Navigating the Variable Landscape of Antibody Selection

par | Déc 6, 2023

Antibodies play a crucial role in life science research and are standard reagents in various imaging applications. In 2022, the global market for research antibodies was valued at approximately $4 billion USD, and it is projected to reach $6.5 billion USD by 2032. Compared to therapeutic antibodies, which require extensive validation, research antibodies can be hit or miss. Despite the significant market, it’s not uncommon to spend hundreds of dollars on micrograms of a reagent that simply does not perform as promised.

As a quick summary, antibodies are either monoclonal and polyclonal. Monoclonal antibodies are derived from clones of a single parent B cell and target a specific epitope.  Polyclonal antibodies, on the other hand, are derived from multiple B cells that target different epitopes in the same antigen. They provide broader epitope coverage but lower specificity

Generally, monoclonal antibodies are preferred for imaging applications due to their high specificity. As polyclonal antibodies are pooled, the exact epitopes targeted are difficult to pinpoint, and their affinity can change by lot. Variability is also a problem for monoclonal antibodies derived from animal immunization.  Increasingly, there is building advocacy for recombinant antibodies with defined and openly accessible protein sequences. 

Open, accessible and searchable databases can help you pinpoint the appropriate antibody. One notable example is citeAb, a database that also provides the number of citations based on PubMed metadata. It also allows the user to filter based on type of antibody, including clonality, reactivity and other properties.

The Antibody Registry is another important resource. It is a publicly accessible database listing more than 2.5 million antibodies and their associated kits. Each reagent listed is given a distinct Research Resource Identifier (RRID). It’s crucial to include this RRID when publishing your research. The RRID is permanent, providing a valuable means of tracking antibodies and related kits, even in cases where they are no longer obtainable such as when a product is discontinued.

While the citeAb and Antibody Registry databases are excellent resources, they provide information and do not guarantee antibody effectiveness. Both provide a list of publications referencing the antibody, but the onus is on the end user to critically review product information and the scientific literature. To assess the likelihood of an antibody working, it’s advisable to evaluate product sheets and any publications where the antibody is used. Further, check that the antibody of interest has been tested and/or validated for the intended application. For example, an antibody that works for western blotting may not work for immunofluorescence or vice versa.  You can also contact colleagues for advice, as often groups do not publish negative data, such as when antibodies do not work. 

If you are working with human cells and tissue, the Human Protein Atlas can also help guide antibody selection. This initiative aims to map and characterize all the proteins encoded by the human genome and provides information on the expression and localization of human proteins. The initiative tests and validates antibodies for various applications, including immunohistochemistry (IHC), Western blotting, and immunofluorescence.

In addition, YCharOS is an organization with the mission to characterize commercial antibodies for the entire human proteome through open science.  As of August 2023, they have presented comprehensive knockout characterisation data for about 812 antibodies and 78 proteins using techniques such as Western blotting, immunoprecipitation, and immunofluorescence.

Other examples of useful databases that provide information about research antibodies include:

Antibodypedia, an open access, online antibody database listing antibodies against human proteins. It provides information about the performance of antibodies in specific applications.

Labome provides information about antibodies that have been validated through knockout studies. 

Once you have narrowed down your choice, and if funds are limited, check for sample sizes to test before making a larger investment. Also, familiarize yourself with the supplier’s return and replacement policies in case the antibody does not meet your expectations. 

Selecting the right antibody is the initial and crucial step in ensuring a rigorous and reproducible experiment. Even when using a well- characterized antibody, however, it is still essential to validate its specificity and sensitivity within the context of your unique biological model. In future posts, we will explore the process of antibody validation, with a particular emphasis on its application in immunofluorescence imaging.

Additional resources for antibody selection

– The DSHB banks and distributes hybridomas and the monoclonal antibodies (mAbs) they produce to the general scientific community.
SAbDab is a database containing all the antibody structures available in the Protein Data Bank.
EuroMAbNet is a European network of academic laboratories sharing expertise and knowledge about antibodies.
Enhanced validation of antibodies for research applications
4th International Antibody Validation Meeting of the Antibody Society
Nature Commentary: Reproducibility: Standardize antibodies used in research

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