[Preparation and characterization of human phage display antibody against peroxiredoxin I of lung adenocarcinoma].
OBJECTIVE
To construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin I (Prx I) of lung adenocarcinoma.


METHODS
The total RNA was isolated from the lymph nodes of lung cancer patients to amplify V(H) and V(L) genes by RT-PCR. V(H) and V(L) were linked with a DNA linker by SOE-PCR to construct the single chain variable fragment gene. The ScFvs were coloned into the phage vector pCANTAB5E. The insert ratio of the ScFv antibody library was identified by PCR, and the products were digested by SfiI/NotI and analyzed with 1% agarose gel electrophoresis. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed, and the positive clones were identified for soluble expression. The soluble antibodies were identified by SDS-PAGE and Western blotting, and ELISA and immunocytochemistry were used to characterize the activity of the antibodies.


RESULTS
A recombination phage antibody library was constructed. The insert ratio of ScFv gene was 77% (23/30), and enzyme digestion identified the target product. The sixth phage harvest resulted in a yield 180 folds of that of the first one. Positive reactions to A549 cells were detected in 6 of 10 random clones, with a positivity rate of 60%. The soluble human ScFvs against Prx I of lung adenocarcinoma were expressed in E. coli HB2151 and confirmed by SDS-PAGE and Western blotting. ELISA and immunocytochemistry demonstrated a relative specific affinity of the soluble antibodies to A549 cells.


CONCLUSIONS
ScFv antibodies against lung adenocarcinoma have been acquired by phage display antibody library technique, and the soluble antibodies have a relative avidity specific to human lung adenocarcinoma A549 cells overexpressing PrxI.
Luo
Yi
Y
Department of Radiation Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. littleroy@163.com
Pang
Hua
H
Li
Shu-jie
SJ
Cao
Hui
H
Li
Shao-lin
SL
Fan
Chun-bo
CB
Wang
Jie
J
D000230:Adenocarcinoma / Q000276:immunology / Q000473:pathology; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008175:Lung Neoplasms / Q000276:immunology* / Q000473:pathology; D019151:Peptide Library; D054464:Peroxiredoxins / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000912:Antibodies, Neoplasm; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; D054464:Peroxiredoxins