ENZYME-LINKED-IMMUNOSORBENT-ASSAY AND ENZYME-LINKED COAGULATION ASSAYFOR DETECTION OF CLOSTRIDIUM-BOTULINUM NEUROTOXIN-A, NEUROTOXIN-B, AND NEUROTOXIN-E AND SOLUTION-PHASE COMPLEXES WITH DUAL-LABEL ANTIBODIES

The measurement of toxins A, B, and E from Clostridium botulinum was a ccomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken anti body 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 fact or X activator of Russell's viper venom (RVV-XA) and a sensitive coagu lation-based assay amplification system known as enzyme-linked coagula tion assay. Complex formation was found to be a slower reaction than b inding to the capture plate, and so the assay used a preincubation ste p to produce the solution-phase complexes before they were bound to th e solid phase. Keeping the concentrations of Russell's viper venom fac tor X activator antibody and capture antibody constant for diluted sam ples and diluting complexes into buffer without keeping labeled antibo dy concentrations constant were equivalent in allowing the detection o f low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, whi ch shows complete resolution of the neurotoxins in addition to the req uisite sensitivity.