Shelf Life and Toxin Development by Clostridium botulinum during Storage of Modified-Atmosphere- Packaged Fresh Aquacultured Salmon Fillets.

Shelf life (onset of sensory spoilage) and the potential for toxin production by Clostridium botulinum type E in retail-type packages of fresh aquacultured salmon fillets packaged in high-barrier film bags under selected atmospheres (100% air, a modified atmosphere containing 75% CO2:25% N2, and vacuum) and stored under refrigeration (4°C) and temperature-abuse conditions (8 and 16°C) were investigated. Chemical spoilage indicators (trimethylamine and surface pH) and microbial populations were compared with sensory spoilage characteristics. Storage temperature influenced the time to onset of both sensory spoilage and toxin development in salmon fillets packaged in all atmospheres. The shelf life of fillets packaged in all atmospheres decreased with increase of storage temperature from 4 to 16°C. Trimethylamine content associated with the onset of spoilage for 100% air-packaged fillets increased as storage temperature increased. However, for modified-atmosphere-packaged fillets, the trimethylamine content associated with the onset of spoilage increased as storage temperature decreased from 8 to 4°C. Surface pH was not a good spoilage indicator for modified-atmosphere-packaged fillets. Toxin development preceded sensory spoilage at 16°C storage for fillets packaged in modified atmospheres. Toxin development coincided with sensory spoilage or was slightly delayed for the fillets packaged in all the atmospheres at 8°C storage. At 4°C none of the fillets packaged in either of the atmospheres developed toxin, even 20 days after spoilage as determined by sensory characteristics.

Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies.

The measurement of toxins A, B, and E from Clostridium botulinum was accomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken antibody or biotinylated horse antibody. The complexes formed in solution phase were captured onto solid phases coated with rabbit anti-chicken immunoglobulin G (chicken antibody) or avidin (biotinylated antibody). The assay was brought to the sensitivity of the mouse bioassay (5 to 10 pg/ml, or 0.03 to 0.07 pM) by employing as labeling enzyme the factor X activator of Russell's viper venom (RVV-XA) and a sensitive coagulation-based assay amplification system known as enzyme-linked coagulation assay. Complex formation was found to be a slower reaction than binding to the capture plate, and so the assay used a preincubation step to produce the solution-phase complexes before they were bound to the solid phase. Keeping the concentrations of Russell's viper venom factor X activator antibody and capture antibody constant for diluted samples and diluting complexes into buffer without keeping labeled antibody concentrations constant were equivalent in allowing the detection of low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, which shows complete resolution of the neurotoxins in addition to the requisite sensitivity.

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.