Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection

Abstract

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It 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.

Vaccination of solid organ transplant recipients previously infected with SARS-CoV2 induces potent responses that extend to variants, including Omicron

“…It is known that the RBD of the CoV2 Spike protein is an immunodominant target and the prime target for virus-neutralizing antibodies, and we therefore focused our study on responses to this region. We used a novel RBD-gp70 fusion protein system to express native RBD as antibody targets; these proteins more efficiently recognize virus-specific antibodies than standard RBD antigens, as previously described 41 . 51 SOTR plasmas were screened for levels of anti-CoV2 -RBD IgG, IgM and IgA induced after a full dose (two vaccinations for Moderna and Pfizer and one for J&J) of COVID-19 vaccines in both CoV2 infected and uninfected subjects.…”

“…We used a novel RBD-gp70 fusion protein system to express native RBD as an antibody target, which recognizes virus-specific antibodies more efficiently than the standard RBD antigens 43 . All 76 SOT recipient’s plasmas were screened for levels of anti-CoV2 -RBD IgG, IgM, and IgA antibodies and 3X background value with pre-pandemic healthy plasma RBD binding (AUC of 10) was considered as cut-off for positive RBD antibody titer.…”

“…RBD-wt antigens were expressed as fusion proteins with the gp70 carrier domain, as previously described 43 . In brief, a gene fragment of the CoV2-Spike gene encoding the RBD was synthesized commercially (Integrated DNA Technologies, Coralville, IA) and cloned at the 3' end of a gene expressing the N-terminal fragment of the Friend ectotropic MuLV (Fr-MuLV) surface protein (SU) gp70 gene in the expression vector pcDNA3.4 (Addgene, Watertown, MA).…”

“…RBD plasma antibodies were detected using an RBD-wt protein-binding ELISA as described earlier 43 . In brief, RBD-wt protein was coated overnight at 4°C at a concentration of 100 ng/well in 50 ૄl of bicarbonate buffer (pH=9.8) using U-shaped medium binding 96-well ELISA plates (Greiner Bio-One; Cat#:650001).…”

Resistance Evolution against Host-directed Antiviral Agents: Buffalopox Virus Switches to Use p38-ϒ under Long-term Selective Pressure of an Inhibitor Targeting p38-α

“…Activated p38 further induces activation of several transcription factors/effector proteins which eventually regulate several cellular processes such as cell cycle, apoptosis, cytoskeleton remodeling as well as inflammatory and immune responses ( Han et al 2020 ; Chander et al 2021 ). A wide variety of viruses are known to directly interact with different isoforms of p38 or their substrates during replication ( Marchant et al 2009 ; Mikkelsen et al 2009 ; Peng et al 2014 ; Su et al 2017 ; Nayak et al 2019 ; Datta et al 2021 ; Higgins et al 2021 ; Mudaliar et al 2021 ; Sugasti-Salazar et al 2021 ). While some of the interactions are pro-viral ( Zachos et al 1999 ; Adamson et al 2000 ; Marchant et al 2009 ; Mikkelsen et al 2009 ; Skaug and Chen 2010 ; Nagaleekar et al 2011 ; Peng et al 2014 ; Su et al 2017 ; Kumar et al 2018b ; Nayak et al 2019 ; Datta et al 2021 ; Higgins et al 2021 ; Mudaliar et al 2021 ; Sugasti-Salazar et al 2021 ), others suppress virus yield ( Penrose et al 2004 ; Schuck et al 2013 ; Wei et al 2013 ).…”