Ambystoma mexicanum/immunology* ; Amputation ; Animals ; Biomarkers/metabolism* ; Blastomeres/immunology* ; Cell Lineage/immunology* ; Connective Tissue Cells/immunology* ; Epithelial Cells/immunology* ; Forelimb ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Immunity ; Peroxiredoxins/immunology* ; Regeneration/immunology* ; Regenerative Medicine/methods* ; Single-Cell Analysis/methods*

OBJECTIVETo construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin I (Prx I) of lung adenocarcinoma.

METHODSThe 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.

RESULTSA 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.

CONCLUSIONScFv 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.

Adenocarcinoma ; immunology ; pathology ; Antibodies, Neoplasm ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cell Line, Tumor ; Humans ; Immunoglobulin Variable Region ; immunology ; Lung Neoplasms ; immunology ; pathology ; Peptide Library ; Peroxiredoxins ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; immunology