Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies

Abstract

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

FIG. 1.

Standard curve for the four botulinum neurotoxin complex serotypes in casein buffer. (A) Standard curve for BoNT/A, BoNT/B, BoNT/E, and BoNT/F. Points represent the mean absorbance values of duplicate wells. Where not visible, the error bars are within the symbol. (B) Relationship between mouse LD₅₀ and concentration.

FIG. 2.

The ELISA determinations of 2 ng/ml BoNT/A (A) and BoNT/B (B) complexes in food samples. The samples were spiked with neurotoxin complex and incubated at 30 min at room temperature. The absorbance of the negative food sample controls was subtracted from the positive food sample absorbance. The toxin extraction procedures are given in Materials and Methods.

FIG. 3.

The ELISA determinations of 2 ng/ml BoNT/E (A) and BoNT/F (B) complexes in food samples. The samples were spiked with neurotoxin complex and incubated for 30 min at room temperature. The absorbance of the negative food sample controls was subtracted from the positive food sample absorbance. The toxin extraction procedures are given in Materials and Methods.