Types of ELISA

Types of ELISA:

The beginning of every ELISA experiment is to fix the antigen to a well in a microtiter plate. There are two ways to perform this immobilization: either via direct adsorption to the surface of the plate, or with a capture antibody.

The capture antibody is chosen based on the antigen and this method is primarily used in sandwich ELISA. Knowing this, ELISA assays can be grouped in four major categories:

Direct ELISA:

This type of ELISA assay is significantly faster than the rest and has fewer steps. With fewer reagents and steps, there is smaller chance of human error when compared to the other techniques.

On the other hand, because the antigen immobilization is not specific, higher background can be seen. This issue is caused by all proteins including the target one binding to the plate, instead of only the protein of interest. Compared to the rest, this method is less flexible because a different conjugated primary antibody is needed for every protein.

There is no signal amplification with direct ELISA, due to the lack of secondary antibody. This leads to a less sensitive assay, making this technique suitable for analysing the immune response to an antigen.

Indirect ELISA:

Being cheaper and more sensitive than direct ELISA, the indirect technique can offer splendid flexibility. With this type of ELISA, several primary antibodies can be used with one labelled secondary one.

But this high sensitivity comes at a price, indirect ELISA takes longer to perform due to the additional incubation of the secondary antibody, which can be cross-reactive and cause background noise.

Sandwich ELISA:

This type of assay utilizes matching antibody pairs, leading to much higher sensitivity than the two previous types of ELISA. With two antibodies participating in capture and detection, the specificity is also higher. Lastly, sandwich ELISA can use both direct and indirect detection, leading to greater flexibility.

On the other hand, optimising antibodies for this procedure is difficult because cross-reactivity can occur between the detection and capture antibodies. Also, a tested antibody pair or a standardised kit is essential for this experiment.

Competitive ELISA:

Also known as blocking ELISA, this technique is the most complex of all the previous. This assay is generally used for measuring the concentration of an antigen via interference detection in signal output. It is less sensitive to sample dilution and matrix effects when compared to sandwich ELISA and out of all the variations, competitive ELISA offers the best flexibility and consistency.

The only drawback to this assay is that it shares the same limitations as a base assay, because all ELISAs can be modified to a competitive variation.