ELISA (enzyme-linked immunosorbent assay) is a plate-based testing technique designed to detect and measure peptides, proteins, antibodies, and hormones. In ELISA, the antigen must be immobilized onto a solid surface and then complexed with an enzyme-linked antibody. The detection process is done by observing a color change due to the enzyme’s activity on the substrate. The most important element of the detection strategy is the highly specific antibody-antigen interaction.

The ELISA examination procedure begins with the process of coating the antigen and/or antibody onto the wells of a plate surface. The next step is blocking the antigen and antibody binding on unspecific sites with a blocking agent. After incubation and washing, the plate is incubated with an enzyme-linked antibody. The plate is then washed and followed by the addition of the substrate resulting in a color change, and the OD (optical density) value is read with an ELISA reader. This substrate changes color upon reacting with the enzyme.

The washing step is an important stage to eliminate antibodies that are not bound to the antigen. Additionally, it is essential to ensure that there is no residual wash fluid left on the plate, as this may affect the subsequent examination stage.

Source: European Union Reference Laboratory

Types of ELISA ELISA can be performed with several modifications to the basic procedures: direct, indirect, sandwich, or competitive. The key step, immobilization of the desired antigen, can be achieved by direct adsorption to the test plate or indirectly through capture antibodies that have been attached to the plate.

Source: BOSTER, ELISA Handbook

1. Direct ELISA This type of ELISA is used to detect and measure the concentration of antigen in a sample. The antigen to be detected will directly bind to the detector antibody (an antibody labeled with an enzyme).

Advantages:

• Fast because it only uses one antibody
• No cross-activity with secondary antibodies

Disadvantages:

• Antibody immunoreactivity may be affected by the enzyme
• Weak signal generated
• Takes a long time for staining each type of antibody

2. Indirect ELISA For indirect detection, the antigen immobilized on the well plate is detected in two stages or layers. First, the unlabeled primary antibody attaches to the specific antigen. Then, the enzyme-conjugated secondary antibody binds to the first antibody.

Advantages:

• Test sensitivity increases with the use of primary and secondary antibodies. Disadvantages:
• Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signals.
• Requires longer incubation time
• Requires more cost than the direct method

3. Sandwich ELISA This ELISA requires two specific antibodies for different epitopes of the antigen. Both antibodies are usually referred to as a matched antibody pair.

Advantages:

• High specificity
• Suitable for use with complex (impure) samples Disadvantages:
• Relatively high cost due to the use of two antibodies

4. Competitive ELISA This method involves a competitive reaction between the sample antigen and the antigen binding to the bottom of the well plate with the primary antibody. In this method, the non-sample antigen is attached to the bottom of the plate. Next, the sample antigen and primary antibody are added to the well. Then, the secondary antibody bound to the enzyme in the well above the plate is added. Both antibodies will compete to bind to the antigen.

Advantages:

• High sensitivity

ELISA Data Interpretation

ELISA testing produces three different types of data output:

1. Quantitative ELISA data can be interpreted by comparing it to a standard curve (serial dilution of a known and purified antigen) to accurately calculate the concentration of antigen in various samples.
2. Qualitative ELISA can also be used to produce a yes or no answer indicating whether a specific antigen is present in a sample, compared to empty wells that do not contain antigen or control antigen that is not related.
3. Semi-quantitative ELISA can be used to compare the relative level of antigen in the test sample because signal intensity will vary directly with antigen concentration.

References:

BOSTER, Antibody and ELISA Expert. ELISA Handbook.

Hidayat, R., & Patricia Wulandari. (2021). Enzyme Linked Immunosorbent Assay (ELISA) Technique Guideline. Bioscientia Medicina : Journal of Biomedicine and Translational Research, 5(5), 447-453.