Rapid quantification of C. difficile glutamate dehydrogenase and toxin B (TcdB) with a NanoBiT split-luciferase assay
Adamson, H. et al. (2022) Rapid quantification of C. difficile glutamate dehydrogenase and toxin B (TcdB) with a NanoBiT split-luciferase assay. Anal. Chem. 94, 8156−8163. DOI: 10.1021/acs.analchem.1c05206
The Gram-positive bacillus Clostridioides (formerly Clostridium) difficile is responsible for most hospital-related infections, with global incidence rising over the past decade. Two large toxins, toxin A (TcdA) and toxin B (TcdB), are responsible for triggering host responses that can result in significant intestinal damage. There is an urgent need for sensitive and rapid tests to detect true C. difficile infection, as distinguished from disease-free carriers.
In this study, the researchers used NanoBiT® technology to design “split-luciferase” assays that could detect TcdB and another C. difficile biomarker, glutamate dehydrogenase (GDH). The assay development strategy used affimers (synthetic nonimmunoglobulin-binding proteins) with high binding affinity for different regions of TcdB and GDH. These affimers are more convenient than antibodies, because they are smaller, stable and easily expressed as fusions with LgBiT and SmBiT tags. Binding to the target antigen in solution reconstitutes functional NanoBiT® enzyme, resulting in a bright luminescent signal. The assay performance with fecal samples was equivalent to that of a current point-of-care (POC) test, but it had the advantages of being quantitative, requiring less user steps, and being able to distinguish clinically relevant TcdB. The researchers conclude that their assay method is suitable for a wide range of biomarkers and offers the potential to develop ultrasensitive and rapid POC tests for many infectious diseases.
Keywords: C. difficile, bioluminescence, NanoBiT, sensors, toxins
