Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli.

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.

Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis.

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.

Generation and characterization of monoclonal antibodies to adenovirus.

Monoclonal antibodies (MoAbs) were generated following immunization of Balb/C mice with adenovirus type 5 grown in Hep2 cell line. Six clones reactive to hexon antigen of the virus were stabilized, of which 4 had mu-heavy chain specificity and 2 were of gamma-heavy chain type. Three of the clones (ADV-1, ADV-3 and ADV-5) had a high ELISA reactivity to the hexon antigen but exhibited differential specificity to the adenovirus types tested. In Western blotting, ADV-1 and ADV-3 reacted with all the adenovirus types tested (types 3,4,5,7 and 8) with reactions at 116 kDa region (hexon antigen), in addition, ADV-3 also had reactivity at 80 kDa region, the penton antigen. Reactivity to these adenoviral types by the 2 MoAbs was demonstrable by dot ELISA. ADV-5 had a type specific reaction only to adenovirus type 5 in dot ELISA with specificity in the hexon region in Western blotting. The reactivity of these 3 clones was not observed to the normal Hep2 tissue culture antigens and to the 3 enteroviruses tested (polio, coxsackie A9 and echo 4).

Use of locally isolated saprophytic Leptospira strain for serological testing of human leptospirosis.

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.

Relative efficacy of egg albumin as blocking agent in dot immunobinding assay for detection of group B coxsackieviruses.

Non specific binding (NSB) is an important factor affecting sensitivity and specificity in dot immunobinding assay (DIA). Several blocking agents e.g. egg albumin, casein, gelatin, milk powder and goat serum were evaluated for their relative efficacy vis-a-vis bovine serum albumin (BSA) for DIA system purported for detection of group B coxsackieviruses (CVB). The results suggest that egg albumin (5%) which is economical and readily available may act as an effective blocking agent in DIA system.