Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay

Abstract

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile , so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

New treatment approaches for Clostridioides difficile infections: alternatives to antibiotics and fecal microbiota transplantation

“…However, their reported use was not for exotoxin neutralization, but rather for diagnostic applications, using a split-luciferase assay in combination with a nanobody. 87 The affimer was fused via a flexible peptide linker to the large subunit of luciferase (18 kDa, termed LgBiT), and the nanobody was tethered to the small subunit (the 11-residue SmBiT). Upon simultaneous binding of both constructs to TcdB, the SmBiT subunit complemented LgBiT to form catalytically active luciferase, thus producing a luminescent signal.…”

Therapeutic drug monitoring of immunotherapies with novel Affimer–NanoBiT sensor construct

“…1 ). With previous work supporting the NanoBiT system as a method of detecting proteins in biological fluids, 17 we expected our Affimer–NanoBiT system to provide a rapid and sensitive alternative for TDM of TmAb levels that could be implemented into a PoC setting.…”

“…Binding of the recognition elements colocalises the two inactive reporter fragments, increasing their effective concentration and prompting re-assembly of the active enzyme, the activity of which can be measured. 17,18,22,27 Here, we describe the development of a homogenous, split enzyme assay for the detection of four routinely used TmAb treatments (adalimumab, ipilimumab, rituximab and trastuzumab). The sensor combines Affimer proteins as recognition elements with an established split-luciferase, NanoLuc® Binary Technology (NanoBiT).…”

“…Binding of the recognition elements co-localises the two inactive reporter fragments, increasing their effective concentration and prompting re-assembly of the active enzyme, the activity of which can be measured. 17,18,22,27…”

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome ofStreptococcus mutans

“…The luciferase enzyme is split and expressed as two separate chimeric protein fusions that are reconstituted into a functional reporter only when the two fused target proteins of interest form stable complexes. SLCA has been used to measure dynamic changes in PPI in numerous studies of viruses and mammalian cells, but thus far has only been rarely employed for prokaryotic genetic research (30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”