Monoclonal antibodies for use in detection of Bacillus and Clostridium spores

Abstract

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C. sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at ؊20؇C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.

Food Sensing: Detection of Bacillus cereus Spores in Dairy Products

“…Quinlan and Foegeding used monoclonal antibodies for spore isotyping [90]. Antibody B183 and B48 reacted very strongly with B. cereus spores, but a light reactivity was also detected for B. subtilis subsp.…”

Identification of Bacillus Spores by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry

“…DNAand RNA-based characterization also requires complex sample preparation. More rapid antibody-based analytical methods have been described, and these methods identify the genus Bacillus; however, they do not distinguish Bacillus spores at the species level (12). Since the genus Bacillus contains Bacillus thuringiensis, an industrially important nonpathogenic pesticide, Bacillus cereus, a noninfectious food pathogen, and Bacillus anthracis, a lethal infectious pathogenic bacterium, rapid discrimination of the spores from each other is necessary for effective intervention and treatment of human disease.…”

Construction and Expression of a Bifunctional Single-Chain Antibody againstBacillus cereusSpores

“…Hybridomas, bacterial spores, and cells. Hybridoma cell lines that produce the monoclonal antibody against B. cereus T spores were previously screened in our laboratory (35). The myeloma cell line X63-Ag8.653 (20) was used as the fusion partner.…”

“…Two functional parameters of the single-chain antibody were checked by a dot blot immunoassay method. The hybridoma cell culture supernatant containing parent native monoclonal antibody (35) was used as a control.…”