ELISA Antibodies: Overview & Applications

ELISA Antibodies: Overview & Applications

ELISA: Overview

Enzyme-linked immunosorbent assay (ELISA) antibodies are crucial elements of the ELISA assay that are used to study proteins. Before proceeding to understand the antibody roles, it’s essential to learn more about the ELISA test.

ELISA is one of the most frequently used assays in labs for the detection and quantitative analysis of soluble substances, such as peptides, antibodies, and hormones. It’s also known as enzyme immunoassay (EIA).

The ELISA assay works on the principle of specific antibody and antigen interaction. It consists of the immobilization of antigen on a solid surface and then complexing it with an antibody attached to a reporter enzyme. Then, using different detection methods, the reporter enzyme activity is measured via incubation with the appropriate substrate.

Though, different types or formats of ELISA assay have been developed by today, which include:

  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA

But, they all follow the same basic steps, consisting of:

  • Adding Capture antibody: The adsorption of the capture antibody (binds the ELISA plate) and a sample containing specific antigen is added to the well plate.
  • Wash Step: Washing microplate wells to remove unbound materials, leaving only antigens of interest.
  • Add Detection Antibody: Binding of the enzyme-conjugated detection antibodies to the different epitope of the antigen of interest.
  • Wash Step: Washing microplates again to remove unbound antibodies.
  • Detection: Involves visualization of the complex using suitable detection methods, such as chemiluminescent, chromogenic, colorimetric, and fluorescent assay. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are the two most commonly used substrates that bind the enzymes to produce a signal.  
  • Read plate: Microplate readers detect colored reaction products and calculate optical density (OD) values, which tell the amount of antigen present in each sample.

The analysis of ELISA reactions is mainly possible because of the power interaction between antigens and antibodies. And, this article expands more on ELISA antibodies, their functions, roles in the ELISA method, and applications in industries.

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ELISA Antibodies: What Are They and Their Types?

ELISA antibodies are one of the main heroes of this method. The antibody pairs with the specific antigen and facilitates the study of specific proteins of molecules in different samples.

By definition, antibodies are proteins that provide organisms immunity against foreign invaders, such as bacteria and viruses (known as antigens). The antibodies are also known as immunoglobulin (Ig).

There are two light chains and two heavy chains in each of the four polypeptides of antibodies. In ELISA, antibodies are classified based on whether they bind directly to antigens or another antibody:

  • Primary Antibody: Antibodies specifically bind to the target antigen
  • Secondary Antibody: Labeled secondary antibodies bind to primary antibodies

These antibodies are also known as matched antibody pairs. To reduce the background noise during the ELISA reaction and produce effective results, it’s recommended to use the dilutions of antibodies.

Today, many companies offer ready-to-use ELISA kits with suitable reagents for the detection of a variety of molecules, such as chemokines, cytokines, and growth factors.

Primary Antibodies

A primary antibody binds directly to the specific antigen, which is immobilized on the surface of the well plate.

To produce primary antibodies, one host species is required, such as a rabbit, mouse, goat, or chicken. In the first step of production, the host will be immunized against an antigen, producing a primary antibody.

Secondary Antibodies

A secondary antibody binds to an antibody-target protein complex. It’s usually labeled and helps in the detection of target antigens. Specifically, this type of assay is crucial for analyzing the number of specific proteins present in small amounts by signal amplification.

A secondary antibody is produced by injecting a primary antibody into another species of host. For example, if the primary antibody is generated in a mouse, the secondary antibodies can be generated in a goat, a rabbit, or a chicken.

Figure: Representation of primary and secondary antibodies.

Both the primary and secondary antibodies can either be monoclonal or polyclonal antibodies.

Monoclonal Antibodies

Monoclonal antibodies are a homogenous population of antibodies that are produced from identical B-cell clones, generated in a single parent cell. It interacts with only a single specific epitope of a specific antigen.

Figure: Generation of monoclonal antibodies.

Polyclonal Antibodies

Polyclonal antibodies are a heterogenous population of antibodies that are produced in different clones of B-cells in the body. They can identify and bind to a variety of epitopes on a single antigen.

Figure: Generation of polyclonal antibodies.

How Do Antibodies Work with Immunoassays?

Immunoassays are powerful techniques used in life sciences labs to study proteins, measure their amount in a given sample, and analyze their functions and localization in organisms. They work on the principle of specific antigen-antibody interaction.

Two immunoassays that detect the antigen-antibody interaction include:

  • Direct immunoassays: In this assay, the enzyme-conjugated primary antibody binds to the target antigen. This antigen-antibody interaction activates a signal molecule for detection.
  • Indirect immunoassays: The primary antibody binds to the target antigen. Then, an enzyme-conjugated secondary antibody binds to the primary antibody for detection of the interaction. Often, the reaction also uses the biotin-streptavidin complex for signal amplification.

What are ELISA Antibodies Used For?

ELISA antibodies have extensive usage in Immunology and Life Sciences labs. Some of the applications are listed below:

  • The primary antibody can be used to detect disease biomarkers of many diseases, such as diabetes, cancer, Alzheimer’s, and Parkinson’s disease. However, to confirm the diagnosis and identify false positives western blotting is required to be performed by researchers.

Additionally, the primary antibodies also have applications in the distribution, metabolism, absorption, and excretion of a variety of therapeutic agents.  

  • Secondary antibodies have profound uses in a range of workflows, including ELISA, Western Blot, immunohistochemistry, immunostaining, and immunocytochemistry.

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ELISA antibodies are proteins involved in the immunoassays to facilitate the studies of proteins, peptides, and hormones in labs. They are two types: primary antibody, which directly binds to the antigen of interest; and secondary antibody, which is conjugated to an enzyme and binds to the primary antibody.

Both, primary and secondary antibodies, can be either monoclonal (binding to one specific epitope of the target antigen) or polyclonal (having the affinity for the same antigen but different epitope) antibodies.

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