[Construction of phage anti-idiotypic antibody library using sensitized in-vitro B-lymphocytes of nasopharyngeal cancer patients].
OBJECTIVE
To construct phage human anti-idiotypic antibody library.


METHODS
Peripheral blood mononuclear cells(PBMCs) of patients with nasopharyngeal cancer(NPC) were sensitized in-vitro and transformed by Epstein-Barr virus(EBV). V(H) and V(L) genes were re-amplified by PCR and combined to single-chain fragment of variable region(ScFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library.


RESULTS
Detection of Sandwich ELISA showed that of 10 NPC patients,8 patients' B cells transformed by EBV could produce anti-idiotypic antibodies to NPC. 5 types of V(H) genes and 7 types of V(L) genes were obtained by PCR re-amplification and then connected with (Gly(4)Ser)(3) linker to form 14 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into MC1061 via electroporation. Phage anti-idiotypic antibody library with sink size being 1.1x10(7) was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 70%.


CONCLUSIONS
A strategy for preparing human single chain anti-idiotypic antibody by means of phage antibody library technique in combination with EBV transformation technique is feasible.
He
Xiao-Juan
XJ
Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha 410078,China.
Li
Guan-Cheng
GC
Zhu
Jian-Gao
JG
Li
Yue-Hui
YH
D001402:B-Lymphocytes; D001435:Bacteriophages / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007963:Leukocytes, Mononuclear; D009303:Nasopharyngeal Neoplasms; D019151:Peptide Library
D007135:Immunoglobulin Variable Region; D019151:Peptide Library