Enzyme immunoassay-Definition, principle ,types,uses

what is Immunoassay?

Immunoassay is a biochemical or bioanalytical technique that utilizes the specific binding interactions between antigens and antibodies to detect and quantify the presence of target analytes in complex samples. It is a powerful tool used in various scientific, medical, and diagnostic applications

Types of Immunoassays On the basis of the nature of labeling compound, there are different types of Immunoassays:

Radioimmunoassay ,fluoroscent immunoassay ,enzyme immunoassay.

What is Enzyme Immunoassay (EIA)?

Enzyme immunoassays also known as ELISA utilize enzymes as labels. The enzyme-labeled antigen or antibody can catalyze a reaction with a substrate to produce a detectable signal, such as a color change or the generation of a fluorescent or chemiluminescent product. Commonly used enzyme labels include horseradish peroxidase (HRP)

Enzyme immunoassay (EIA) is a highly sensitive and specific analytical technique used to detect and quantify the presence of an antigen or antibody in a sample.

How does EIA work?

Basic ELISA test works as follows :

  1. Coating the surface

The first step in an ELISA test is to coat a solid surface, such as a plastic plate, with antigens or antibodies. The antigens or antibodies that are used will depend on the analyte that is being detected.

  1. Adding the sample

The next step is to add the sample to the plate. If the analyte is present in the sample, it will bind to the antigens or antibodies that are coated on the surface.

  1. Adding the detecting antibodies

A second set of antibodies, called the detecting antibodies, are then added to the plate. These antibodies are specific to the analyte that is being detected. If the analyte is present in the sample, the detecting antibodies will bind to it.

4.Adding the enzyme

The detecting antibodies are often conjugated to enzymes. Enzymes are proteins that can catalyze chemical reactions. When the enzyme is added to the plate, it will catalyze a reaction that produces a measurable signal, such as a color change

5. Measuring the signal

The intensity of the color change is proportional to the amount of analyte in the sample. This allows the concentration of the analyte to be determined.The more bindings are formed, the more color is released. The more color, the more a specific substance is present in the sample.

Types of ELISA

There are two main types of EIAs: direct and indirect. In a direct EIA, the first antibody is already conjugated to an enzyme. In an indirect EIA, the first antibody is not conjugated to an enzyme, and a second antibody that is conjugated to an enzyme is used.

  1. Direct ELISA: In a direct ELISA, the antigen of interest is directly immobilized onto the solid surface (e.g., a microplate) and then detected using a labeled antibody that directly binds to the antigen. This method is relatively simple and straightforward but may have limitations in terms of sensitivity and specificity.
  2. Indirect ELISA: Indirect ELISA is a more commonly used variation. In this method, the antigen is immobilized onto the solid surface, and then a primary antibody specific to the antigen is added. After washing to remove unbound primary antibodies, a secondary antibody labeled with an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) is introduced, which binds to the primary antibody. The enzyme-labeled secondary antibody allows the detection and quantification of the antigen through an enzymatic reaction with a substrate.
  3. Sandwich ELISA: Sandwich ELISA is designed to detect and quantify antigens that have two or more specific binding sites. The sample is first coated with a capture antibody that binds to the target antigen. Then, a detection antibody, labeled with an enzyme, is added, which binds to a different epitope on the antigen. The antigen is “sandwiched” between the capture and detection antibodies. This method is highly specific and sensitive and is commonly used for detecting proteins or large molecules.

4.Competitive ELISA: Competitive ELISA is used when the antigen in the sample competes with a labeled antigen for binding to a limited amount of specific antibodies. The amount of labeled antigen bound to the antibodies is inversely proportional to the concentration of the antigen in the sample. This variation is useful for measuring small molecules, such as hormones or drugs..

Uses of ELISA

ELISA s are used to detect a wide variety of antigens and antibodies, including those involved in infectious diseases, autoimmune diseases, and allergies. Some of the most common uses of ELISAs include:

Infectious disease testing: ELISA plays a crucial role in the detection of infectious agents, such as viruses, bacteria, and parasites. It is utilized in screening blood donations for infectious diseases like HIV, hepatitis, and Lyme disease. ELISA can also diagnose specific infections, including HIV, hepatitis B and C, dengue, malaria, and sexually transmitted infections (STIs).Detecting HIV

Pregnancy testing: ELISA-based pregnancy tests detect the presence of human chorionic gonadotropin (hCG), a hormone produced during pregnancy. These tests provide a simple and rapid way to determine pregnancy status.

Allergy testing : ELISA is used for for determining Allergen in allergic conditions

Hormone Analysis : ElLISA is used for hormone analysis like Thyroid profile, growth hormone,insulin

Autoimmune disease : ELISA is used for ANA testing, DsDNA antibodies, and other autoantibodies identification as well as quantification

Advantages of EIAs

EIAs are highly sensitive and specific, and they can be used to detect very low levels of antigens or antibodies. They are also relatively easy to perform, and they can be automated.

Disadvantages of EIAs

EIAs can be time-consuming, and they can be expensive to set up. They can also be susceptible to interference from other substances in the sample. like biotin, heterophile antibodies

Here are some additional resources that you may find helpful:

I hope this blog post was helpful!

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