[Expression and identification of phage display library for Fab fragments of colorectal cancer-related antibodies].
OBJECTIVE
To express the original human Fab antibody phage display library with positive recombined bacterium XL1-blue-Pcomb3 and identify its specific binding activity with colorectal cancer cells in vitro after screening with human colorectal cancer-related antigens.


METHODS
The recombination rate of Fd fragment of the heavy chain and insertion of kappa chain of the antibodies was determined with PCR, and the original Fab library was expressed. The antigens were extracted from 3 sensitized colorectal cancer tissues previously used for construction of the original Fab library and from 13 non-sensitized colorectal cancer tissues, along with the antigens from LoVo, HT-29 and LS-174T cells cultured in vitro. The original Fab antibody library was screened with the 3 groups of mixed antigens derived in preceding procedure and 3 tertiary Fab antibody libraries were obtained, which were then mixed in equal volume for subsequent tests of binding activity with human colorectal cancer tissues and cells in vitro using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Specimens of gastric and esophageal carcinomas and normal intestinal mucosa, together with liver cancer cells and gastric cancer cells were utilized as control.


RESULTS
The recombination rate of Fd and kappa chain were 40 % and 70 % respectively, and the rate of their simultaneous insertion into Pcomb3 vector was 28%. The capacity of library for Fab fragment genes was 2.1x10(6), and the original antibody libraries screened with the 3 groups of mixed antigens were enriched to varied degrees, which all displayed relatively specific binding activity with human colorectal cancer tissue and cells in vitro.


CONCLUSIONS
Colorectal cancer-related antibody Fab fragments are obtained through screening phage display library, which show relatively specific binding activity with human colorectal cancer tissues and cells.
Sun
Xun
X
Institute of Digestive Diseases of PLA, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Wu
Bao-Ping
BP
Xiao
Bing
B
Zhang
Zhen-Shu
ZS
Lai
Zhuo-Sheng
ZS
D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology; D015179:Colorectal Neoplasms / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D019151:Peptide Library
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library