A Rapid Assay for SARS-CoV-2 Neutralizing Antibodies That Is Insensitive to Antiretroviral Drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. METHODS: The ability of 4 commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 patients who were SARS-CoV-2 PCR positive, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 patients who were SARS-CoV-2 negative. Serology results were compared to a cell-based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. RESULTS: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received 2 doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme NAbs assay correlated with the PSV SARS-CoV-2 median infective dose (ID50) neutralization titers (R2 = 0.70), while correlation of the Roche S-antibody assay was weaker (R2 = 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received 2 doses of the Moderna vaccine (ID50, 597) compared to individuals who received a single dose (ID50, 284). CONCLUSIONS: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Commercial Serology Assays Predict Neutralization Activity against SARS-CoV-2.

BACKGROUND: It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. METHODS: A neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR. RESULTS: The SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively. CONCLUSIONS: These 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

Multi-Platform Comparison of SARS-CoV-2 Serology Assays for the Detection of COVID-19.

BACKGROUND: COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that is responsible for the 2019 coronavirus pandemic. Acute infections should be diagnosed by polymerase chain reaction (PCR) based tests, but serology tests can demonstrate previous exposure to the virus. METHODS: We compared the performance of the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays using 179 negative participants to determine negative percentage agreement (NPA) and in 60 SARS-CoV-2 PCR-confirmed positive patients to determine positive percentage agreement (PPA) at 3 different time frames following a positive SARS-CoV-2 PCR result. RESULTS: At ≥15 days, the PPA (95% CI) was 100 (86.3-100)% for the Diazyme IgM/IgG panel, 96.0 (79.7-99.9)% for the Roche total Ig assay, and 100 (86.3-100)% for the Abbott IgG assay. The NPA (95% CI) was 98.3 (95.2-99.7)% for the Diazyme IgM/IgG panel, 99.4 (96.9-100)% for the Roche total Ig assay, and 98.9 (96.0-99.9)% for the Abbott IgG assay. When the Roche total Ig assay was combined with either the Diazyme IgM/IgG panel or the Abbott IgG assay, the positive predictive value was 100% while the negative predictive value remained greater than 99%. CONCLUSIONS: Our data demonstrates that the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays have similar clinical performances. We demonstrated a low false-positive rate across all 3 platforms and observed that false positives observed on the Roche platform are unique compared to those observed on the Diazyme or Abbott assays. Using multiple platforms in tandem increases the PPVs, which is important when screening populations with low disease prevalence.