User Guide Rapid Affinity-Based SARS‑CoV‑2 Virus Neutralization Assay Published on April 12th, 2021 This workflow and its products described here are for research use only and is not to be used for any other purposes, including, but not limited to, in vitro diagnostics, clinical diagnostics, or use in humans. The document and its content are proprietary to Fluidic Analytics and is intended for use only in connection with the products described herein and for no other purposes. download user guide 1. Intended Use This User Guide describes a rapid affinity-based virus neutralization assay to detect virus‑neutralizing antibodies directly in COVID-19 patient serum samples by use of the Fluidity One-W Serum instrument. 1.1. Background COVID-19 is a respiratory disease caused by the coronavirus SARS-CoV-2. In order to infect a human cell, SARS-CoV-2 utilizes spike proteins on its surface to bind to the human angiotensin‑converting enzyme 2 (ACE2) receptor protein which is typically found on the surface of human lung cells. Upon entry into the cell, the virus replicates causing the immune system to respond by producing different types of antibodies. These include virus‑neutralizing antibodies (NAbs) which are crucial to protect the human body from future infections. While conventional cell-based neutralization assays offer a well-established way to determine the presence of neutralizing antibodies, these assays are cumbersome and lengthy and often require live biological materials and strict biosafety regulations. To overcome these limitations we have developed a rapid affinity-based virus neutralization test that mimics the virus neutralization process by measuring the binding interaction between the ACE2 receptor and the SARS-CoV-2 spike RBD protein, as well as the subsequent displacement of the spike protein in the presence of virus-neutralizing antibodies (NAbs) directly in patient serum. 2. Rapid Affinity-Based SARS-CoV-2 Virus Neutralization Assay This User Guide describes the use of a rapid affinity-based virus neutralization assay as an alternative to conventional cell-based neutralization assays. The assay can be performed in approximately 1 hour and requires no prior knowledge of antibody concentrations. For the assay, labeled ACE2 is mixed with unlabeled SARS-CoV-2 RBD-Fc protein and patient serum is added followed by an incubation of 30 min at 4 °C. In the presence of virus-neutralizing antibodies (NAbs), the SARS-CoV-2 RBD-Fc protein will dissociate from the ACE2/SARS-CoV-2 RBD-Fc complex and bind to the neutralizing antibodies (Figure 1). As a result, the hydrodynamic radius (Rh) will decrease compared to the Rh measured for the complex. In the absence of NAbs, ACE2 remains bound to SARS-CoV-2 RBD-Fc protein resulting in an unchanged Rh. Figure 1: Labeled ACE2 is mixed with unlabeled SARS-CoV-2 RBD-Fc protein. After complex formation, patient serum is added. In the presence of virus-neutralizing antibodies (NAbs), the SARS-CoV-2 RBD-Fc protein will dissociate from the ACE2/SARS-CoV-2 RBD-Fc complex and bind to the neutralizing antibodies. As a result, Rh will decrease compared to Rh measured for the complex. In the absence of NAbs, ACE2 remains bound to SARS-CoV-2 RBD-Fc protein resulting in an unchanged Rh. 3. Before You Start Good laboratory practice when working with serum samples Serum or plasma collected from COVID-19 patients (whether newly infected or recovered) as well as “uninfected” individuals who could still harbor infectious agents must be handled using a high level of precaution at the recommended biosafety level (dependent on national legislation, will vary between countries). For safety reasons we would recommend handling all serum samples under the assumption that they contain infectious agents. For the full protocol download the user guide below download user guide Results After data submission, background subtraction will be performed for each measurement. If virus-neutralizing antibodies (NAbs) are present in the sample, the SARS-CoV-2 RBD-Fc protein will dissociate from the ACE2/SARS-CoV-2 RBD-Fc complex and bind to the neutralizing antibodies. As a consequence, the Rh will decrease compared to Rh measured for the complex in the negative control, as exemplified in Figure 2. In the absence of NAbs, ACE2 remains bound to SARS-CoV-2 RBD-Fc protein resulting in an unchanged or increased Rh compared to the negative control. Figure 2: Example data of the rapid neutralization assay. Five convalescent SARS‑CoV‑2 serum samples, ACE2-only control and ACE2/SARS-CoV-2 RBD-Fc complex control were tested. Three of the serum samples exhibited neutralization potential resulting in Rh lower than for the ACE2/RBD-Fc complex. Two of the tested convalescent serum samples exhibited Rh higher than for the ACE2/SARS-CoV-2 RBD-Fc complex, suggesting antibody binding without complex disruption. The assay principle has been described in Schneider et al. and in Fiedler et al.. Download user guide Fill out the form below to download the full user guide "Rapid Affinity-Based SARS‑CoV‑2 Virus Neutralization Assay". We hope that this user guide is useful to all in some capacity to help with the COVID‑19 pandemic.