The direct cloning of the immunoglobulin VH genes from primary cultured B cells specific for a short peptide.
A new and simple method was devised to obtain immunoglobulin VH genes directly from primary cultured B cells specific for a short peptide. Peptide-specific B cells were separated from splenocytes of peptide-immunized BALB/c mice with antigen-coated magnetic beads, and were cloned by a limitedly diluted culture in the presence of lipopolysaccharide, recombinant interleukin (rIL) -2, rIL-4 and rIL-5, and 3T3 fibroblasts as filler cells for 7 days. Seventeen clones were obtained from 3 x 10(3) fractionated cells by screening the positive wells containing anti-peptide antibody-secreting cells by an enzyme-linked immunosorbent assay (ELISA). The VH cDNAs of these clones were amplified by a set of primers; a primer complementary to the mu-chain constant region gene and the other with high complementarity to most of the VH genes. This is the first report of success in obtaining unknown VH genes directly from primary B cell clones, after their antigen-specificity has been confirmed by ELISA. This new method will provide a powerful tool for designing specific recombinant antibodies.
Takahashi
Y
Y
Department of Applied Biological Chemistry, University of Tokyo, Japan.
Ametani
A
A
Totsuka
M
M
Kaminogawa
S
S
D000818:Animals; D000941:Antigens; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification; D005260:Female; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics*; D018189:Immunomagnetic Separation / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics
D000941:Antigens; D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D012333:RNA, Messenger