Monoclonal Antibodies

Monoclonal antibodies (mAbs) are each derived from a single B cell clone, meaning that every antibody within the same preparation shares the same isotype and specificity. They were first reported in 1975, when Kohler and Milstein discovered hybridoma technology. To generate hybridomas, an animal is immunized with the protein of interest to stimulate the production of specific B lymphocytes. The B lymphocytes are then isolated from the spleen and fused in the presence of hypoxanthine aminopterin thymidine (HAT) medium with immortal myeloma cells containing non-functional hypoxanthine guanine phosphoribosyl transferase (HGPRT) genes. This ensures that only the hybridomas survive. After several weeks in culture, the hybridomas are transferred to microtiter plates via the limiting dilution method and the expressed antibodies are screened by enzyme-linked immunosorbent assay (ELISA). This allows for identification of the best-performing clones, which are expanded in vitro. The resultant antibodies may be purified from the growth medium for use in various immunoassay techniques. Alternatively, the hybridomas can be injected intraperitoneally into mice and the ascites fluid collected. While ascites fluid can be used directly, it is recommended that the antibodies be purified to prevent their degradation by endogenous proteases. The in vitro approach is preferred to using ascites fluid as a source of monoclonal antibodies as it both reduces animal use and minimizes the risk of contaminating mouse immunoglobulins in the final product. Although hybridoma technology was originally developed using mice, it has since been adapted for rats, hamsters, and rabbits. In addition, monoclonal antibodies are now produced using various recombinant methods. These include sequencing an existing hybridoma, or screening a phage display library for desirable antibody fragments, before introducing the antibody-coding DNA into a mammalian cell line for in vitro expression – generating recombinant monoclonal antibodies. An important advantage of monoclonal antibodies is that they are highly specific for their antigenic targets since they recognize just a single epitope. This means that, compared to polyclonal antibodies, monoclonal antibodies are less likely to produce unwanted background signal by cross-reacting with non-target proteins. Monoclonal antibodies also demonstrate consistent experimental performance and are available in unlimited long-term supply. A limitation of monoclonal antibodies is the length of time taken for their production, which is typically in the region of 6-9 months. This can prohibit their use when a new target, such as SARS-CoV-2, is first discovered. In contrast, novel polyclonal antibodies may be developed in just a matter of weeks. Producing monoclonal antibodies for small peptides and protein fragments may also be problematic when using mice, due to a lack of immunogenicity, but this issue has largely been addressed by developing hybridoma technology for other species, and generating rat monoclonal antibodies and rabbit monoclonal antibodies.