A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.

Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing.

The previous serological algorithm for Zika virus (ZIKV) comprised screening by anti-ZIKV IgM capture ELISA (MAC-ELISA) for samples collected within 3 months postexposure or onset (MPEO). Samples positive by MAC-ELISA and samples collected beyond 3 MPEO were tested by the confirmatory plaque reduction neutralization test (PRNT), which proved laborious and time-consuming during the 2015 outbreak. Thus, we evaluated several ZIKV ELISAs to establish an anti-IgM and anti-IgG combination for use as a screening tool for all samples prior to PRNT confirmation. The MAC-ELISA or InBios-M in combination with the Euroimmun-G demonstrated sensitivities of 99.1% and 97.2%, respectively, and nonflavivirus specificity of 96.0%. Their cross-reactivities were 71.4% and 50.0%, respectively, for sera positive for Dengue virus antibodies. Due to near-perfect interrater agreement with PRNT and excellent detection of samples collected beyond 3 MPEO, these combinations were recommended as a screening protocol in a new high-throughput algorithm with special considerations for ZIKV diagnostics.

Clinical utility of commercial enzyme immunoassays during the inaugural season of West Nile virus activity, Alberta, Canada.

West Nile virus (WNV) has spread rapidly across North America, creating a need for rapid and accurate laboratory diagnosis on a large scale. Immunoglobulin M (IgM) capture enzyme immunoassays (EIA) became commercially available in the summer of 2003, but limited data are available on their clinical performance. Consolidated human WNV diagnostic testing for the province of Alberta, Canada, at the public health laboratory permitted a large-scale evaluation of the assays, covering a wide clinical spectrum. Two thousand nine hundred sixty-nine sera were tested, from 2,553 Alberta residents, and 266 cases were identified. Sensitivities of the Focus assay and first-generation Panbio IgM capture EIA were 79 and 80%, respectively. During the first week of illness only 53 to 58% of cases were positive, but sensitivity was 96 to 97% after day 8. Sensitivity for neurological cases was 92% overall. Specificity was high for the Focus kit at 98.9%, but only 82.9% for the first Panbio kit. A positive Focus WNV IgG result with a twofold rise in IgG index was a reliable indicator of acute flavivirus infection (67/67 WNV). Agreement between the IgG test and hemagglutinin inhibition titers in paired sera was at least 82%. Commercial IgM and IgG EIA proved useful for WNV diagnosis, provided follow-up sera were collected after 8 days of illness.

Phylogenetic analysis of North American West Nile virus isolates, 2001-2004: evidence for the emergence of a dominant genotype.

The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus.

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.