Detection of Botulinal Neurotoxins A, B, E, and F by Amplified Enzyme-Linked Immunosorbent Assay: Collaborative Study

Abstract

An amplified enzyme-linked immunosorbent assay (amp-ELISA) was compared with the AOAC Official Method 977.26 for detection of Clostridium botulinum and its toxins in foods. Eleven laboratories participated and the results of 10 laboratories were used in the study. Two anaerobic culture media, tryptone peptone glucose yeast extract (TPGY) and cooked meat medium (CMM) were used to generate toxic samples with types A, B, E, and F botulinal strains. Nonbotulinal clostridia were also tested. The toxicity of each botulinal culture was determined by the AOAC method, and the cultures were then diluted, if necessary, to high (about 10 000 minimal lethal dose [MLD]/mL) and low (about 100 MLD/mL) test samples. The overall sensitivity of detection in TPGY and CMM cultures with the amp-ELISA was 94.7% at about 100 MLD/mL and 99.6% for samples with ≥10 000 MLD/mL toxicity. The amp-ELISA detection sensitivity for low toxin samples was 92.3% in TPGY and 99.4% in CMM. The false-positive rate ranged from 1.5% for type A to 28.6% for type F in TPGY, and from 2.4% for type A to 11.4% for type F in CMM. Most of the cross-reactivity was due to detection of other botulinal types, especially in high toxin samples. The amp-ELISA could be used to screen suspect cultures for botulinal toxins. Positive amp-ELISA samples would be confirmed by the AOAC reference method.

Increased Internal and External Bacterial Load during Drosophila Aging without Life-Span Trade-Off

“…The values obtained for the six flies were averaged, and standard deviations are indicated in the figures. Nutrient agar was used for aerobic culture media, and TPGY (tryptone peptone glucose yeast extract) was used for anaerobic culture media (Ferreira et al, 2003). For bacterial counts inside flies, each fly used above was surface sterilized by immersion in…”

DNA vaccines targeting heavy chain C-terminal fragments ofClostridium botulinumneurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge

“…The coated plates were absorbed with the respective purified botulinum neurotoxin and the non-specific binding sites were pre-blocked according to established methods. 32 Plates were incubated with serial dilutions of immunized and na€ ıve sera (37 C, 1.5 h). Plates were washed with PBS-T (0.05% Tween 20 in PBS) and incubated with goat antimouse IgG-HRP (Santa Cruz Biotechnology, Inc.., sc-2055) at a 1:5000 dilution in 1% FBS in PBS-T (0.05% Tween 20 in PBS).…”

Monoclonal antibodies and reagents for botulinum research

“…Detection of BoNT relies on use of the 'gold standard' mouse bioassay, a highly sensitive method with detection limits of 10-20 pg/mL (Ferreira et al, 2003;Wictome et al, 1999). However, the mouse bioassay is time consuming (up to four days), requires the use of large numbers of animals, has virtually no dynamic range, and relies on death as an endpoint.…”