What Are The Principles Of Elisa?

ELISA, or EIA, which stands for Enzyme-linked Immunosorbent Assay, may be a valuable test for detecting and measuring the antibodies, antigens, proteins, and glycoproteins present in biological samples. Antibodies protect the body from foreign invaders (i.e., antigens), while antigens are alien invaders, including chemicals, bacteria, viruses, or pollen. ELISA is one of the several other immunosorbent assays, which include: RadioImmunoAssay (RIA), Fluoroimmunoassay (FIA), Counting Immunoassay (CIA), and many more.

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The explained principle of ELISA, and its procedure

As earlier mentioned, ELISA kits (i.e., the most extensively used technology in enzyme immunoassay technology) are mainly for diagnosing the antibodies and antigens present in the body of plants and animals. It is helpful in applications such as microbiology, blood screening, veterinary, and immunology. To increase the sensitivity and precision of ELISA, the multi-well (96 or 384 well) plate is coated with antibodies that are high in affinity. The step by step procedure on how to do elisa and how ELISA is used to carry out a blood test includes:

First step

This involves attaching a primary antibody with a high affinity towards bacteria, antibodies, and hormones to a multi-well plate. One can add the antigen to the multiwell plate next.

Second step

This step involves filling a microtiter that has an antigen coat with the antigen-antibody combination. The next step is to remove any free antibody by washing.

Third step

Another antibody (i.e., secondary antibody) specific to the primary antibody must be conjugated with an enzyme before adding it to the plate. The next step is to remove any free enzyme-linked secondary antibody.

Fourth step

The last step involves adding a substrate. The substrate change in color is used to measure the antigen in the blood sample. The color change is measured using spectrophotometry.

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The more the substance that is tested for is present, the more reaction that takes place, which results in a decrease in linked enzymes that can bind to the solid surface.

The types of ELISA techniques

The working mechanism behind ELISA is the combination of antigens that are immobilized to a solid surface and antibodies that are linked to an enzyme. The antibodies must interact with the antigens for detection to happen. The types of interaction formed are dependent on the type of ELISA technology used. The kinds of ELISA include:

1. Direct ELISA

The antibody specific to the immobilized antigen bound to the multi-well plate interacts with the antigen. For direct ELISA, the antibody is conjugated to the Horseradish peroxidase (HRP) or other detection molecules. The advantages of using the Direct ELISA technique includes: 

• It has a short procedure; it saves time and the use of reagents.

• It does not require cross-reactivity from secondary antibodies.

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2. Indirect ELISA

This technique requires a two-step process to detect antigens. There are two antibodies involved, one is labeled primary antibody (i.e., specific for the antigen), and the other, against the primary antibody's host species, is labeled secondary antibody. 

The primary antibody that is specific for the antigen first binds to the target. The second step involves the secondary antibody binding to the primary antibody for detection. This is unlike the direct ELISA technique, where the antibody is bonded to HRP.  This method is helpful for detection in specific serum samples. 

Some of the advantages of this technique may include:

• Many secondary antibodies will bind to the primary antibody, and as a result, the signal is amplified. 

• There is flexibility because one secondary antibody can conjugate with several primary antibodies

Some disadvantages may include:

• When compared to direct ELISA, it is more time-consuming and has a more extended protocol.

• There is a possibility for cross-reactivity from secondary antibodies to happen.

3. Sandwich ELISA

Sandwich immunoassay is the most common ELISA technique. It requires two specific antibodies for the epitopes of the antigen. This technique looks like a sandwich; it can compare to a trap where two antibodies trap the antigen inside. 

The antibodies are referred to as matched antibody pairs. The first antibody serves as a capture antibody; it coats the surface of the multiwell plate to speed up the immobilization of antigen, while the second antibody conjugates with the antigen for detection purposes.

Some advantages of sandwich ELISA may include:

• It is suitable for detection in complex samples.

• It can make use of both direct and indirect methods.

• It has high sensitivity.

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4. Competitive ELISA

Competitive immunoassay, also known as inhibition ELISA, detects and measures the presence of an antigen by detecting signal interference. Each of the types discussed earlier can be implemented here. There is a competition between the sample antigen and reference antigen for binding to a particular amount of labeled antibody. 

The first step involves pre-coating the reference antigen to a multi-well. The second step is pre-incubating the sample antigen with the labeled antibody before adding it to the multi-well. The amount of reference antigen detected depends on the antigen in the example —the more antigens in the sample, the fewer free antibodies to combine with the reference antigens. One of the advantages of competitive ELISA advantages includes its suitability for minor antigens.

Conclusion

There are three factors to consider before performing ELISA. These factors are accountable for the erroneous results in ELISA, and they include specimen factor, reagent, factor, and operating factor. To reduce the effect of these factors on the results, one must thoroughly understand the principle of ELISA and not ignore any of the factors.