Elecsys anti sars cov 2 s immunoassay

The neutralizing effects of samples against pseudotyped viruses were examined by the htCRNT, as previously described (12 ). Briefly, serum diluted 100-fold with Dulbecco’s Modified Eagle Medium (DMEM, Nacalai Tesque, Inc., Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum was incubated with pseudotyped SARS-CoV-2 for 1 h. After incubation, VeroE6/TMPRSS2 cells (JCRB1819) were treated with DMEM-containing serum and pseudotyped virus. The infectivity of the pseudotyped viruses was determined by measuring luciferase activity after 24 h of incubation at 37°C. The value of samples without pseudotyped virus was defined as 0% infection and 100% inhibition, while the value of samples with pseudotyped virus but without serum was defined as 100% infection and 0% inhibition. A score with ≥50% inhibition of viral infection was considered positive for neutralization.
For the commercial assay, serum samples were tested at Toyama University Hospital using the Elecsys Anti-SARS-CoV-2 S immunoassay (Roche Diagnostics GmbH, Basel, Switzerland) to quantitatively measure antibody levels against SARS-CoV-2 RBD. The manufacturer’s cutoff value (COV) was 0.8 U/ml and the minimum value was expressed as <0.4 U/ml.

The neutralizing effects of samples against pseudotyped viruses were examined by the htCRNT, as previously described (12 ). Briefly, serum diluted 100-fold with Dulbecco’s Modified Eagle Medium (DMEM, Nacalai Tesque, Inc., Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum was incubated with pseudotyped SARS-CoV-2 for 1 h. After incubation, VeroE6/TMPRSS2 cells (JCRB1819) were treated with DMEM-containing serum and pseudotyped virus. The infectivity of the pseudotyped viruses was determined by measuring luciferase activity after 24 h of incubation at 37°C. The value of samples without pseudotyped virus was defined as 0% infection and 100% inhibition, while the value of samples with pseudotyped virus but without serum was defined as 100% infection and 0% inhibition. A score with ≥50% inhibition of viral infection was considered positive for neutralization.
For the commercial assay, serum samples were tested at Toyama University Hospital using the Elecsys Anti-SARS-CoV-2 S immunoassay (Roche Diagnostics GmbH, Basel, Switzerland) to quantitatively measure antibody levels against SARS-CoV-2 RBD. The manufacturer’s cutoff value (COV) was 0.8 U/ml and the minimum value was expressed as <0.4 U/ml.

Elecsys Anti-SARS-CoV-2 S immunoassay (Roche Diagnostics, Basel, Switzerland) was used to measure anti-spike immunoglobulin G (IgG) SARS-CoV-2 antibody titers on a Roche Cobas e411 analyzer (Roche Diagnostics) according to the manufacturer’s instructions. Samples with a titer >250 U/mL were serially diluted until the titer became ≤250 U/mL, according to the manufacturer’s protocol. Anti-nucleocapsid antibodies (Elecsys Anti-SARS-CoV-2, Roche Diagnostics) were measured according to the manufacturer’s protocol.

SARS-CoV-2 specific antibodies were analysed using Elecsys Anti-SARS-CoV-2 S immunoassay (Roche Diagnostics International Ltd, Switzerland) on Cobas e6000 instrument. The test detects antibodies specific to SARS-CoV-2 spike (S) protein receptor binding domain (RBD) in human serum and plasma. The assay uses a recombinant protein representing the RBD of the S antigen in a double-antigen sandwich assay format, which favors detection of high affinity antibodies (IgG, IgA, IgM) against SARS-CoV-2 in a quantitative manner. The method is based on double-antigen sandwich principle using electrochemiluminescence for quantitative determination of antibodies. The limit of quantification is 0.4 U/ml. Results below 0.8 U/ml are considered negative and results equal to or above 0.8 are considered positive due to the test description (11 (link)).

A complete description of the materials and methods is provided as a supplement (S1 File and S1 Fig). Participants with documented PCR-positive SARS-CoV-2 infection (SARS-CoV-2 positive) and participants without knowledge of previous infection (SARS-CoV-2 negative) were invited to participate in the study and blood samples were collected by venipuncture and capillary microsampling. The samples were tested on Roche Elecsys Anti-SARS-CoV-2 S immunoassay as a reference assay, a commercial ELISA Euroimmun SARS-CoV-2 anti-S IgG, and on a microfluidic nano-immunoassay [22 (link)]. For the Roche Anti-SARS-CoV-2 S reference assay, studies performed independently showed high specifictity and sensitivity of 99.95% (95% confidence interval [CI]: 99.87–99.99; 7876/7880) and 97.92% (95% CI: 95.21–99.32; 235/240), respectively, for samples ≥14 days post-PCR [23 (link)]. This study was approved by the local ethics committee (CCER project numbers 2020–00516 and 2020–02323) and registered (NCT04329546) prior to initiation. For all participants, a written informed consent was obtained.