The HISCL SARS-CoV-2 Antigen assay kit is a chemiluminescent enzyme immunoassay that uses a HISCL automatic immunoassay analyzer (Sysmex, Kobe, Japan). Number?5 presents a schematic illustration of the procedure: (1) the samples were incubated with biotinylated SARS-CoV-2 antibodies (R1: antibody 2C3) at 42?C for 3?min; (2) the mixtures were incubated with streptavidin-bonded magnetic particles at 42?C for 2?min; (3) after protein separation and washing, alkaline phosphatase (ALP)-bound SARS-CoV-2 antibodies (R3: antibody 2C12) were added and the mixtures were incubated at 42?C for 3?min; (4) after magnetic separation and washing again, the chemiluminescent substrates were added and the mixtures were incubated at 42?C for 5.5?min; and (5) chemiluminescence signals (CDP-Star, C0712, Sigma-Aldrich, St. of 95.4% and 16.6% in samples with copy numbers ?100 and ?99, respectively. The kit did not cross-react with human being coronaviruses causing seasonal common chilly 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 weight ( ?50 copy numbers) were diagnosed as negative. Therefore, using HISCL antigen assay packages may reduce overdiagnosis compared with RT-qPCR checks. The quick 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. cycle threshold, standard deviation. HISCL SARS-CoV-2 Antigen assay kit As explained in the Methods section, recombinant SARS-CoV-2 proteins (ACRO Biosystems, Newark, DE) were incubated with biotinylated SARS-CoV-2 antibodies (R1: ab 2C3) and alkaline phosphatase (ALP)-bound SARS-CoV-2 antibodies (R3: ab 2C12). After washing out the unbound proteins, chemiluminescent substrates (CDP-Star, C0712, Sigma-Aldrich, St. Louis, MO, USA) were added, and fluorescence signals were measured using the picture counter of HISCL-800 (Sysmex, Kobe, Japan). The level of SARS-CoV-2 antigens was indicated as the cut-off index (COI), determined from the difference in the luminescence intensities in the buffers with and without the SARS-CoV-2 antigens. The COI was defined as: Cut-off index (C.O.I.)?=?(light intensity inside a samplelight intensity with the bad control) / (light intensity at a determined cut-off valuelight intensity with the bad control). Reproducibility To check the reproducibility of Dimethyl 4-hydroxyisophthalate the HISCL SARS-CoV-2 Antigen assay kit, we measured one sample comprising recombinant SARS-CoV-2 antigens (109.5??36.5?pg/mL, ACRO Biosystems, Newark, DE) (positive control sample) and 1 sample containing only buffers (negative control sample). The checks were repeated ten instances for each sample and all experiments were carried out in triplicate. Table ?Table11 shows the COI ideals for both samples. The bad control samples showed 100% agreement with COI of zero and the positive control samples showed the average COI of 27.7 to 28.8 having a coefficient of variation (CV) percentage of 1 1.3C2.5. Table 1 Within-run reproducibility. The COI ideals of samples without or with SARS-CoV-2 antigens were measured a total of 30 instances (three units of 10 consecutive measurements). cutoff index, standard deviation, coefficient of variance. To check reproducibility between measurements in five different days, we performed assays using the negative and positive control samples twice daily for 5?days. Table ?Table22 demonstrates the COIs of the negative and positive control samples were consistent (CV%? ?10). Table 2 Between-run reproducibility. cut-off index, standard deviation, coefficient of variance. Cross-reaction To check for specificity, numerous corona and influenza viral antigens were used to measure Dimethyl 4-hydroxyisophthalate COIs in the HISCL assay. Table ?Table33 summarizes the COI measurements. The COI showed high ideals for SARS-CoV at a concentration of 0.25?ng/mL and positive ideals for MERS-CoV at a low concentration. In contrast, the COI Dimethyl 4-hydroxyisophthalate ideals stayed in the bad range for additional coronaviruses and influenza viruses, indicating that the HISCL SARS-CoV-2 Antigen assay kit does not react with seasonal common chilly and Dimethyl 4-hydroxyisophthalate flu viruses. Table 3 Cut-off index (COI) ideals in bad controls. human Rabbit Polyclonal to CDC25B (phospho-Ser323) being coronavirus, Middle East respiratory syndrome-related coronavirus, severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2. Dedication of the cut-off value To optimize the cut-off antigen level for accurate diagnoses, we 1st determined the relationship between COI and viral lots using a total of 84 nasopharyngeal swabs collected from 17 individuals at Kobe City Medical Center General Hospital and 67 commercially available samples (Cantor Bioconnect, Santee, CA, USA): 30 positive samples and 54 bad samples. Chemiluminescence counts were measured by HISCL SARS-CoV-2 Antigen assay kit using these samples and recombinant SARS-CoV-2 proteins (0, 20, and 100?pg/mL)..