Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility

Abstract

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

Tracking the incidence and risk factors for SARS-CoV-2 infection using historical maternal booking serum samples

“…This assay has a specificity greater than 99.6% (95% CI 99.6-100), defined by testing 825 serum samples that predated the COVID-19 pandemic, 100 of which were historic antenatal booking serum samples from 2018; and a sensitivity of 98.9% (95% CI 96.8-99.8) when evaluating 276 serum samples from individuals with RT-PCR-confirmed SARS-CoV-2 infection [16]. Further details are provided in Rosadas et al [17].…”

“…As set out in Rosadas et al [17], the cutoff for seroreactivity was established by adding 0.1 to the average of optical density (OD) obtained for three negative controls assayed in each run. The signal-to-cutoff value, known as the binding ratio (BR), for each sample was determined by dividing the sample OD by the cutoff OD.…”

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein

“…We then conducted ELISAs using deglycosylated S1 Spike proteins to determine if cross-reactivity was affected by the glycosylation of the S1 protein. Next, we compared the results of our SARS-CoV-2 S1 Spike ELISAs to results of other SARS-CoV-2 ELISAs using the receptor binding domain of Spike, the Nucleocapsid protein, and a combined S2 Spike and Nucleocapsid.We also tested whether cross-reactivity can be reduced with the Microimmune SARS-COV-2 Hybrid Double Antigen Bridging Assay (DABA) 16 or with an ELISA protocol modification incorporating an avidity wash with urea 14 . To test whether pre-pandemic samples reactive to S1 Spike correlated to immune responses to other antigens, we tested sample reactivity to a very large number of peptide and protein arrays using Rapid Extracellular Antigen Profiling and Serimmune.…”

“…The Hybrid DABA measures reactivity to S1 and RBD antigens in a two-step double antigen binding format to reduce non-specific binding and was performed according to manufacturer instructions 16 The next day, patient sera dilutions were prepared in concentrations of 1:10-1:1280 and were pre-incubated with Spike-expressing VSV pseudovirus for 1 h at room temperature with gentle agitation. Pseudovirus-sera mixtures were added to the adhered Vero-E6 cells at a final virus concentration of 1:10 volume/volume and incubated at 37 • C for 24 h. Cells were lysed using the Renilla Luciferase Assay System (Promega) according to manufacturer instructions.…”

“…Having identified that the molecular target of the cross-reactive malaria-associated antibodies was sialic acid, we hypothesized that these might represent low-avidity interactions that could be reduced with experimental modifications or alternatives. To this end we explored two alternative assays, an avidity assay with urea wash 14 and a hybrid DABA assay 16 . Twenty COVID-19 inpatient samples all tested positive by S1 subunit ELISA and by S1 subunit ELISA with a 4 M urea wash (Fig.…”