title: 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.
authors.affiliation: 1 Department of Experimental Medicine and Immunotherapy, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University , Aachen, Germany .
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bialon
authors.forename: Ludmila
authors.initials: L
authors.lastname: Schellenberg
authors.forename: Nicolas
authors.initials: N
authors.lastname: Herzog
authors.forename: Stefan
authors.initials: S
authors.lastname: Kraus
authors.forename: Hannah
authors.initials: H
authors.lastname: Jörißen
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
authors.forename: Jörg
authors.initials: J
authors.lastname: Nähring
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.forename: Christiane
authors.initials: C
authors.lastname: Püttmann
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology; D017422:Sequence Analysis, DNA
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins