[Cloning and expression of human anti-colonic cancer single-chain Fv fragment].
We report a new strategy for the generation of human anticolon cancer monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs derived from peripheral blood mononuclear cells (PBMC) that were transformed by EBV. The immortalized B cells secreting tumor-specific antibodies were identified by HRT-18 cell ELISA and cloned by limiting dilution. Heavy- and light-chain VH-CH1 (gamma) and V kappa-C kappa cDNAs were rescued from ELISA-positive cells wells by RT-PCR. VH and V kappa were amplified by 2nd PCR and linked them together by 3rd PCR assembly with the use of a (Gly4Ser)3 linker. The ScFv cDNAs were then cloned into the fUSE 5 vector and displayed on phage. Phage clones were selected on HRT-18 cells and normal human PBMC. ELISA tested phage clones randomly picked after each panning step. > 80% of the clones showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting anticolon cancer antibodies. This approach offers an effective method by combining in vitro immunization, B-cell expansion for enrichment of specific B-lymphocytes, PCR gene cloning and phage display to make high-affinity human anticolon cancer monoclonal antibody molecules.