Generation And Characterization Of Monoclonal Antibodies Against Bacillus Anthracis PA₁₅ And Establishment Of AC-ELISA Detection System

Anthrax is an acute lethal infectious disease caused by Bacillus anthracis. Because of higher mortality rate of anthrax and their spores have strong ability to survive in harsh environments, Bacillus anthracis has served as a biological warfare agent used in the manufacture of biological weapons and biological terrorist attacks. Early in infection, there is no obvious specificity symptomst, thereby making the diagnosis is more difficult. Currently, treatment of anthrax is mainly dependent on penicillin and symptomatic treatment.Anthrax toxin is composed of three protein components:protective antigen (PA), lethal factor (LF) and edema factor (EF). PA combined with either LF or EF, constitutes lethal toxin (LeTx) or edema toxin (EdTx), respectively. PA plays a critical role in the pathogenic process of anthrax. Hence, the preparation of PA is essential for anthrax-related researches such as developing antitoxic agents. Besides, PA is the most immunogenic factor of the three proteins and all licensed anthrax vaccines or vaccine candidates in development rely on PA as the main antigenic component currently. The monomeric PA has four domains, domain4is a carboxy-terminal receptor-binding domain and the most neutralizing epitope of PA. We provided an effective method to purify the soluble of rPA63with the natural activity, and developed a mAb against rPA63by hybridoma technique. Data obtained using clinical samples showed that protective antigen could be detected by AC ELISA detection system using the3D7Mab. ObjectivesTo obtain the anthrax protective antigen in E.coli, prepare its monoclonal antibody and establish an antigen capture ELISA (AC ELISA) detection system and test the efficiency by using clinical samples.Methods1. To construct prokaryotic recombinant vector, expression and purification of Bacillus anthracis PA63in E.coli and analysis of biological activity of rPA63.2. To produce antibodies against PA with hybridoma technology, and analysis the characterization of a mAb against rPA63-3. The MAb, whose titer was higher for antigen, was used to establish an antigen capture ELISA (AC ELISA) detection system and test the efficiency by using clinical samples.Results1. Prokaryotic recombinant vector pCold II-PA63was constructed and transformed. rPA63was purified by FPLC of His-trap HP. The soluble rPA63showed natural biological activities in vitro.2. A cell line named3D7, which could secrete monoclonal antibody against PA15was selected. Anti-PA15monoclonal antibody was purified, which the titration is about1:12800.3. Data obtained using clinical samples showed that protective antigen could be detected by AC ELISA detection system using the3C7Mab.ConclusionsWe have developed murine monoclonal antibody against PA. It was potentially useful for the further development of highly sensitive, easily handled, and relatively" rapid detection kits/tools for anthrax surveillance in those areas where Bacillus anthracis is endemic.