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

Nidhi Puranik; Manoj Kumar; Nagesh K Tripathi; Vijai Pal; Ajay Goel

doi:10.14429/dlsj.5.15591

Nidhi Puranik DRDO-Defence Research and Development Establishment, Gwalior - 474 002, India https://orcid.org/0000-0003-3306-058X
Manoj Kumar DRDO-Defence Research and Development Establishment, Gwalior - 474 002, India https://orcid.org/0000-0003-0312-0676
Nagesh K Tripathi DRDO-Defence Research and Development Establishment, Gwalior - 474 002, India https://orcid.org/0000-0003-3533-3262
Vijai Pal DRDO-Defence Research and Development Establishment, Gwalior - 474 002, India https://orcid.org/0000-0002-8955-9286
Ajay Goel DRDO-Defence Research and Development Establishment, Gwalior - 474 002, India https://orcid.org/0000-0001-7930-6974

DOI: https://doi.org/10.14429/dlsj.5.15591

Keywords: Bacillus anthracis, Protective antigen, purification, ELISA

Abstract

Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purified and used for the development of a sandwich ELISA for detection of PA. The detection sensitivity of ELISA was found to be 3.9 ng/ mL PA.