Production and characterization of monoclonal antibodies against the Acinetobacter baumannii antigens NcsP, TonB-R, Pase1 and Pase2

• Main Authors: Pei-Ning Li, 李珮寧
• Other Authors: Chiou-Ying Yang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/37851650967377267273

碩士 === 國立中興大學 === 分子生物學研究所 === 104 === Acinetobacter baumannii, is an opportunistic pathogen that can easily survive in different environments. It may cause respiratory infection, urinary tract infection, wound infection, even can cause bacteremia and lead to death. The leading way to treat A. baumannii infection now is using antibiotics. However A. baumannii can easily acquire antibiotic resistant genes, and pan-drug resistant A. baumannii has emerged. In this study I tried to produce and characterize monoclonal antibodies (mAbs) against four A. baumannii proteins, NcsP, TonB-R, Pase1, Pase2, the protective antigens identified in our laboratory. I got 6 clones applying hybridoma technique, selection by ELISA and Western blot and I named the mAbs N1, P1a, P1b, P1c, P2, TPP. N1 recognizes NcsP; P1a, P1b, P1c recognize Pase1; P2 recognizes Pase2; and TPP recognizes TonB-R, Pase1, Pase2 at the same time. All 6 antibodies are IgG1 isotype. To differentiate the binding site of the three Pase1 antibodies, competition ELISA was carried out. The results showed that P1b and P1c can compete with each other, implicate they may recognize the same epitope. P1a and P1b or P1a and P1c showed no competing on each group. Sequencing of VH and VL regions of mAbs, P1b and P1c exhibited high identity. Next, in vivo and in vitro assay were carried out to test the efficacy of 6 mAbs. To test in vitro efficacy, I mixed TPP or N1 with A. baumannii, incubated for 4 hours and counted the number of colonies. Compared with negative control, N1 and TPP groups displayed no significant decrease of the colony count. Then I mixed N1 with pre-immune serum to test if complement could coordinate with N1 to kill A. baumannii. The results showed whether the complement was inactivated or not there was no significant difference. Passive protective assay was carried out to test in vivo efficacy of 6 mAbs. Mice were injected with mAbs from tail vein 2 hours prior to inoculate intraperitoneally (i.p.) with A. baumannii. The survival rate showed no significant difference between experimental and control groups. Compared bacterial burdens in blood, lung and spleen, there were no significant difference between experimental and control groups. Finally, I injected mice with a mixture of P1a, P1c, P2, N1, TPP, and inoculate i.p. with A. baumannii. There were also no significant difference between experimental and control groups in blood, spleen and lung. In conclusion, 6 mAbs did not display good efficacy either in vivo or in vitro, but they can apply to study and detect target proteins of A. baumannii.