Screening Specifical Antibody From Phage Display Library And The Study Of It's Growth Inhibition On Lung Adenocarcinoma Xenograft Mice

Objective: To screening specific antibodies from phage display library, and detect its efficiency. Label the antibodies with radionuclide 131I and make radio-immunoimaging study on xenograft in mice. Observe distribution of antibodies in the mice. Study against effect to lung adenocarcinoma. This study will lay the foundation for lung cancer radio-immunotherapy, and provide useful treatment to lung cancer patients.Methods and results:Part 1 Phage antibody library experienced mute solid phase screening1. Negativity screening: Normal human bronchial epithelial cells (HBE16) were used to deplete the phage library of nonspecific binders.2. Cell line screening: The phage library were screened on lung adenocarcinoma cells A549 3 rounds. Obtain the specific antibody against lung adenocarcinoma. The number of eluted phages increased after 3 rounds panning.3. Foregone antigen screening: The phage library after 3 rounds screening on A549 cells were then selected on PrxⅠ3 rounds. After these 3 rounds anti-PrxⅠwere obtained.4. Expresed soluble antibody: E. coli HB215l was infected with colonies which giving high signals against PrxⅠfor soluble expression. With IPTG. Induce cultivation, then fragments were purified, obtain the soluble antibody.5. Assess soluble antibodies: The soluble antibodies were identified by SDS–PAGE. A clear 30 ku band was observed, confirming the scFv fragments had been soluble expressed in E. coli HB215l. The soluble antibodies were analyzed by ELISA and immunocytochemistry, The result showed that the anti-PrxⅠantibodies specific purified with high affinity to A549 cells.Part 2 Study effect of anti-PrxⅠantibodies in vitro1. Internalize experiments: The anti-PrxⅠantibodies were labeled with radionuclide 131I by the chloramine T method(131I-PrxⅠantibodies). The A549 cells were incubated with labeled antibodies at 37°C. The temperature control was at 4°C. The cells were washed, then the cells were lysed. The acid washes and cell lysate radioactivity was measured on aγ–counter. The result showed that, compared with the control group, anti-PrxⅠantibodies the can combine with A549 cells and be internalized effectly.2. Effect of anti-PrxⅠantibodies in vitro: After anti-PrxⅠantibodies incubation with A549 cells 72 hs. MTT analysis showed growth inhibition to A549 cells. The apoptosis of A549 cells were analyzed by flow cytometry. The anti-PrxⅠcaused a significant apoptosis contrast the control group.3. PrxⅠprotein analysis: After 72 hs incubation with anti-PrxⅠantibodies, expression of PrxⅠprotein in A549 cells was examined with Western blot. Contrast control group, anti-PrxⅠantibodies caused a decrease in A549 cells.Part 3 Effect of anti-PrxⅠantibodies in vivo1. Text the anti-PrxⅠantibodies: 131I-PrxⅠantibodies purified by Sephadex G200 column. The labeling yield, specific activity, radiochemical purity and the stability of 131I- PrxⅠantibodies in serum were tested. The results showed that the labeling yield was up to the standard. After incubation with fresh human serum 48 hs, the radiochemical purity of the 131I–PrxⅠantibodies showed no significant depression Contrast to 1h group.2. Biodistribute of anti-PrxⅠantibodies in vivo: The xenograft mice were injected via the tail vein with 131I-PrxⅠantibodies. At various times, the tumor and organs were removed, and were weighed and counted on aγ-counter. The injected xenograft mice were fixed on boards for radio-immunoimaging analysis at various times. An index of the evaluation is percent injection dose per gram (%ID/g). The results showed that at 48 h tumor/blood and tumor/muscle values reached the peak of 4.06±0.13 and 5.17±0.97. The radioactivity was thicknessed at tumor locations and the tumor imaging was clearly showed.3. Against tumors of anti-PrxⅠantibodies in vivo: The xenograft mice were injected via the tail vein with 131I-PrxⅠa ntibodies. NS, 131I-IgG as control. One time per 2 days, and 14 times in all. Four weeks later, execute the mice. The tumors were removed, and were weighed. Calculate inhibition neoplasms efficiency was 56.8%. Pathology analysis of tumors after treatment showed that more tumor cells died, karyorrhexis and caryolysis in treat of anti-PrxⅠa ntibodies.Conclusion: We screening the anti–PrxⅠantibodies from phage display technology. Expressed soluble antibody and the specificity and proliferation ability were analyzed. Labeled the anti–PrxⅠantibodies with radionuclide. The study of the anti–PrxⅠantibodies against lung adenocarcinoma A549 cell line in votro. The result showed that the lung adenocarcinoma specificity human scFv antibodies have been prepared successfully and inhibition growth of A549 cell line. Biodistribution study and SPECT imaging showed that the anti–PrxⅠantibodies showed strong anti-proliferative effects on lung adenocarcinoma cells. The satisfactory radio-immunoimaging and specific affinity to target tumor implicates its potential application in lung adenocarcinoma imaging diagnosis and targeted therapy.