Abstract：
: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.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Takahashi Y,Ametani A,Totsuka M,Kaminogawa Sdoi
10.1016/0168-1656(96)01506-4subject
Has Abstractpub_date
1996-08-20 00:00:00pages
201-10issue
1-3eissn
0168-1656issn
1873-4863pii
0168-1656(96)01506-4journal_volume
49pub_type
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pub_type： 杂志文章
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