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Cloning Murine Antibody V-genes with Non-degenerate Primers and Conversion to a Recombinant Antibody Format

: Bialon, M.; Schellenberg, L.; Herzog, N.; Kraus, S.; Jörißen, H.; Fischer, R.; Stein, C.; Nähring, J.; Barth, S.; Püttmann, C.


Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 33 (2014), Nr.6, S.369-377
ISSN: 2167-9436 (online)
Fraunhofer IME ()

Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR.