Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool.
The B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen-selected B cells as a source of V gene libraries. Mice were immunized with (4-hydroxyl-3-nitrophenyl)acetyl (NP)-chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen-coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the V lambda 1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4-hydroxy-3-iodo-5-nitrophenyl)acetyl-bovine serum albumin. The frequency of antigen-binding clones was much greater (greater than 50 fold) in the library from the DNA of antigen-selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen-binding clones revealed that they almost invariably used the V-186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.
Hawkins
R E
RE
MRC Laboratory of Molecular Biology, Cambridge, Great Britain.
Winter
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006241:Haptens; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007156:Immunologic Memory; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data
D006241:Haptens; D007135:Immunoglobulin Variable Region; D004247:DNA