IMMUNOLOGICAL DETECTION OF CLOSTRIDIUM-BOTULINUM TOXIN TYPE-A IN THERAPEUTIC PREPARATIONS

The potent neurotoxins produced by strains of Clostrudium botulinum ac t by blocking the release of acetylcholine from peripheral nerve junct ions. This specific action of the botulinum neurotoxins is now being e xploited therapeutically to treat a variety of conditions involving in voluntary muscle spasms. We aimed to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA) which may be used in paralle l with the currently accepted mouse bioassay test for the determinatio n of botulinum neurotoxin type A in therapeutic preparations. High tit re polyclonal antitoxins and their biotin derivatives, highly labelled horseradish peroxidase (HRP)-antibody conjugates, and streptavidin-bi otin-HRP complexes were prepared and used in a sandwich ELISA for the detection of pure neurotoxin and neurotoxin in therapeutic material. T he ELISA utilized either a monoclonal or polyclonal antibody as captur e agent and HRP-labelled IgG or F(ab')(2) fragment as second antibody. The limit of detection was 4-8 pg of purified toxin/ml (gcv < 13%), e quivalent to 1-2 mouse bioassay units/ml. The assay was used to evalua te therapeutic preparations and the results compared with the mouse bi oassay. The lower limit of detection for a therapeutic preparation of BoTxA was 2-5 mouse bioassay units/ml. Although across different manuf acturers and bulk products there was no correlation between immunologi cally detected neurotoxin and its biological activity in different the rapeutic preparations (r = -0.44, p = 0.34, n = 8), the assay could be used to quantify neurotoxin in therapeutic preparations derived from the same bulk concentrate and manufacturer. The assay is relatively si mple, and may be readily adapted to routine monitoring of BoTxA conten t in therapeutic preparations.