Detection of antibodies against botulinum toxins

Abstract

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 LD(50) 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.

Long-term efficacy and safety of incobotulinumtoxinA injections in patients with cervical dystonia

“…The other samples taken during the study were only tested if the patient had a positive FIA result at the trial termination visit. As the FIA cannot discriminate between neutralising and non-neutralising antibodies, FIA-positive samples were subsequently tested using the mouse hemidiaphragm assay (HDA) 23 24. The Investigator Global Assessment of Tolerability for each treatment cycle was rated on a 4-point Likert scale ranging from 1 (very good) to 4 (poor) at injection visits 2–5 or the trial termination visit.…”

Sustained efficacy and safety of repeated incobotulinumtoxinA (Xeomin®) injections in blepharospasm

“…Blood samples for laboratory tests and determination of antibodies against botulinum toxin were collected at all injection visits and the TTV. Samples were initially screened for botulinum neurotoxin antibodies using a fluorescence immunoassay (FIA); as the FIA cannot discriminate between neutralizing and non-neutralizing antibodies, positive FIA samples were subsequently tested using a mouse hemidiaphragm assay (HDA) (Göschel et al 1997; Sesardic et al 2004). …”

Bioassays for evaluation of medical products derived from bacterial toxins

“…against tetanus or botulism) are designed to confirm functional neutralising activity by measuring the ability of the antitoxin to Bioassays for evaluation of medical products Sesardic 311 protect animals from the specific toxic effects [16]. The assay comprises the mixing of fixed predetermined concentration of specific toxin with a graded concentration of a test and a reference antitoxin sample and injecting into animals to observe toxin-specific effect.…”