ELISA to select mice / rats for fusion and screening ELISA for hybridomas.
Our standard monoclonal antibody development service includes ELISA testing to determine which mice (or rats) are ready for fusion and ELISA screening of the resulting hybridomas to determine the best cell lines to move forward with full characterization and subcloning. ELISA results are provided to researchers after each test bleed and are meant only to track the progression of the immune response to antigens. This data in conjunction with end application testing (e.g. IHC, FACS, etc.) against native protein is considered to complete the screening and select final clones for immortalization.
Titration ELISA for Test-bleeds:
Following the 5-week immunization schedule, test-bleeds of each mouse (or rat) are tested using the our standard titration ELISA assay. ELISA titers and IgG-vs-IgM isotyping are determined by screening each sample in two sets of replicates: one set with an anti-IgG secondary antibody and one set with an anti-IgM secondary antibody. By monitoring both the IgG and IgM levels we are able to assess when secondary isotype switching has occurred and determine the optimal time for fusion. This is extremely important to get the highest possible affinity antibodies and to limit the number of IgM monoclonals produced during fusion.
Once hybridoma supernatants are obtained, they are again screened, this time by single-point dilution ELISA to measure the antigen specific reactivity. IgG and IgM secondary antibodies are again used to ensure that IgG hybridomas are selected where possible. Complete reports are provided to researchers at each stage and conference calls are set up to discuss the data and chart the desired course based upon their needs.
Screening is perhaps the most crucial aspect to successfully generating monoclonal antibodies. Many researchers elect to screen antibodies themselves, but we can screen the antibodies against native antigens in applications for you if needed. For more information on our validation services, contact a project specialist.