After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact, the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin‐specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD50 bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of <0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype‐specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells. © 2004 Movement Disorder Society

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