Using Split Luminescent Biosensors for SARS-CoV-2 Antibody Detection in Serum, Plasma, and Blood Samples.

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days.

Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme.

Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C-H activation and cyclization of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution.