and quick method but is less sensitive than other types.

2. Indirect ELISA :
 - In an indirect ELISA, an unlabeled primary antibody is used to bind to the antigen, followed by a
labeled secondary antibody that recognizes the primary antibody. This method amplifies the signal
and is more sensitive than the direct approach.

3. Sandwich ELISA :

- A sandwich ELISA is used to detect antigens (rather than antibodies) and is particularly useful for
detecting antigens present in low concentrations. Two antibodies are used: one is coated on the
plate, and the other is labeled and used for detection.

4. Competitive ELISA :

- Competitive ELISA is used when there is a need to quantify the amount of antigen or antibody in a
sample. A labeled antigen competes with the sample antigen for binding to a limited amount of
antibody.

Applications of ELISA:

ELISA has a wide range of applications in immunology and other fields:

1. Disease Diagnosis : ELISA is commonly used to diagnose various diseases, including HIV, hepatitis,
and autoimmune disorders.

2. Vaccine Development : It is used to evaluate immune responses to vaccines and determine

antibody titers.

3. Allergen Testing : ELISA can identify specific allergens in patient samples to aid in allergy
diagnosis.

4. Cancer Biomarker Detection : Researchers use ELISA to detect specific cancer biomarkers in blood
or tissue samples.

5. Drug Screening : ELISA is used to screen for the presence of drugs or their metabolites in clinical
samples.

6. Environmental Monitoring : It is employed to detect pollutants or contaminants in environmental

samples.
Performing a Sandwich ELISA:

A sandwich ELISA is a commonly used format for detecting antigens. Here are the steps involved:

1. Coating : Coat the microtiter plate wells with the capture antibody specific to the antigen of
interest.

2. Blocking : Block any uncoated sites to prevent non-specific binding.

3. Sample Incubation : Add the sample containing the antigen to the wells and incubate, allowing
the antigen to bind to the capture antibody.

4. Washing : Wash the plate to remove unbound antigens.

5. Secondary Antibody : Add a labeled secondary antibody that is specific to a different epitope on
the antigen. This secondary antibody binds to the antigen, forming a "sandwich" with the capture
antibody.

6. Washing : Wash the plate again to remove unbound secondary antibodies.

7. Enzyme Reaction : Add the substrate for the enzyme conjugated to the secondary antibody.

8. Signal Detection : Measure the enzymatic reaction's intensity, which is proportional to the
amount of antigen present in the sample.

In conclusion, the Enzyme-Linked Immunosorbent Assay (ELISA) is a vital tool in immunology with
various types and applications. It plays a crucial role in disease diagnosis, vaccine development, and
biomarker detection. The sandwich ELISA, in particular, is a powerful technique for detecting
antigens with high specificity and sensitivity, making it a cornerstone in immunological research and
clinical diagnostics. Understanding the procedure and significance of ELISA is fundamental for
researchers and healthcare professionals in the field of immunology.