CONSTRUCTION AND SCREENING OF HUMAN CEA SPECIFIC PHAGE ANTIBODY COMBINATORIAL LIBRARY

Objective To construct human phage antibody library and produce human monoclonal antibodies to CEA.Methods Example of human phage antibody library obtained from lymphonode of ten patients with colon carcinoma.The concentration of plasma CEA of every patient was more than 10 ng/mL.Wuth routine RT-PCR with sets of oligonucleotide primers designed by Kang,the genes of human immunoglobulin Fd and κlight chains were amplified and cloned into phagemids(p3MH) to generate human phage antibody library.The affinity selection and ELISA were adopted for identification of specific phase antibodies to CEA.Results A phage antibody library of κchain containing 1.4×10~7members was constructed and a phage antibody library of Fab had 5.2×10~6 members.The recombinant frequency of the Fab was 30% determined by digesting DNA extracted from 10 clones.Five rounds of panning produced 30-fold amplification in eluted phage,indicating enrichment for specific antigen-binding clones and four positive clones showed clear reactivity to CEA but not with BSA or HBsAg by ELISA.Conclusion The success of isolating human anti-CEA Fab proves the usefulness of phage display system in human antibody preparation.Human monoclonal antibodies to CEA may be useful as molecular tools to study the pathogenesis of tumor expressing CEA,as well as for diagnostic and therapeutic purposes.