A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

Abstract

Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health importance. It is imperative to develop a simple system which can detect and differentiate B. anthracis from other closely related species. The surface array protein (Sap), which is secreted during the early growth phase of bacteria can be an important biomarker for detection of B. anthracis. In the present study, we have developed a rapid flow through membrane ELISA for detection of B. anthracis. Polyclonal antibodies were used to develop a sandwich plate ELISA, which could detect 3.9 ng/ml of recombinant Sap. B. anthracis bacteria grown in culture broth could be detected after 5 h of growth. Finally, a rapid flow through membrane ELISA was developed which can be accomplished just within 2 minutes, instead of 3-4 h as required in sandwich plate ELISA. The results established that the developed flow through membrane ELISA may be used for detection of B. anthracis. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture. 2. Walsh, J.J.; Pesik, N.; Quinn, C.P.; urdaneta, V.;Dykewicz, C.A.; Boyer, A.E., et al. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. clin. Infect. Dis., 2007, 44(7), 968-971. 7. Jimenez, G.; urdiain, M.; Cifuentes, A.; Lopez-Lopez, A.; Blanch, A.R.; Tamames, J., et al. Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations. syst. appl.

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

“…New Zealand white rabbits and BALB/c mice were used for production of anti-PA polyclonal antibodies as described elsewhere 30 . Final bleeding of rabbit and mice was carried out on day 60.…”

“…The purified PA was run on 12 % SDS-PAGE and transferred on PVDF membrane and incubated with 1:2000 dilution of anti-PA mouse or rabbit serum as described earlier 30 . The membranes were incubated with anti-mouse or anti-rabbit IgG HRP conjugate (1:10000) for 1 h at 37 °C, and developed with 3,3′-Diaminobenzidine/H 2 O 2 substrate solution for 2 min at room temperature.…”

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

“…Detection of B. anthracis spores is established by employing various methods including culturing followed by biochemical characterisation, immunofluorescence 13 , ELISA and other immunological systems [14][15][16][17] , PCR 18 , and surface plasmon resonance 19 . However, these methods are time consuming, laborious and require sophisticated laboratory and infrastructure.…”