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

Abstract

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 (RW-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 aliowing 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

Sensitive assay for measurement of antibodies to Clostridium botulinum neurotoxins A, B, and E: use of hapten-labeled-antibody elution to isolate specific complexes

“…ELISA-ELCA for botulinum toxin. In previous reports, we have described the details of the development of the ELISA and the enzyme-linked coagulation assay (ELCA) (1,3,4,9).…”

“…These conjugates are used to prepare detectable complexes with solid phases coated with capture antibody (4) or in solution phase with affinity-purified chicken antitoxin antibody or biotinylated antibody (1) or with fluoresceinated horse antibody (3). When solution-phase complexes are formed, capture of the complex with appropriate capture matrices such as anti-chicken IgY for chicken antibodies (1) (IgY is the common term used to describe the IgG fraction which is isolated from egg yolks), avidin, or streptavidin for biotinylated antibody (1) and antifluorescein for fluoresceinated antibodies (3) is an effective method for isolating and measuring the complexes. The antibodies used in these previous studies were from horses immunized at the Fort Detrick (Frederick, Md.)…”

“…We recently developed an amplified immunoassay for botulinum toxin whose sensitivity matched the sensitivity of the mouse bioassay (1,3,4,9). We were able to demonstrate that titers of human and horse antisera could be measured by competition of binding with the labeled complexes (2).…”

Detection of C. Botulinum Types in Honey by mPCR

“…The mouse bioassay has remained the standard procedure for determining the presence of BoNTs (Hatheway and McCroskey ; FDA ). Moreover, ELISA have been developed for detecting BoNTs (Doellgast and others ). Recently, PCR‐based analysis methods for detecting the (BoNT) gene have been introduced in an attempt to replace time‐consuming conventional methods (Campbell and others ; Fach and others ; Szabo and others ; Hielm and others ; Aranda and others ).…”

“…The mouse bioassay has remained the standard procedure for determining the presence of BoNTs (Hatheway and McCroskey ; FDA ). Moreover, enzyme‐linked immunosorbent assays (ELISA) have been developed for detecting BoNTs (Doellgast and others ). Detection of BoNT gene fragments by PCR has become a rapid alternative method for the detection and typing of BoNT‐producing C. botulinum (Aranda and others ).…”