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.

Detecting SARS-CoV-2 neutralizing immunity: highlighting the potential of split nanoluciferase technology.

The coronavirus disease 2019 (COVID-19) pandemic has progressed over 2 years since its onset causing significant health concerns all over the world and is currently curtailed by mass vaccination. Immunity acquired against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be following either infection or vaccination. However, one can never be sure whether the acquired immunity is adequate to protect the individual from subsequent infection because of three important factors: individual variations in humoral response dynamics, waning of protective antibodies over time, and the emergence of immune escape mutants. Therefore, a test that can accurately differentiate the protected from the vulnerable is the need of the hour. The plaque reduction neutralization assay is the conventional gold standard test for estimating the titers of neutralizing antibodies that confer protection. However, it has got several drawbacks, which hinder the practical application of this test for wide-scale usage. Hence, various tests have been developed to detect protective immunity against SARS-CoV-2 that directly or indirectly assess the presence of neutralizing antibodies to SARS-CoV-2 in a lower biosafety setting. In this review, the pros and cons of the currently available assays are elaborated in detail and special focus is put on the scope of the novel split nanoluciferase technology for detecting SARS-CoV-2 neutralizing antibodies.