ABSTRACT
BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies. RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction. INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.
Subject(s)
Animals , Anthrax Vaccines/immunology , Antigens, Bacterial , Bacillus anthracis/metabolism , Bacterial Toxins/blood , Biocompatible Materials/pharmacology , Blotting, Western , Cattle , Chromatography, Liquid , Durapatite/pharmacology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Viper Venoms/metabolismABSTRACT
Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.