Researchers develop assay for qualitative assessment of neutralizing SARS-CoV-2 antibodies

Researchers develop assay for qualitative assessment of neutralizing SARS-CoV-2 antibodies

Extensive testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been a primary containment measure to contain the ongoing pandemic. The infection causes coronavirus disease 2019 (COVID-19), which has so far claimed over 2.7 million lives worldwide. The need for effective therapeutic solutions is great, also given the ongoing vaccination programs against SARS-CoV-2 in many parts of the world. One such plasma-based therapeutic approach is the use of neutralizing antibodies from the blood of convalescent patients.

To address the need for a high-quality neutralization test against SARS-CoV-2, researchers at the National Institute of Allergy and Infectious Diseases (NIAID) modified and optimized a previously established fluorescence reduction neutralization (FRNA) test to enable qualitative assessment of a large number of infected cells through the use of a high-content imaging system.

In this study, the team described a semi-high-throughput, highly quantitative neutralization assay for SARS-CoV-2 based on the Operetta high-content imaging system. The authors claimed that similar devices (which may not be available in many laboratory settings) could be equally effective with appropriate validation.

They reported that as of February 2021, SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

"While there will always be variability in live virus assays due to the nature of the biological system, using a rigorous statistical approach to inform data acceptability can mitigate the potential negative effects of poor infection efficiency, pipetting errors, edge effects, and inconsistent staining."

COVID-19 is a heterogeneous disease that often manifests as severe respiratory diseases, cardiovascular diseases and neurological diseases. During an acute illness there is a rapid change in the antibody class from immunoglobulin M (IgM) to IgG and IgA. The researchers note that the antibody isotype is just as important in controlling the disease as the viral target protein.

The researchers observed that disease severity is associated with more robust and longer-lasting antibody responses to the viral nucleoprotein (N). Therefore, the presence of anti-SARS-CoV-2 antibodies in the blood is believed to be a good measure of the protective immunity of a vaccine candidate. This requires rapid methods that are reliable and sensitive to detect SARS-CoV-2 neutralizing antibodies.

Immunofluorescence staining of SARS-CoV-2-infected cells.A. Uninfected cells stained with Hoechst nuclear stain (blue). B. Cells infected with SARS-CoV-2 and examined with a SARS-CoV N protein-specific antibody and an Alexa594 secondary antibody (red). Cells were counterstained with Hoechst nuclear stain (blue).

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article.View sources

The FRNA is a highly specific, quantifiable and rigorous test for evaluating neutralizing antibodies in a test sample. The assay quantifies individual cells infected with the initial addition of virus and does not require multiple rounds of virus replication.

As an atypical test, it does not rely on the subjective determination of cell cytopathic effects nor on the development of multicellular plaques or immune foci using small numbers of infectious particles per well. The researchers examined the virus control and cell control observations for outliers and assessed the variability and specificity of the assay.

Because the assay uses 12-step dilution schemes and four-parameter logistic analysis to quantify a specific NT50, it allows for a more precise understanding of neutralization capacity, particularly in the case of monoclonal antibodies or nanobodies.

In this study, researchers described the development of a semi-high-throughput SARS-CoV-2 neutralization assay that leverages the capabilities of a high-content imaging system to quantify the number of infected cells in individual wells. Importantly, the researchers showed that the test could be quickly adapted for use with multiple virus variants. Such tests help immensely and accelerate preclinical vaccine studies and clinical trials.

During the COVID-19 crisis, the US Food and Drug Administration (FDA) initially approved an Expanded Access Program (EAP) to treat COVID-19 patients with plasma from individuals with a neutralization titer of 1:160 or higher. This program has treated over 94,000 patients across the United States. Last August, the FDA issued an Emergency Use Authorization (EUA) to allow therapeutic plasma treatment of COVID-19 patients outside of clinical trials.

The researchers claim that this test does not require subjective interpretation and is therefore more precise than most other tests for neutralizing wild-type viruses.

“Although there will always be variability in live virus assays due to the nature of the biological system, using a rigorous statistical approach to inform data acceptance can mitigate the potential negative effects of poor infection efficiency, pipetting errors, edge effects and inconsistency stains.”

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article.View sources

Article revisions

• 5. April 2023 – Die vorab gedruckte vorläufige Forschungsarbeit, auf der dieser Artikel basiert, wurde zur Veröffentlichung in einer von Experten begutachteten wissenschaftlichen Zeitschrift angenommen. Dieser Artikel wurde entsprechend bearbeitet und enthält nun einen Link zum endgültigen, von Experten begutachteten Artikel, der jetzt im Abschnitt „Quellen“ angezeigt wird.

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