Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.
We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.
Chardès
T
T
CNRS UMR 9921, Faculté de Pharmacie, Montpellier, France. chardes@pharma.univ-montpl.fr
Villard
S
S
Ferrières
G
G
Piechaczyk
M
M
Cerutti
M
M
Devauchelle
G
G
Pau
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D017931:DNA Primers; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region