2.1 Evaluation of the LoD
ImmunoArrow® SARS-CoV-2 (ImmunoArrow®, TOYOBO Co., Ltd., Osaka, Japan) was provided by TOYOBO Co. Ltd. In addition to ImmunoArrow®, ESPLINE® SARS-CoV-2 (ESPLINE®, Lot K4B1I020, Fujirebio Inc., Tokyo, Japan), QuickNavi™-COVID19 Ag (QuickNavi™, Lot 1041081, Denka Co., Ltd., Tokyo, Japan), ImmunoAce® SARS-CoV-2 (ImmunoAce®, Lot 191211098, TAUNS Laboratories, Inc., Izunokuni, Japan), Panbio™ COVID-19 Ag Rapid Test Device (Panbio™, Lot 41ADG542A, Abbott Diagnostics Medical Co., Ltd., Tokyo, Japan), and SARS-CoV-2 Rapid Antigen Test (Rapid Antigen Test, Lot QC0391058I, Roche Diagnostics K.K., Tokyo, Japan), were evaluated for their LoDs. To prepare the testing solution, heat-inactivated wild-type SARS-CoV-2 (USA-WA1/2020) (ZeptoMetrix, NY, USA), and its variants including the Alpha (B.1.1.7) (ZeptoMetrix), Beta (B.1.351) (ZeptoMetrix), Gamma (P.1) (ZeptoMetrix), Delta (B.1.617.2) (ZeptoMetrix), Kappa (B.1.617.1) (ZeptoMetrix). To make testing solutions, the heat-inactivated viruses were dissolved by D-PBS and serially diluted at the concentration of 2.5 × 105 to 7.9 × 101 TCID50/mL by a half-logarithmic dilution. Then, 50 μL of the testing solution were introduced into the recommended volume of lysis reagent for each test (sample-reagent mixture). The indicated volume of the sample-reagent mixture was applied into each test cartridge according to each manufacturer's protocol. The reaction was judged by two judges and repeated additional one experiment. The LoD was defined as the minimum concentration of the sample solution in which at least one judge judged positive. The confirmation of the control line was requested for all judgement to ensure testing.
2.3 Evaluation of the Omicron variant
The recombinant SARS-CoV-2 nucleocapsid antigens of the wild type (RayBiotech, GA), the Omicron variant B.1.1.529 (Acro BIOSYSTEMS, DE), and the Omicron variant BA.2 (Acro BIOSYSTEMS) were used for the evaluation of the LoD of ImmunoArrow® to compare its performance to detect them. The reason for using recombinant proteins for this evaluation is that the inactivated viruses of Omicron variants were not available because they appeared during the period of the evaluation.
Each recombinant antigen was diluted by the lysis reagent. The level at which two individuals judged positive in triplicate was defined as the 1 × LoD of the wild type. Three judges examined single test independently. The confirmation of the control line was requested for all judgement to ensure testing.