Portable bioluminescence test for botulinum neurotoxin

Summary | Background | Current state of the art |What could your solution be used for? Collaboration | 3Rs impact

This Solution received CRACK IT Solutions funding. For further information about the project and impacts see our science pages


Summary

The BLB-Test is a portable, automated assay for detecting BoNT as a replacement for the MLB. The BLB-Test is integrated into a microfluidic platform that is controlled automatically by a battery or mains-powered reader for use at point of need. The microfluidic disk contains all the reagents for the assay, and programmed operation of the BLB-Test is done from a laptop. The assay can be performed in half an hour, or longer if greater sensitivity is required. It can distinguish and quantify all the known disease causing BoNT serotypes in buffer and complex media.


Background

Botulism is a potentially fatal paralysis caused by BoNT which blocks neurotransmitter release. With an estimated LD50 of 1 ng/kg body weight, the family of eight BoNT serotypes, subdivided into a total of more than 40 subtypes, are the most toxic naturally occurring substances known.

The BLB-Test detects the endopeptidase activity of the light chain domain of BoNT, thereby detecting only active BoNT and avoiding potential false positives caused by inactive BoNT. The assay [1], consists of immobilized polypeptides that tether a reporter molecule. The polypeptides are cleaved by BoNT, releasing the reporter molecules which are detected by an optical readout. The BLB-Test can detect and distinguish all of the disease-causing BoNT serotypes: A, B, C, D, E and F, as well as mixed types H/F5, A/B, A/F, B/F, B/H, and the closely related tetanus neurotoxin (TeNT).

The polypeptides used in the BLB-Test are based on the naturally occurring substrates of BoNT that form the SNARE complex of neurons. Different BoNT serotypes have specific cleaveage sites on the SNARE complex, and so modifications in the amino acid sequences of these polypeptides allow the various serotypes to be distinguished. Cross reactivity with untargeted serotypes is negligible, thereby providing highly specific detection required for targeted therapy of botulism patients.

BoNT detection with the BLB-Test is automated and can be performed with minimal training/expertise: liquid samples or extractions from solids are dispensed into a microfluidic disk which is then loaded into the disk reader [2]. During operation, a metered amount of sample is distributed to each reaction chamber where it is analysed after an incubation period depending on the required sensitivity. After analysis the single use disk containing the specimens can be safely destroyed.


Current state of the art

The most widely accepted method for BoNT detection is the MLB [3]. This has a sensitivity, depending upon the BoNT type, of 10-100 pg/mL [3].  In this assay, mice are injected intraperitoneally with a solution that is suspected to contain BoNT. Observation of symptoms such as laboured breathing, weakness of limbs, and finally death due to asphyxiation are required to confirm the presence of BoNT. The MLB requires a long readout time of 1-4 days; uses a large number of mice (> 100) for serotyping and precise quantitation; and the toxicity depends on the age and strain of mice used, leading to variability in sensitivity [3].  To overcome these limitations, in vitro BoNT assays have been developed. The most commonly used of these are ELISA-based.

Although some laboratory-based immunoassays are highly sensitive and specific, they require a large panel of specific high-affinity antibodies to reliably detect the increasing number of known subtypes, currently more than 40, and might miss novel serotypes, thereby producing false negative results [3]. No single immunoassay has been produced to detect all BoNT subtypes. Although some immunoassays are highly sensitive with readout times of 4-6 hours, they are unlikely to discriminate active from inactive BoNT [3]. In contrast, the BLB-Test can detect  the proteolytic activity of BoNT subtypes in a single measurement [1].

Other in vitro BoNT assays include the endopeptidase assays and cell-based assays, both of which can detect BoNT with sensitivities exceeding the MLB. However, these assays are laboratory based and require highly trained personnel to provide reliable results due to the need for highly sophisticated equipment and techniques [3] [4].


What could your Solution be used for?

The high sensitivity and ability to detect and distinguish the disease-causing BoNT serotypes should make the BLB-Test useful for analysis of samples from human and animal, foodstuff and environmental samples. The BLB-Test can detect BoNT in complex matrices without the need for sample pre-treatment. Matrices successfully tested so far include blood plasma, milk, bouillon and carrot juice. It could therefore be applied to a wide range of security, food safety and health care services. Veterinary, hospital or local testing government laboratories, emergency services personnel and basic research scientists will likely be the initial beneficiaries of the BLB-Test, where questions about BoNT contamination or intoxication can be addressed safely, rapidly and economically and without the use of animals.

The BLB-Test format is flexible and can be adapted to different conditions. For use by emergency services that need to quickly test for BoNT in environmental samples, the disk would incorporate the fluorescence based assay, which does not require a substrate. In emergency work, the portability of the BLB-Test would also be important. Where sensitivity is of paramount importance, the luciferase based assay would be more applicable. Disk designs with 2 -12 channels are available for detecting and quantifying individual serotypes.


Need for collaboration

The sensitivity of the BLB-Test in various matrices is still being assessed for all of the known disease causing serotypes. However, the limit of detection to BoNT/A is 40 fM (6 pg/mL) [1], which is comparable to the sensitivity of the MLB [3]. We seek partnerships with end-users who are interested in helping to thoroughly validate the BLB-Test on a broad range of complex matrices that could be contaminated with BoNT. These include patient specimens, environmental samples, food and beverages, animal feed, gardening products etc. Collaborators would ideally have expertise in testing for BoNT in veterinary or farm settings, hospital laboratories, pharmaceutical companies, government testing labs, or with military or emergency services.

We also hope to form partnerships with companies interested in producing and commercializing the BLB-Test, particularly those with experience in production, distribution and marketing ´point of need´ diagnostics or biological assays.

Finally, we seek institutional collaborators with expertise in the biochemistry of cellular membranes and receptors who are interested in joining us to apply for research grants. Our proposal would be to extend the BLB-Test to detect BoNT binding to membrane receptors, and translocation of the proteolytic light chain domain of BoNT through pores formed in endosome membranes.


3Rs impact assessment

The MLB is used extensively for testing products and samples that are suspected to contain BoNT. According to German standard DIN 10102 and AOAC official method 977.26 [3], in vivo testing strategies requires up to 20 mice per BoNT containing sample (i.e. >140 animals to fully serotype each specimen). It was estimated that approximately 600,000 mice were used worldwide in 2009 for diagnostics and pharmaceutical purposes [5]. The test method causes severe distress to the animals, due to death by slow asphyxiation over a period of up to 4 days.

In its present form, the BLB-Test can be used to replace the MLB in most circumstances because it can detect, distinguish and quantify all the serotypes (BoNT A-F) that are known to cause disease in humans and animals. BoNT/G, which is not linked with disease [3], cannot currently be detected with the BLB-Test at the concentrations presently available. However, since BoNT-G cleaves the same substrate polypeptide as BoNT-B, it should in principle also be detectable at millimolar concentrations. In summary, with the ability to distinguish the main BoNT serotypes in complex matrices the BLB-Test has the potential to dramatically reduce the number of mice used in testing and serotyping BoNT specimens.


References

  1. Stevens GB et al. (2013). Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity. Analyst 138(20): p. 6154-62.
  2. van Oordt T et al. (2013). Rapid and highly sensitive luciferase reporter assay for the automated detection of botulinum toxin in the centrifugal microfluidic LabDisk platform. RSC Advances 3(44): p. 22046-22052.
  3. Dorner MB et al. (2013). Complexity of botulinum neurotoxins: challenges for detection technologyCurr Top Microbiol Immunol 364: p. 219-55.
  4. Pellett S (2013). Progress in cell based assays for botulinum neurotoxin detection. Curr Top Microbiol Immunol 364: p. 257-85.
  5. Bitz S (2010). The Botulinum Neurotoxin LD50 Test - Problems and Solutions. Altex-Alternatives to Animal Experimentation. 27(2): p. 114-116.

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Solution provider

University of Freiburg
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