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Cloning murine antibody V-genes with non-degenerate primers and conversion to a recombinant antibody format.

ABSTRACT: 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.

SUBMITTER: Bialon M

PROVIDER: S-EPMC4278175 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Cloning murine antibody V-genes with non-degenerate primers and conversion to a recombinant antibody format.

Bialon Magdalena M Schellenberg Ludmila L Herzog Nicolas N Kraus Stefan S Jörißen Hannah H Fischer Rainer R Stein Christoph C Nähring Jörg J Barth Stefan S Püttmann Christiane C

Monoclonal antibodies in immunodiagnosis and immunotherapy 20141201 6

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 ...[more]

PMID: 25545205

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