Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

ABSTRACT

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.