Real-time cell analysis: A high-throughput approach for testing SARS-CoV-2 antibody neutralization and escape.

Real-time cell analysis (RTCA) enables high-throughput, quantitative kinetic measurements of cytopathic effect (CPE) in virus-infected cells. Here, we detail a RTCA approach for assessing antibody neutralization. We describe how to evaluate the neutralizing potency of monoclonal antibodies (mAbs) and identify viral escape mutants to antibody neutralization for severe respiratory syndrome coronavirus 2 (SARS-CoV-2). For complete details on the use and execution of this protocol, please refer to Zost et al. (2020) and Suryadevara et al. (2021).

A touch transfer assay to determine surface transmission of highly pathogenic viruses.

Transmission via fomites poses a major dissemination route for many human pathogens, particularly because of transfer via fingertips. Here, we present a protocol to investigate direct transfer of infectious agents from fomites to humans via naked fingertips. The protocol is suitable for pathogens requiring highest biosafety levels (e.g., SARS-CoV-2). We used an artificial skin to touch a defined volume of virus suspension and subsequent quantification of infectious entities allows quantitative measurement of transfer efficiency and risk assessment. For complete information on the generation and use of this manuscript, please refer to Todt et al. (2021).

SARS-CoV-2 proteome microarray for COVID-19 patient sera profiling.

The immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome is largely unknown. Here we describe a protocol for analyzing sera samples with SARS-CoV-2 proteome microarray. The proteins were expressed by either E. coli expression system or eukaryotic cell expression systems and obtained by affinity purification. The protocol includes microarray fabricating and sera profiling, which will be used to build an antibody response landscape for IgG and IgM. The protocol may help to facilitate a deeper understanding of immunity related to SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Li et al. (2021c).

Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis.

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.

Fluorogenic in vitro activity assay for the main protease Mpro from SARS-CoV-2 and its adaptation to the identification of inhibitors.

This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET (Fluorescence Resonance Energy Transfer) substrates. The protocol is based on an aminomethyl coumarin substrate. High sensitivity, specificity, and an easily detectable fluorescent read-out are the advantages offered by this rapid assay, which allows high throughput screening of new Mpro inhibitors.