Your Hybridomas Are Monoclonal, But Are They Monospecific?
Scientific research has been revolutionized by the use of antibodies in basic research, diagnostics and as biopharmaceuticals. Antibodies were first described by Emil von Behring and Kitasato Shibasaburō in 1890 when they developed a serum “antitoxin” after injecting diptheria and tetanus toxins into animals. However, it was the seminal work in 1975 by César Milstein and Georges Köhler (who shared the 1984 Nobel prize with Niels Kaj Jerne) in developing the methodology to create monoclonal antibodies (bind to a single epitope) that lead to a quantum leap forward in the utility of antibodies. To create a hybridoma, an antibody producing plasma cell is fused to a myeloma cell which is then cloned to the single cell. This single cell produces one antibody clone and is thus monoclonal, or so the dogma tells us. The advantages of hybridoma derived monoclonal antibodies are that they provide an immortal source of antibody of a single specificity with highly reproducible and scalable batches and low variability.
However, a recent publication by Bradbury, ARM et al (mAbs Volume 10 (2018) – Issue 4) provides compelling evidence that although hybridomas may be monoclonal they are not necessarily all monospecific. Indeed of 185 hybridomas tested, 59 (31.9%) were found to contain one or more additional productive heavy or light chain. This is significant as the expression of additional chains resulted in reduced quality of the antibodies when assessed by ELISA and IHC (reduced specificity, binding and signal to noise ratio). Indeed, the authors go on to advocate producing antibodies recombinantly to benefit from improved sensitivity and specificity, long term stability and improved reproducibility when working from a known sequence rather than a hybridoma.
Traditional methods for sequencing antibodies (RACE, V region PCR) typically rely on prior knowledge of the heavy (α, β, ε, γ or μ) and light chain (λ or κ) with the use of specific leader sequence primers. Next generation sequencing, such as that offered by Absolute Antibody, relies on random primers with no inherent bias and can be performed without prior knowledge of the expected heavy and light chains to identify all possible sequences. Once the sequences have been identified, different combinations of heavy and light chains can then be produced and tested at small scale to identify the pair with the desired characteristics.
Sequencing of hybridomas is a prerequisite if you intend to switch to recombinant production or additional antibody engineering (changing species, isotype, subtype etc) as well as to secure the sequence in case of hybridoma loss. However, this research suggests that sequencing hybridomas is also important to identify any issues with hybridoma monospecificity which may affect antibody quality and reproducibility in the longer term.