Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19

Abstract

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

Figure 1

Western blot analysis. Anti-SARS-CoV antibodies (left two strips) and anti-His antibodies (third strip form the left) reacting with SARS-CoV-2 antigens. The far-right strip shows negative control (without antigens). All strips were cropped from a membrane shown in Supplemental Figure 1. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; His: histidine.

Figure 2

Standard curve for relations between Ct values and copy numbers. Error bars indicate SD. Ct cycle threshold, SD standard deviation.

Figure 3

Receiver operating characteristic (ROC) curve analysis. An ROC curve was drawn using human samples with and without SARS-CoV-2.

Figure 4

A scatter plot of HISCL SARS-CoV-2 Antigen (COI) and copy numbers measured by RT-qPCR tests. Data are plotted as logarithmic scales and the plots were fitted with a power approximation.

Figure 5

Assay protocol of the HISCL SARS-CoV-2 Antigen assay kit. The biotinylated SARS-CoV-2 Ag antibody (R1) was allowed to react with the sample at 42 °C for 3 min. Then, streptavidin-bonded magnetic particles (R2) were added and allowed to react at 42 ℃ for 2 min. After protein separation and washing, ALP-bound SARS-CoV-2 Ag antibody (R3) was added and reacted at 42 °C for 3 min. After another magnetic separation and washing, buffer solution (R4) and the chemiluminescent substrate (R5) were allowed to react at 42 °C for 5.5 min, then the luminescence intensity was measured. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; ALP: alkaline phosphatase; Ag: antigen. The illustration was drawn by A.K. using Microsoft PowerPoint 17.0 (Microsoft Corporation, Redmond, Washington: https://www.microsoft.com/en-us/microsoft-365/powerpoint).