Plasma phase was aspirated carefully from the top. compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings. strong class=”kwd-title” Keywords: COVID-19, Seroepidemiology, Microsampling, Breast milk 1.?Introduction Diagnosis of infection with the novel coronavirus SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, has relied on two classes of assays. One comprises the methods for detecting the presence of the virus in upper respiratory specimens, either by viral nucleic acid amplification tests (NAAT) or immunodetection of viral antigen. NAATs based on Real-time PCR represent the gold standard for diagnosis of acute SARS-CoV-2 infection while the antigen tests, which are comparatively less sensitive, are critically important for public health purposes, since they have a very rapid turn-around and detect infectious cases (Mallett et al., 2020; Mina et al., 2020; Ravi et al., 2020; van Kasteren et al., 2020; Mina and Andersen, 2021; Yce et al., 2021). The second class of assays comprises methods for detecting virus-specific antibodies in peripheral blood. These antibodies are reliable indicators of viral exposure, since they become detectable approximately two weeks after initiation of productive infection and typically persist for 6C12?months or longer, well beyond the time in which virus detection assays return to negativity (Fig. 1 ). Thus, antibody-based assays are most valuable as metrics of infection burden in the population for epidemiological purposes and large-scale studies. Open in a separate window Fig. 1 Time course of key biomarkers in SARS-CoV-2 infection, adapted from The solid green Rabbit polyclonal to PHF7 line represents a typical trajectory of the RT-PCR data for viral nucleic acid from respiratory samples, while the broken purple line indicates a typical virus-specific antibody trajectory in peripheral blood, relative to time of infection, as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Antibody-based assays for SARS-CoV-2 infection are based on two SARS-CoV-2 antigens. One is Spike (S), a two-subunit protein that decorates the surface of the virion and establishes contact with the host cell receptor, angiotensin-converting enzyme 2 (ACE2), through the WIKI4 receptor-binding domain (RBD) in the S1 subunit, thus determining host range and tissue tropism (Li, 2016). The second viral antigen is the Nucleocapsid (N), which interacts with the viral genomic RNA inside the viral envelope. Both antigens have been used for SARS-CoV-2 antibody detection, with an initial preference for the N antigen in most commercial antibody detection assays utilized in clinical settings (for example, (Kohmer et al., 2020; Padoan et al., 2020)). The S protein has been adopted as antibody capture antigen in research settings since the beginning of the pandemic]e.g., (Amanat et al., 2020; Robbiani et al., 2020; Gaebler et al., 2021)], primarily because the S1 RBD region is particularly immunogenic and the dominant target of neutralizing (protective) antibodies (Ju et al., 2020; Robbiani et al., 2020; Shi et al., 2020). Moreover, mutations in RBD, which are important factors in the evolution of all major SARS-CoV-2 variants, increase affinity for the ACE-2 receptor and lead to resistance to monoclonal and polyclonal antibodies developed in response to infection or vaccination (Starr et al., 2020; Greaney et al., 2021a, Greaney et al., WIKI4 2021b). More recently, use of S1 RBD for antibody testing has been extended to commercial assays and clinical applications (see list of emergency use authorized serology tests at since the introduction of SARS-CoV-2 vaccines, which contain S but not N (Jackson et al., 2020; Polack et al., 2020). Thus, it becomes increasingly important to identify all potential uses of WIKI4 S-based serological assays for SARS-CoV-2 infection. Here we describe key characteristics of our serological assay utilizing a novel S1 RBD antigen and its suitability for antibody detection from minimal (l scale) amounts of remotely collected peripheral blood, which is critical for seroepidemiological and large-scale studies conducted outside of health care settings. We also show that the assay is equally.