Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials

Abstract

To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10^-19 moles/assay for recombinant S1 proteins and 2.6 × 10⁶ RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 10⁴ RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19.

Keywords: COVID-19; SARS-CoV-2; antigen test; spike protein; thio-NAD cycling; ultrasensitive ELISA.

Figure 1

Schematic representation of a thio-NAD cycling ELISA for SARS-CoV-2 S1 proteins. Two antibodies used in ELISA specifically target the S1 protein. The first antibody is used to immobilize the protein, whereas the second antibody is labeled with alkaline phosphatase, which hydrolyzes a substrate containing phosphate. The hydrolyzed substrates are used in the thio-NAD cycling that employs a main enzyme (dehydrogenase) and its coenzymes (NADH and thio-NAD). Thio-NADH accumulates in a triangular manner and can be measured at 405 nm.

Figure 2

Linear calibration curves of SARS-CoV-2 S1 proteins by thio-NAD cycling ELISA. Three datasets measured by 3 investigators are presented as (A–C). The absorbance was obtained from a 40 min cycling reaction time. Each investigator performed 3 replicate experiments. The dots show the measured plots, and the lines show those obtained by the least-squares method. The antigen was applied in the range of 31.25–1000 pg/mL.

Figure 3

Detection of S1 proteins in UVB-inactivated SARS-CoV-2. Because a SARS-CoV-2 virus contains single-stranded RNA, the RNA copies in the x-axis correspond to the number of viruses. Three datasets measured by 3 investigators are presented as (A–C). The absorbance was obtained from a 60 min cycling reaction time. Each investigator performed 3 replicate experiments. Error bars indicate the standard deviation. The 3 investigators consistently obtained signals that were higher than the blank at concentrations over 2.6 × 10⁶ RNA copies/assay. * p < 0.05, ** p < 0.01 compared with the value of blank by one-way ANOVA with a post-hoc Tukey test.