PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts.
Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.
Department of Ophthalmology, Flinders University of South Australia, Adelaide, SA 5042, Australia.
Yazad
Y
Irani
Melinda
M
Tea
Ronald G
RG
Tilton
Douglas J
DJ
Coster
Keryn A
KA
Williams
Helen M
HM
Brereton
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015321:Gene Rearrangement; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A