Split luciferase-based assay to detect botulinum neurotoxins using hiPSC-derived motor neurons

Abstract

Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). In vitro assays for BoNT testing using rodent cells or immortalized cell lines showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP-25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split NanoLuc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, with a detection sensitivity of femtomolar concentrations of toxin, and can be used to study the different steps of BoNT intoxication.

Development of Luminescent Biosensors for Calprotectin

“…To establish a user-friendly calprotectin detection method, we focused on adapting the Fab binders with the NanoBiT-based technology, in which the two binding modules are fused to LgBiT or SmBiT (VTGY­RLFE­EIL), two fragments of split NanoLuc luciferase (NanoLuc) and reconstitute an active luciferase enzyme upon dual binding to calprotectin, due to the close proximity of split NanoLuc fragments . NanoBiT-based technology has recently been developed with various synthetic scaffolds and applied for different tests, including but not limited to SARS-CoV-2 (COVID-19), , Clostridium difficile infections, , and botulinum neurotoxins . While efforts have been reported in combining NanoBiT with full-length antibodies, − the most common and versatile protein-based binder so far, such antibody engineering usually relies on chemical conjugation of antibody with LgBiT or SmBiT, which necessitates not only the costly production of antibodies, but also extensive purifications due to the heterogeneity of products.…”

“…28 NanoBiT-based technology has recently been developed with various synthetic scaffolds and applied for different tests, 41 including but not limited to SARS-CoV-2 (COVID-19), 42,43 Clostridium dif f icile infections, 44,45 and botulinum neurotoxins. 46 While efforts have been reported in combining NanoBiT with full-length antibodies, 47−53 the most common and versatile protein-based binder so far, such antibody engineering usually relies on chemical conjugation of antibody with LgBiT or SmBiT, which necessitates not only the costly production of antibodies, but also extensive purifications due to the heterogeneity of products. While primary efforts suggested Fab binder tagged with a short peptide (peptide β9 from NanoLuc) is compatible with the NanoBiT system, 54 we reasoned that directly fusing NanoBiT with Fab could address the challenges associated with antibody conjugation, thereby fully leveraging Fab's versatility.…”