Antibodies play a central role in the
ELISA application,
driving specific target binding and signal detection.
Pre-packaged ELISA kits are widely available and contain all the necessary antibodies and other reagents.
However, there are situations in which a new or customized assay will need to be developed, such as the investigation of rare or novel targets.
This guide describes the antibody types used in ELISAs and what prospective buyers should consider during their selection.
Choosing a capture antibody
The capture antibody, a defining component of sandwich ELISAs, is used for coating the assay plate and binding the target antigen.
1
Choosing a capture antibody with high affinity and selectivity for the target is important.
Serving primarily an antigen-binding role, these antibodies do not need to have conjugated enzyme labels.
While both monoclonal and polyclonals can be used as a capture antibody, polyclonals are often used due to their ability to bind multiple epitopes.
This maximizes the potential to immobilize the target antigens on the plate surface.
Finally, it is best practice to choose a capture antibody that is properly validated for use in ELISA applications.
Choosing a detection antibody
The detection antibodies are used to bind the target antigen and generate the detection signal via an enzyme-substrate reaction.
In direct ELISAs, this is carried out with a single enzyme-conjugated primary antibody.
Indirect ELISAs, meanwhile, use an unconjugated target-specific primary antibody followed by a labeled
secondary antibody.
When choosing a primary antibody, it is important to select antibodies that will recognize the target antigen’s epitope of the appropriate species.
Meanwhile, the secondary antibody must be reactive to the host species of the primary antibody and selective to the isotype of the primary.
The enzyme labels most commonly conjugated to detection antibodies are alkaline phosphatase (AP) or horseradish peroxidase (HRP).
These enzymes react with
substrates
to produce colorimetric signal readouts.
For lower abundance targets, chemiluminescent substrates may increase the detection sensitivity.
2
For these substrates, choose an antibody conjugated to HRP.
Matched antibody pairs
For certain targets, antibody suppliers may offer matched antibody pairs, two different antibodies that serve as the capture and detection antibodies for use in a sandwich ELISA.
3
These pre-selected antibodies have been determined by the manufacturer to be suitable for both capturing the antigen and generating a detection readout.
Using a
matched antibody pair
can help speed the development of new ELISA experiments.
Validation for ELISA antibodies
Early in the antibody selection process, users should assess whether candidate antibodies are validated to work in the ELISA application.
Antibody validation,
proof that a given antibody recognizes its specific target under specific experimental conditions, should be provided by the supplier in the product documentation.
A provided protocol, if present, should also be reviewed.
Antibody performance depends on several experimental variables and even a highly validated antibody can perform poorly if the wrong protocol is utilized.
4
Other useful information on the datasheet include a listing of known cross-reactivities and any links to references that cite the use of the antibody.
Finally, check to see if certain antibodies have been mentioned in the literature, particularly for monoclonal or recombinant antibodies.
A published figure using a similar sample type or experimental condition may lead to finding an ideal product, or—inversely—rule out a potentially problematic antibody.
The Biocompare Antibody Search Tool lets users search for products with citations, figures, or reviews.
See if your protein of interest has been used by others for ELISA or other immunoassays.
References
1. Alhajj M, Zubair M, Farhana A. Enzyme Linked Immunosorbent Assay. [Updated 2023 Apr 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK555922/
2. Easthope E. ELISA Detection Strategies. Biocompare. 2022 Dec 20 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/591588-ELISA-Detection-Strategies/
3. Easthope E. Factors to Consider for ELISA Development. Biocompare. 2022 May 24 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/584575-Factors-to-Consider-for-ELISA-Development/
4. Easthope E. The Importance of Antibody Validation. Biocompare. 2023 Sep 19 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/599564-The-Importance-of-Antibody-Validation/