Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test

Abstract

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified BoNT/D under nonreducing (lane 2) and reducing (with dithiothreitol [DTT] treatment) (lane 3) conditions, which separate the heavy and light chains. kD, kilodaltons.

FIG. 2.

Disposable immunoaffinity assay column (65 mm by 7 mm). Three sintered polyethylene disks are at the bottom of the column. The assay disk in the middle is coated with the capture reagent (capture antibodies).

FIG. 3.

Assay setup. The sample is preincubated with monoclonal anti-BoNT/C and D and biotinylated polyclonal anti-BoNT/C and D (anti-BoNT/C+D) antibodies. During preincubation, a complex with BoNT is formed, which is captured by the anti-mouse IgG coated on the assay disk of the column. Streptavidin-poly-HRP binds to the biotinylated antibody and precipitates the substrate on the assay disk.

FIG. 4.

Variations of antibody concentrations and incubation times and their effect on the sensitivity and assay performance for the detection of BoNT/C and D. The best assay sensitivity is achieved with 0.3 μg ml⁻¹ monoclonal antibodies and 0.1 μg ml⁻¹ biotinylated polyclonal antibodies for BoNT/C and D. With BoNT/D, 4-h incubations showed the highest sensitivity; with BoNT/C, 16-h incubations showed the highest sensitivity.

FIG. 5.

Dilution of standardized BoNT/D culture supernatant in the optimized assay (toxicity is given as MLD per milliliter). Note the slight background in the negative (neg.) control.

FIG. 6.

Assay specificity tested with C. botulinum and C. tetani culture supernatants. BoNT/C and D cultures gave positive results. The other culture supernatants were below the assay cutoff.