ELISA assay test or Enzyme-Linked Immunoassay is a technique utilized for quantifying and detecting substances such as antibodies, protein, hormones, and peptides. The test detects the immune system components such as IgM antibodies or immune responses generated by our body such as an infection.

The antigen and enzyme (protein used for catalyzing the reaction) utilized in the ELISA assay test forms the basis for the results of the tests. If the antibody and antigen react, the test results come out as positive, otherwise, negative.

This article will discuss how an ELISA assay test works and the variations of ELISA assay test available in the industry. Based on this guide, you can select the suitable ELISA assay test format for your purpose.

Working of ELISA assay test

In the ELISA assay test, an antigen is attached to a solid surface plate, usually a 96-well or 384-well microplate. Then, an enzyme-linked antibody is complexed to this solid surface. Immobilizing the antibody on the microplate allows separation of non-bound and bound material during the experiment. The non-bound material is washed away and remaining analytes are measured with high precision.

For detection, a substrate is added to the enzyme and the resulting solution is incubated. After incubation, the antibody-antigen reaction is assessed and detected. Sometimes, a secondary antibody is utilized to link the detection enzyme to the primary antibody such as alkaline phosphatase (AP) and horseradish peroxidase (HRP). The substrate selection is conducted based on the required sensitivity and specificity of the assay.

Types of ELISA assay test

Several factors impact the selection of the ELISA technique for various purposes. For instance, hapten detection, which is a small molecule, requires a competitive ELISA assay test. However, on the other hand, a large protein containing various epitopes like cytokine requires sandwich ELISA assay test.

Apart from the type and size of the molecule, the availability of the antibody also influences the selection of the ELISA assay test. If you have only one antibody for the antigen, the competitive or direct ELISA assay is utilized.

Below we have explained the working of common ELISA assays.

Direct ELISA assay test

The direct ELISA assay uses only the primary antibody to directly bind it to the antigen. This antigen is either complexed or immobilized to the microplate's solid surface. A substrate of relevant sensitivity is added to the solution, which results in a visible reaction between the enzyme in the antibody and substrate. The antigen present in the solution is detected with visible markers.

Indirect ELISA

The indirect ELISA uses two antibodies. The primary antibody is conjugated to the antigen on the microplate and the secondary antibody is labeled to the enzyme.

After the conjugation of primary antibody, the secondary antibody is added, which causes the primary antibody to attach to the secondary antibody. The substrate reacts with the enzyme in the secondary antibody to create a visible, measurable signal.

Sandwich ELISA

In both the above-mentioned techniques, the antigen was conjugated or immobilized to the microplate. However, in sandwich ELISA, the antibody is complexed to the plate, commonly referred to as capture antibody. The detection of antibodies involves primary antibody (unlabeled) and secondary antibody (enzyme-labeled).

In the test, the antigen is bound to the plate with the help of the capture antibody. Then, the primary antibody used for detection connects to the antigen. This is followed by binding of secondary antibody to the primary antibody used for detection. Lastly, a substrate is added to create a visible, measurable signal.

Competitive ELISA

From all the processes used for ELISA assay, competitive ELISA is comparatively complex due to inhibitor antigen. This is also why competitive ELISA is often referred to as inhibitor ELISA.

Here, both the antigen and inhibitor antigen compete to bind with the primary antibody. Below we have explained the stepwise procedure.

Firstly, a sample of antigen is incubated with the primary antibody (unlabeled). This leads to the formation of an antibody-antigen solution. However, the antibody is more in comparison to antigen, hence, free antibodies are present in the solution.

Secondly, the antibody-antigen solution is added to plate which has inhibitor antigen, which also has the ability to connect to the primary antibody. The free antibody present in the mixture combines to inhibitor antigen. However, the antibody-antigen solution doesn’t bind to the inhibitor antigen, so these are washed away.

Thirdly, the secondary antibody (enzyme-labeled) is added to the mixture. It combines to the primary antibody on the plate which is already connected to inhibitor antigen.

Lastly, after the addition of a substrate, a visible signal is produced for detection.

However, it is important to note, that the final signal for detection is dependent on the antigen amount. More antigen will produce a weaker signal.

Optimization of ELISA

Prefer utilizing microplate with <5% CV (coefficient of variation) value and at least 400 ng/cm2 protein binding ability.

Detect the microplate before use to eliminate scratches as it can lead to aberrations.

The type of plate is dependent on the detection signal. The colorimetric signals require polystyrene plates with a flat bottom. The chemiluminescent signals require white opaque plates.

Conclusion

ELISA assay test is utilized in various domains such as the pharmaceutical industry, medical testing, and cosmetic testing. The reason behind the high popularity of this experiment is highly specific and sensitive detection when compared to other available detection procedures. However, it is necessary to evaluate all the factors before selection of any assay to achieve accurate and precise results.