Adding the heterologous OC43 S as a competitor resulted in?<10% inhibition for all proteins, except structurally similar SARS-CoV-1 S, where some level of cross-reactivity was expected and observed

Adding the heterologous OC43 S as a competitor resulted in?<10% inhibition for all proteins, except structurally similar SARS-CoV-1 S, where some level of cross-reactivity was expected and observed. Determination of optimal reagent concentrations and protocol parameters. We tested three different protein concentrations (50, 5, and 0.5?g of protein per 1.25??106 beads) and two secondary antibody concentrations (1:125 and 1:62.5). assays, the IgG antibody measurement geometric mean ratios Acrizanib were between 0.895 and 1.122, and no evidence of interference was observed between antigens. Lower and upper IgG concentration limits, based on accuracy (between 80% and 120%), precision (percent relative standard deviation, 25%), and sample dilutional linearity (between 75% Acrizanib and 125%), were used to establish the assay range. Precision was established by evaluating 24 individual human serum samples obtained from vaccinated and SARS-CoV-2-infected individuals. The assay provided reproducible, consistent results with typical coefficients of variation of 20% for all assays, irrespective of the run, day, or analyst. Results indicate the assay has high sensitivity and specificity and thus is appropriate for use in measuring SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. IMPORTANCE The SARS-CoV-2 pandemic resulted in the development and validation of multiple serology tests with variable performance. While there are multiple SARS-CoV-2 serology tests to detect SARS-CoV-2 antibodies, the focus is usually either on only one antigen at a time or multiple proteins from only one SARS-CoV-2 variant. These tests usually do not evaluate antibodies against viral proteins from different SARS-CoV-2 variants or from other coronaviruses. Here, we evaluated a multiplex serology test based on Luminex technology, where antibodies against multiple domains of SARS-CoV-2 wild type, SARS-CoV-2 mutants, and common coronavirus antibodies are detected simultaneously in a single assay. Rabbit polyclonal to ZNF490 This Luminex-based multiplex serology assay can enhance our understanding of the immune response to SARS-CoV-2 infection and vaccination. KEYWORDS: Luminex-based multiplex assay, SARS-CoV-2, serology, COVID-19 INTRODUCTION Coronavirus disease (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 genome sequence analysis has provided unprecedented opportunities for tracking variants. Compared with the original Wuhan strain, SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) and other known variants (Eta, Kappa, Lambda, Epsilon, Zeta, Theta, and Mu) have been associated with severe disease or increased transmission (1,C3). Licensed vaccines protect people from serious illness and death caused by COVID-19 (2). Protection against SARS-CoV-2 infection is mediated by functional, neutralizing antibodies specific to SARS-CoV-2. Hence, monitoring antibody responses to infection and immunogenicity of vaccines is critical as it provides important insight into the progression of regional outbreaks, rates of exposure/infection, and viral transmission dynamics. Given the evolution of SARS-CoV-2 over the last 3 years, the development of an agile and dynamic multiplex serology-based assay is important to quickly respond to and monitor immune responses to new viral variants. SARS-CoV-2 belongs to the Acrizanib genus of the (CoV) family, which is closely related to the severe acute respiratory syndrome (SARS) coronavirus and the Middle East respiratory syndrome CoV (MERS-CoV). Certain coronaviruses are common human pathogens; two types of alphacoronaviruses (229E and NL63) and two types of beta-coronaviruses (OC43 and HKU1) circulate in humans and cause the common cold. More pathogenic coronaviruses for humans include SARS-CoV-1, MERS-CoV, and now SARS-CoV-2, which likely emerged from animals to humans (4). The genome of CoVs (27 to 32?kb) is a single-stranded positive-sense RNA larger than any other RNA viruses. SARS-CoV-2 contains four structural proteins (spike [S], envelope [E], membrane [M], and nucleocapsid [N] proteins) and 16 nonstructural proteins (nsp1 to nsp16) (4). The N-terminal part of the S protein (S1) contains a receptor-binding domain (RBD), which is the structural part of SARS-CoV-2 involved in viral attachment to host cells and immunity by specifically recognizing the angiotensin-converting enzyme 2 (ACE2) receptor. The total nucleotide sequence similarity between SARS-CoV-2 and SARS-CoV is approximately 79% and is about 50% between SARS-CoV-2 and MERS-CoV (5). Here, we describe the development and validation of a 15-plex Luminex bead-based assay to measure the concentration of IgG antibodies specific to SARS-CoV-2 proteins and against six other coronaviruses. We evaluated the assay performance characteristics to detect the presence of antibodies directed to the S, N, and RBD (wild type [WT] and variants) proteins of SARS-CoV-2 in SARS-CoV-2-infected and vaccinated individuals. RESULTS Selection of appropriate proteins for the Luminex multiplex assay. Multiple SARS-CoV-2-related proteins were.