Hemi-Diaphragm Assay (HDA) for detection and quantification of botulinum neurotoxins (BoNT)

The Hemi-Diaphragm Assay (HDA) is an ex vivo assay using phrenical hemidiaphragm preparations from mice for detection and quantification of botulinum neurotoxins (BoNT).

BoNT inhibit the release of acetylcholine from peripheral nerve endings and this inhibition can be detected in isolated nerve-muscle preparations, e.g. phrenical hemidiaphragm preparations. The tissue preparation is maintained in an organ bath. Via ring electrodes the nerve is stimulated by an electric pulse followed by muscle contraction. The contraction amplitude is recorded and decreases when BoNT acts in the nerve endings. The time period between application of toxin and half maximum contraction amplitude (inflection point) is read out parameter. The variants of BoNT can be identified by specific antibodies.

The submitter claims that the HDA test is applicable to analyse food and environmental samples for contamination with BoNT, human or veterinarian samples (blood and stool) in case of suspicious intoxications (e.g. bioterrorism), to test cosmetic and pharmaceutical products containing BoNT for batch release, to analyse human sera for specific anti-BoNT antibodies that may appear after treatment with BoNT and the test is a valuable tool in research. In many cases, mice are used for this purpose, developing Botulism disease in the presence of BoNT and subsequently die due to respiratory failure.

The phrenical hemidiaphragm preparations are derived from humanely killed mice. Apart from the refinement aspect, the submitter claims that the HDA has reduction potential.