iGIST—A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

作者：Valeriy M. Paramonov, Cecilia Sahlgren, Adolfo Rivero‐Müller, Arto T. Pulliainen · 发表于：ACS Sensors · 年份：2020 · DOI：10.1021/acssensors.0c01340 · 被引用次数：17 · 研究领域：Receptor Mechanisms and Signaling、Nicotinic Acetylcholine Receptors Study、Neuropeptides and Animal Physiology

High Resolution Image Download MS PowerPoint Slide Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require thousands of mice annually. Here, we describe I nterference in G α i -mediated S ignal T ransduction (iGIST), an animal-free kinetic bioassay for detection of PTX, by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells coexpressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high-affinity agonist octreotide; and a luminescent 3′5′-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in the pg/mL range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e., a commercial PTX-toxoid-containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including id...