Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F

Abstract

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.

Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection

“…7C, D, and A, respectively). These data agree with previous findings by Poli and coworkers [6] and Szilagyi and coworkers [7]. We also observed that this recognition was different for serotype E (Fig.…”

“…One problem with such assays is the susceptibility of the assay substrates to nonrelated proteases and the consequent risk of false positives. A variety of antibody-based assays that are both sensitive and serotype specific have been proposed [5][6][7][8]. A drawback with all immunoassays is their inability to distinguish biologically active toxin or toxin fragments from biologically inactive ones.…”

“…It is important to evaluate any assay against the complexed forms as well because these are most likely to be found in real intoxication situations. It is difficult to accurately compare both forms on a mass unit basis because the native pure form will be more toxic per unit of protein than will the complex form [6,7]. The relationship of toxicity to protein toxin concentration was determined by the manufacturer and noted in the certificate of analysis provided with each serotype as described in Materials and methods.…”

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…The experiment usually takes 5-6 hours to complete, and is significantly faster than mouse lethality assay. ELISAs with an experimental setup analogous to this were reported to have a wide range of sensitivities, depending on the specific antibody and reporter system used, and have been employed for detection of all BoNT serotypes [48,49,50,51]. …”

“…ELISA methodologies have been successfully employed in the detection and quantification of purified botulinum toxin [49,51,59], in C. botulinum cultures that produce the toxin [60,61,62], in an extensive variety of food samples [52,54,63,64,65] (both contaminated food and food artificially spiked with the toxin), and in clinical samples such as serum [49] and feces [66]. Some foods tend to interfere with ELISAs and decrease their sensitivity; therefore, results should be confirmed by mouse lethality assay.…”