Tuesday, 07 May 2019 14:42

Animal-free Botulinum toxin diagnostics possible Featured

For the diagnosis of botulinum toxin poisoning in clinical samples and food, the Robert Koch Institute in Berlin has presented an alternative method for the test on mice.

The LD50 test with mice for batch testing of the wrinkle remover botulinum toxin (Botox) is well-known. For batch tests of each of their products, various manufacturers such as Allergan, Ipsen or Merz have developed a replacement method with cells, which is also approved by the authorities. However, it is less well known that the painful mouse test, in which samples are injected into the abdominal cavity of mice making that the animals have to suffer from respiratory paralysis, has also been used so far to diagnose food poisoning. The Robert Koch Institute has just presented an animal-free procedure to replace this mouse bioassay.

It is based on the fact that the toxin of the bacterium Clostridium botulinum and other related bacteria cuts certain target proteins substrates SNAP-25 or VAMP-2) in the synapse of the nerve cell into two parts. This prevents the fusion of neurotransmitter-filled transport vesicles with the synaptic membrane thereby preventing the release of the neurotransmitter. Seven different toxin serotypes are known, named A-F and recently also a serotype H, which might play a role in botulinum poisoning. They cut the target protein at different sites so that they become distinguishable.

The RKI researchers have developed special antibodies with which the individual fragments  cut by serotypes A to F can be recognised with high specificity. Intact synaptic target proteins are not considered. The test can detect all common botulinum toxin serotypes. The method is designed to test clinical patient as well as food samples for botulinum toxins.

The researchers are in good society with their development. Several scientists are developing an in vitro botulinum test method. Prof Gerhard Püschel from the University of Potsdam for instance presented a method for botulinum detection in food in 2015. However, he used cells for this purpose. Living systems are expensive and need care. The RKI method uses antibodies on a single platform. Such a procedure is easy to maintain and can be implemented on a large scale.

The scientists presented their test in the Journal Scientific Reports.

Laura von Berg, Daniel Stern, Diana Pauly, Stefan Mahrhold, Jasmin Weisemann, Lisa Jentsch, Eva-Maria Hansbauer, Christian Müller, Marc A. Avondet, Andreas Rummel, Martin B. Dorner & Brigitte G. Dorner (2019). Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology. Scientific Reports 9: 5531. https://www.nature.com/articles/s41598-019-41722-z

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