The specificity of antibody-antigen binding underlies the versatility of antibodies. In biological systems, antibody-antigen binding enables antibodies to target foreign agents for elimination by triggering several adaptive immune mechanisms. In the lab, as tools for discovery, antibody specificity supports a broad range of applications, including Western blot, ELISA, immunoprecipitation, and immunochemical staining (e.g., immunocytochemistry and immunohistochemistry). Albeit the remarkable specificity of antibodies, investigators face different challenges when utilizing these reagents in various applications.
Antibody, Antigen, Epitope Basics
Antibodies are generated by B cells as part of the adaptive immune response involved in identifying and eliminating foreign agents such as infectious pathogens. Soluble antibodies secreted by B cells are endowed with unique specificities, mostly determined by the amino acid sequences and motifs forming their antigen-binding domains or complementary determining regions (CDRs) (Janeway et al. 2001, Sela-Culang et al. 2013). Therefore, the specificity of antibody-antigen interactions is shaped by unique paratope-epitope contacts. Epitopes may be defined as the immunogenic or antigenic regions of a protein. Simply put, an epitope is formed by the amino acid sequences, domains, or motifs of an antigen that directly interact with an antibody’s binding site or paratope.
Adapted from “Antigen Recognition by Antibodies” by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates
A single protein may be recognized by a range of antibody types, each of which may bind unique epitopes. Chemically, the interactions between epitope and paratopes supporting antigen-antibody binding are non-covalent and instead involve a series of weaker forces (Qaraghuli et al. 2020).
Technical Challenges of Antibody-Based Assays
There is no doubt that antibodies are indispensable tools for discovery in the lab. Yet, investigators may encounter several challenges in the process of optimizing their use across applications such as ELISA, Western blot, and cell or tissue immunostaining. For example, investigators often find that antibody binding is susceptible to the assay’s conditions. Thereby, an antibody may specifically bind to its antigen in a Western blot assay but fail to detect its target protein in situ through IHC or ICC, once proteins have been exposed to fixatives (Uhlen et al. 2016). Generally, conformational changes are to blame, as they frequently lead to epitope masking (Saper, 2008).
Adapted from “Sandwich ELISA” by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates
A definite advantage of the sandwich ELISA lies in its specificity and sensitivity, conferred by two antibodies. Therefore, sandwich ELISAs are suitable for analyzing antigen concentrations in crude biological samples commonly used in diagnostics.
Another challenge may be presented by applications needing antibodies to recognize multiple epitopes on a single antigen. In sandwich ELISA, “capture” and “detection” antibodies are used to specifically detect an antigen within crude conditions typical of complex biological samples such as urine and serum. To this end, investigators may need to do extensive troubleshooting to identify an optimal capture-detection “antibody pair.” The main challenges to overcome in this type of assay are ensuring that antibody pairs are not cross-reactive and that antibody-access to each epitope is unhindered. Often, investigators’ optimization efforts are complicated by antibody availability in the market.
Lastly, although antibodies are highly specific reagents, problems with unexpected reactivity or off-target binding frequently occur. Thereby it has become increasingly clear that investigators must extensively validate antibody reagents to ensure specificity and reproducibility (Uhlen et al. 2016). A recommended validation strategy by the International Working Group for Antibody Validation (IWGAV) consists of the use of independent antibodies. This strategy proposes, “Use two or more independent antibodies that recognize different epitopes on the target protein and confirm specificity via comparative and quantitative analyses” as a validation method.
Uncovering a Broader Antibody Repertoire
Discovery workflows providing more opportunities to identify a broad range of antibodies with unique specificities are desirable and provide an edge when developing and validating analytical and diagnostic assays, including sandwich ELISA, Western blot, and immunostaining protocols. While hybridoma technology has been the standard in monoclonal antibody discovery efforts, immortalization processes unavoidably result in the loss of antibody-producing clones. The estimated survival rate for B cells undergoing fusion is, on average, 1/5000 (Winters et al. 2019). In contrast, because antibody discovery strategies based on B cell cloning bypass the need for immortalization, this technology provides investigators more opportunities to screen a greater number and diversity of antibody-producing cells.
Given the challenges that investigators confront with assay optimization, technologies that maximize discovering a broad repertoire of antigen-specific antibodies provide a real advantage in finding the best reagents for a wide range of applications.