Feasibility of using polyclonal antibodies in an enzyme-linked immunosorbent assay (ELISA) to estimate bacterial spore populations in raw foods or food ingredients

Polyclonal antibodies were produced using inactivated Bacillus cereus T and Clostridium sporogenes PA 3679 spores and a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of bacterial spores. B. cereus spore antibody detected, with varying sensitivity, the spores of five species or varieties of Bacillus (B. megaterium, B. subtilis, B. stearothermophilus, B. coagulans, B. subtilis var. globigii) and one species of Clostridium spores (C. botulinum 1304E). C. sporogenes spore antibody cross-reacted with C. botulinum 41B among all Bacillus and Clostridium spores which were tested in this study. Neither spore polyclonal antibody cross-reacted with any vegetative cells of Bacillus, Clostridium or other cells (Listeria monocytogenes F5069, Salmonella typhimurium ATCC 14028, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 33625, Pseudomonas aeruginosa ATCC 27853, Lactobacillus acidophilus NCK 56 N2, Lactococcus lactis ME2). Aspergillus flavus ATCC 15547 and Penicillium atramentosum ATCC 10104 spores showed cross-reaction with both the Bacillus and Clostridium antibodies. Thus, because of immunological similarities of bacterial spores, development of a rapid and simple method for estimation of bacterial spore loads may be feasible. However, detection required about 10{dollar}sp6{dollar}-10{dollar}sp7{dollar} spores/ml.; Because the detection limit of the sandwich ELISA developed here was not sensitive enough to apply in food analysis, a biotin-avidin amplification system was evaluated to increase the sensitivity of the assay. At absorbance values of 0.25, the detectable concentration of B. cereus spores was decreased by about 0.35 log cycle or the absorbance value was increased 2- to 4-fold when compared with the prototype sandwich ELISA. Using this amplification system, B. cereus T spores were detected at concentrations above 10{dollar}sp6{dollar}spores/ml. Possible interference by food components in the biotin-avidin ELISA was checked. Gelatin and starch did not interfere with the assay. Raw milk decreased the sensitivity of the ELISA so that the spore population required for detection was 0.7 log cycle greater at an absorbance value of 0.25.; To determine the antigenic sites in the spores, immunocytochemical localization was used. Ultrathin cryosections of B. cereus T spores were labeled with rabbit anti-B. cereus T spore serum and protein A-gold conjugate, and viewed with a transmission electron microscope. Antigens were located throughout the exosporium of spores and also in the spore coat and protoplast.