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NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research

Evaluating a microfluidic assay for botulinum neurotoxin testing

We have funded a collaboration between Dr Greg Stevens and eight partners including academics, small companies and government organisations to validate an in vitro test for detecting botulinum neurotoxin.

Research details

Solution provider: Dr Greg Stevens
Organisation: Albert Ludwig University of Freiburg, Germany
Start date: 2016
Duration: 12 months
Amount: £30k
Project partner: toxogen, Germany

Case study

The Botulinum neurotoxin LabDisk-Test (BLD-Test) was developed by Greg at Albert Ludwig University of Freiburg. It is a microfluidic assay for detecting botulinum neurotoxin (BoNT), a potent toxin, produced by the bacterium Clostridium botulinum, which inhibits neurotransmitter release resulting in potentially fatal paralysis. The BLD-Test detects bioluminescent or fluorescent reporter molecules that are cleaved from naturally occurring substrates of BoNT by the enzymatic activity of the toxin, with the intensity of the reporter signal reflecting the amount of BoNT present in the test sample.

The BLD-Test is automated and can detect the six major botulism-causing BoNT serotypes including in human sera, animal, foodstuff and environmental samples. It has the potential to replace the mouse bioassay currently used, which can cause significant suffering. However, the BLD-Test requires validation to a standard where end-users and regulators can have confidence in the assay’s ability to reliably and reproducibly detect, distinguish and quantify BoNT contaminants in complex biological samples. Working with eight partners, including four from Germany – toxogen, QIAGEN Lake Constance, the research institute Hahn-Schickard and public health Robert Koch Institute – as well as the UK National Institute for Biological Standards and Control, Greg characterised and validated the performance of the BLD-Test. In an international ring trial of the BLD-Test, five participating laboratories could identify BoNT-containing samples (without serotyping) in different concentrations in serum, bean juice and carrot juice in only three hours compared to up to 24 hours for the mouse bioassay.

Around 600,000 mice are used annually for screening and serotyping BoNT specimens, and for assessing the safety of botulinum products for therapeutic purposes. Applying the BLD-Test could substantially reduce the number of mice used for diagnostics. Based on the findings of the ring trial Greg, Hahn-Schickard and toxogen continue to collaborate to further improve the sensitivity, specificity and stability of the BLD-Test, with the aim of marketing it as an alternative to the mouse bioassay. The BLD-Test is protected by European and US patents.

In-kind contributions

A range of in-kind contributions were provided. This included: the production of assay components and tests in the ring trial from toxogen; Hahn-Schickard enabled the microfluidic implementation of the assay; QIAGEN Lake Constance provided processing devices; tests in the ring trial were carried out by five partners, including Robert Koch Institute and National Institute for Biological Standards and Control.

This case study was published in our 2019 CRACK IT Review.