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The Different Types of ELISA Assay And Their Benefits

ELISA Assay

ELISA assays are one of the most employed assays in bioanalytical studies. They are quick and easy to carry out. Also, because of its ability to handle many study samples, they have become the primary choice of assays for evaluating research and diagnostic compounds. However, ELISA method development and validation are vital while developing a new analysis method. The current article outlines the different types of ELISA formats and dives deep into ELISA benefits.

Direct ELISA

Direct ELISA uses an enzyme-labeled primary antibody for detection. As the name suggests, the antibody binds directly to the immobilized target analyte. The enzyme reacts with its substrate and produces a signal proportional to the analyte concentration present in the sample.

The above principle shows that direct ELISA is much simpler and faster to perform than other ELISA types. Moreover, direct ELISA has fewer steps and requires fewer reagents. Hence, they are less prone to errors and potentially have no cross-reaction, as a secondary antibody is unnecessary.

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Indirect ELISA

Indirect ELISA uses two antibodies; primary and secondary antibodies. But here, instead of labeling the primary antibody, a secondary antibody is conjugated with an enzyme. First, the primary antibody conjugates with the target immobilized antigen, and then the secondary antibody binds to the primary antibody. Finally, the enzyme conjugated with the secondary antibody interacts with the substrate to produce a signal for measuring the analyte.

As indirect ELISA uses two antibodies, it is more sensitive than direct ELISA. Moreover, multiple labeled secondary antibodies may bind to the primary antibody, and therefore fewer labeled antibodies are required in indirect ELISA. It makes indirect ELISA more economical and flexible.

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Sandwich ELISA

In sandwich ELISA, , a capture antibody is immobilized on the solid surface instead of an antigen. Sandwich ELISA also uses a primary unlabeled and a secondary labeled antibody. Here, the capture antibody captures the target antigen. The primary antibody then binds to the target antigen, followed by binding a secondary labeled antibody to the primary antibody. Lastly, similar to all ELISAs, the enzyme reacts with its substrate and produces a signal for estimating the analyte of interest.

Sandwich ELISA is around 2-5 times more sensitive than direct or indirect ELISAs. Also, two antibodies offer higher specificity for detecting the target antigen. Besides, both direct and indirect formats can be used in sandwich ELISA making it highly flexible. However, in the absence of a standardized kit or antibody pairs, ELISA development and validation becomes slightly tedious. Hence, sponsors must focus more on ELISA assay validation to reduce cross-reactivity among different antibodies.

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Competitive ELISA

Competitive ELISA involves an inhibitor antigen; it is relatively more complex than the other three ELISA formats. However, each ELISA format can be modified into a competitive ELISA. The term competitive indicates that the target antigen and the inhibitor antigen compete for conjugation with the primary antibody.

The primary advantage of competitive ELISA is that crude samples can directly be used for analysis. They are highly robust assays being less prone to sample matrix effects or sample dilution. Additionally, less variability is observed among samples. To conclude, they offer maximum flexibility as all three formats can be used in competitive ELISA.

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