Antibody Specificity and Epitopes
Antibodies have the ability to attach to a particular antigen epitope. It is crucial to remember that every epitope might be found in several protein antigens. It indicates that if two or more proteins are highly similar and have the same epitope, a specific antibody can recognize them both (Sethi, Endo, and Brandis, 2010, p. 193).
The Hybridoma Approach
In order to identify and choose the monoclonal antibody, the hybridoma approach uses four main steps: vaccination, somatic cell fusion, selection and screening, and subcloning.
Vaccination
An animal, such as a mouse, is immunized during vaccination in order to produce a particular and potentially highly affine antibody response. This requires repeated immunization to ensure exposure to the antigen, a process that increases specificity (Todd, Nadler and Schlossman 2011, p. 1442).
Somatic Cell Fusion
The somatic cell fusion is performed in which the splenocytes from the mouse are fused with myeloma cells line. The step is mediated with polyethylene glycol (PEG) which causes the spleen and myeloma cells to combine leading to generation of a hybridoma (Sethi, Endo, and Brandis, 2010, p. 193).
Selection and Screening
HGPRT reagent is added which allows selection of B lymphocytes hybridoma cells lines that produce an antibody with a specificity of interest, for example, BFV-X. The cells are selected using the HAT which allows survival of hybridoma and not non-fused B or myeloma cells (Todd, Nadler and Schlossman 2011, p. 1442). These are then screened for production of an antibody specific to BFV-X. An immunoassay is carried out to enable detection of antibody specific for the antigen of interest. This leads to the identification of cell population that contains the intended antibody.
Subcloning
Sub-cloning is the final step that involves scaling up for large quantity generation of the specific monoclonal antibodies (Todd, Nadler and Schlossman 2011, p. 1442). To achieve the goal of removing the unwanted cells in this phase, the procedure if sub-cloning entails one or more rounds of limiting dilution using 96-well plate which results in production of antibody specific to the original antigen because the plate may only contain one cell specific to the antigen of interest (Todd, Nadler and Schlossman 2011, p. 1442).
References
Sethi, K.K., Endo, T. and Brandis, H., 2010. Hybridomas secreting monoclonal antibody with specificity for Toxoplasma gondii. The Journal of parasitology, pp.192-196.
Todd, R.F., Nadler, L.M. and Schlossman, S.F., 2011. Antigens on human monocytes identified by monoclonal antibodies. The Journal of Immunology, 126(4), pp.1435-1442.
