Abstract:

Abstract:Objective
To sieve out the fully human,Peroxiredoxin Ⅰ (Prx Ⅰ) -specific single chain variable fragment (scFv) antibodies from the lung adenocarcinoma-related phage antibody library and analysis the biodistribution of the antibodies in vivo. Methods The insert ratio of scFv antibodies library was identified by polymerase chain reaction (PCR) and the products digested by Sfi Ⅰ/ Not Ⅰ were analyzed on 1% agarose gel. Panning against lung adenocarcinoma cell line A549 and Prx Ⅰ were performed three rounds,separately. The positive clones were chosen for soluble expression and analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE),enzyme-linked immunosorbent assay (ELISA) and competition ELISA. The tumor-beared athymic mice were injected via the tail vein with purified 131I-labeled scFv. The biodistribution analysis and radioimmunoimaging were taken to evaluate the specificity and distribution of antibodies in vivo. Results The ratio of recombinant bacteria inserted with scFv was 77% (23/30) and the enzyme digest reaction showed the aim products on 1% agarose gel. From the first to the sixth round of panning,the obtained phages number increased 180 times. The positive reactions to A549 cells were detected in 6 of 10 clones. The positive ratio was 60% (6/10). The human scFv vs  against Prx Ⅰ of lung adenocarcinoma were confirmed by SDS-PAGE and ELISA. Competition ELISA showed significant,concentration-dependent affinity of scFv antibodies to A549 cells. The radiochemical purity of 131I-labeled scFv was (95.6±3.7)% and the specific activity was (2.8±0.2) TBq/g. The radiolocalization indexes (RI) of tumor/serum and tumor/muscle were gradually increased,reaching its peak (4.06±0.13 and 5.17±0.97,respectively) at 48 h postadministration. The radioactivity was aggregated in tumor locations and the tumor imaging was clearly observed by single photon emission computed tomography (SPECT) imaging. The T/NT value at 48 h (3.73±0.20) was significantly higher than those at 12 h (1.26±0.15),24 h (2.18±0.16) and 72 h (2.85±0.18) (all P<0.01). Conclusion The scFv fragments against Prx Ⅰ of lung adenocarcinoma are acquired by screening the phage antibody library successfully. The soluble scFv antibodies have bonding avidity specifically in vivo.

Key words: phage antibody library;single chain variable fragment;Peroxiredoxin Ⅰ;radioimmunoimaging;adenomatosis,pulmonary