Concordance was high with a Gwet’s Ac1 of 0.992. measured by Chorus SARS-CoV-2 Neutralizing Ab: 1 false negative and 2 false positives. Values of sensitivity and specificity were very high: percent positive agreement (sensitivity) 99.6% (95% CI: 97.7 C 99.9) and percent negative agreement (specificity) 99.6% (95% CI: 98.0 -99.9). Concordance was high with a Gwet’s Ac1 of 0.992. No significant differences were observed between the alpha and original variants. Conclusions The Chorus SARS-CoV-2 Neutralizing Ab test was highly sensitive and specific, and varies from most other currently available tests since it analyzes only antibodies with viral-neutralizing capacity. strong class=”kwd-title” Keywords: SARS-CoV-2, COVID-19, Neutralizing antibodies, Diagnostic assay, Immunoassay 1.?Introduction Antibodies against SARS-CoV-2 play a central role in clearing the virus from infected patients. To prevent COVID-19, antibodies should be able to engage the S1 subunit of SARS-CoV-2 spike protein, which contains the receptor binding domain (RBD) to angiotensin-converting enzyme (ACE) 2, and neutralize the virus [1]. It has been extensively reported that the degree of the antibody response correlates with the severity of COVID-19 and that the quantity of neutralizing antibodies declines rather rapidly with time [2], [3], [4]. Serological assays for SARS-CoV-2 play a role in diagnosis of COVID-19, in understanding viral epidemiology and screening convalescent sera for therapeutic and prophylactic purposes, to better understand the immune response to the virus, and to assess the degree and duration of the response of specific antibodies [5], [6], [7]. Moreover, serological assays are of major importance in monitoring immunization status in those previously infected with COVID-19 and in individuals vaccinated against SARS-CoV-2 over time. Tests designed to measure antibodies to SARS-CoV-2 antigens have been developed, but most have not been adequately evaluated and validated [8]. Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies, but there are limited data correlating the results from commercial assays that measure neutralizing antibodies [9, 10]. Diesse Diagnostica Senese SpA (Siena, Italy), in collaboration with the Spallanzani Institute (Rome, Italy), has developed serological diagnostic kits to rapidly detect IgM, IgG, and IgA antibodies using indirect ELISA. Unlike other commercial serological tests based on specific recombinant viral antigens, such as N or Iproniazid phosphate S proteins, these assays are based on whole inactivated virus from crude extracts of SARS-CoV-2 with the aim of increasing the possibility of specific antibody detection [11]. Among the many neutralizing antibodies from COVID-19 survivors, it has been shown that the most potent recognize the spike protein RBD, followed by antibodies that recognize the S1 domain, spike protein trimer, and S2 subunit [12]. Monitoring the total anti-S1 antibody titer can thus serve as an indication of immune Iproniazid phosphate protection for post-COVID-19 patients or for vaccinated individuals. The ability to monitor neutralizing antibody titer is especially relevant given the emerging viral variants as neutralizing antibodies from both prior infection or vaccines may be less amenable to bind the spike protein in variants [13]. Herein, we present the results obtained with an automated diagnostic kit applied to the Chorus TRIO Diesse instrument based on a competitive enzyme immunoassay for the quantitative determination of anti-S1 SARS-CoV-2 total neutralizing antibodies. Data comparing the accuracy of the Chorus SARS-CoV-2 Neutralizing Ab test with the micro-neutralization assay versus the original and Alpha variants are also presented. 2.?Materials and methods 2.1. Chorus SARS-CoV-2 neutralizing AB test The Chorus SARS-CoV-2 “Neutralizing” Ab test was performed on Diesse Diagnostica Senese Chorus system, a fully automated instrument capable of processing 30 samples in about 90?min. The format is a monotest device containing Iproniazid phosphate all the reagents necessary to carry out the test, which is processed automatically by the instrument by reading a bar code. No operator intervention is required except to place the sample in the device well. The Chorus SARS-CoV-2 “Neutralizing” Ab assay is a competitive binding immunoenzymatic Iproniazid phosphate assay. SARS-CoV-2 anti-S1 total antibodies present in the test sample (IgG, IgM and IgA) compete with the tracer (peroxidase-conjugated SARS-CoV-2 anti-S1 therapeutic recombinant antibody) to occupy binding sites, available in limited numbers, of the UKp68 antigen fixed on the solid phase. Details of antigen production have been previously described [11]. The recombinant tracer.