Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.
We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.
Orlandi
R
R
Division of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Güssow
D H
DH
Jones
P T
PT
Winter
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics*; D002678:Chimera; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA