Enhanced Production of Recombinant Extractable Antigen (EA1) an Extracellular Protein and its use in Detection of Spores of Bacillus anthracis the Causative Agent of Anthrax

Abstract

Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in environment is cumbersome due to the presence of spores of other closely related Bacillus species. The Extractable Antigen 1 (EA1), an extracellular protein is considered as a biomarker for detection of B. anthracis spores. In the present work, we have cloned and expressed the recombinant EA1 protein in soluble form in Escherichia coli. Optimisation of culture conditions and cultivation media was carried out to achieve enhanced soluble expression of recombinant EA1 protein. Further, the batch fermentation process was also developed using optimised conditions for scale up production of recombinant EA1 protein. The final yield of protein purified employing affinity chromatography was 42.64 mg/l of culture during batch fermentation process. The polyclonal antibodies were raised against recombinant EA in rabbit and mice and used to develop an ELISA for detection of B. anthracis spores. The specificity of the developed assay was ascertained with spores of other Bacillus species. The results corroborated that the EA1 could be a suitable biomarker for detection of B. anthracis spores.

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

“…A sandwich ELISA was developed for detection of PA as described earlier 32 . Briefly, ninety-six-well ELISA plates were coated overnight with purified rabbit anti-PA antibodies (200 ng/well) in carbonate-bicarbonate coating buffer (0.06 M Na 2 CO 3 and 0.14 M NaHCO 3 ), pH 9.5 at 4 °C.…”