A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA.
The amplification of variable regions of immunoglobulins by reverse transcription polymerase chain reaction (RT-PCR) has become an invaluable technique either for the cloning of monoclonal antibodies (mAbs), or for the building of single-chain fragment variable (ScFv) libraries. Numerous applications have been described either for studying the antigen-antibody interactions or for medical purposes, with the recent development of recombinant antibodies for therapeutic use. Several publications by different groups have reported primer sequences to perform such amplification, but the strategy used to design these primers, and particularly the way of performing the necessary alignments, generally appear poorly detailed. In the present work, we propose a rational method of designing primers in order to amplify the variable region of heavy chain (VH) and variable region of light chain (VL) domains for framework 1 (FR1) of immunoglobulins. The described sets of primers have been designed to hybridize with the entire VH and VL mouse repertory without modification of amino acids since amino acids of framework 1 play a role in the folding, and thus in the functionality, of recombinant antibody. These primers have been applied to the cloning of monoclonal antibodies previously produced in the laboratory. This approach can be extended to other species or members of the immunoglobulin superfamily.
Essono
S
S
Service de Pharmacologie et d'Immunologie, DRM/DSV, CEA-Saclay, Cedex 91191, Gif sur Yvette, France.
Frobert
Y
Y
Grassi
J
J
Créminon
C
C
Boquet
D
D
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000145:classification / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000145:classification / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains