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. 2020 Aug 14;10(8):594.
doi: 10.3390/diagnostics10080594.

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Yuta Kyosei  1 , Mayuri Namba  1 , Sou Yamura  1 , Rikiya Takeuchi  2 , Noriko Aoki  2 , Kazunari Nakaishi  3   4 , Satoshi Watabe  2   4 , Etsuro Ito  1   4   5
Affiliations

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Yuta Kyosei et al. Diagnostics (Basel). .

Abstract

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10-18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10-20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

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

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Conflict of interest statement

R.T., N.A., K.N., and S.W. are employees of TAUNS Laboratories, Inc. E.I. received research expenses and a patent royalty from TAUNS Laboratories, Inc. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Principle of the ultrasensitive detection of SARS-CoV-2. The SARS-CoV-2 spike proteins were measured by a sandwich ELISA coupled with thio-NAD cycling using alkaline phosphatase, androsterone derivatives, and 3α-hydroxysteroid dehydrogenase (3α-HSD) and its coenzymes (NADH and thio-NAD). During this cycling reaction, thio-NADH accumulated in a triangular-number fashion. Accumulated thio-NADH was measured directly at an absorbance of 405 nm without interference from the other cofactors.
Figure 2
Figure 2
Linear calibration curves obtained from the absorbance. (A) Cycling reaction time of 60 min. This curve is expressed as y = 8.0 × 10−4x, R2 = 1.00 in the range of 63–500 pg/mL. (B) Cycling reaction time of 60 min. This curve is expressed as y = 8.0 × 10−4x, R2 = 1.00 in the range of 10–1000 pg/mL.
Figure 3
Figure 3
Linear calibration curve obtained from the absorbance at 10 min of thio-NAD cycling and the relation between the period of thio-NAD cycling and the LOD for SARS-CoV-2 spikes. (A) Cycling reaction time of 10 min. This linear calibration curve is expressed as y = 3.0 × 10−5x, R2 = 1.00 in the range of 63–2000 pg/mL. (B) The relation between the period of thio-NAD cycling and the LOD for SARS-CoV-2 spikes. This relation is expressed as y = −1.0 × 10−18x + 7.0 × 10−17, R2 = 0.86 in the range of 10–60 min of thio-NAD cycling.
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