One of the big stories of the coronavirus pandemic has been COVID-19 neutralizing antibodies (NAbs). NAbs are produced by B cells within the immune system and are characterized by stopping infections through incapacitation of the invading pathogen.
Coronavirus NAbs were heralded as a possible treatment and diagnostic target from the moment of the announcement of their discovery in May 2020.
NAbs from SARS-CoV-2 usually form approximately 1-2 weeks following infection. Serologic tests cannot diagnose acute infections but can expand the time for the testing window to beyond the acute infection phase.
The assays are used to assist in the tracking of infection chains and can also be used to answer epidemiological, virological, and clinic questions, including, but not limited to, whether a robust antibody response has occurred and how many infected individuals have remained asymptomatic.
The research use-only (RUO) designation has been applied to almost all coronavirus NAb assays. This designation carries no warrantee except for their contents and also does not indicate any level of adherence to the quality or regulatory directives.
In cases where there is provided validation, documentation, or certifications of quality or purity, it is to differentiate products from each other, not as a regulatory requirement.
Conversely, IVDs, or in vitro diagnostics, are highly regulated and approved medical devices that are developed in accordance with ISO 13485 guidance and following jurisdictional regulations like the European In Vitro Diagnostic Regulation and CE marking.
An exercise in risk management
The journey from RUO to IVD can be challenging due to patient variability, and this still applies to coronavirus NAbs. The Director of International Sales and Marketing for EUROIMMUN, a PerkinElmer company, has remarked, “the concentration of NAbs in the blood varies greatly from person to person.”
The correlations between disease immunity status and antibody level become less clear when there is a wide range of NAb titers; this can be a problem in the world of health regulation where this lack of clarity is equated to risk.
Regulators of IVDs are concerned mainly with risk, and that the device does what it promises, nothing more and nothing less. The performance data required depends on the complexity and type of test. Which include clinical data supporting limits of detection, reproducibility, and performance in the presence of interfering substances. Commercializing an IVD is not trivial. It requires significant capital plus a network of teams working towards a common goal. At its core, IVD approval is a task in risk management.”
Marcus Manocha, Custom Services Manager, Absolute Antibody
Regulatory bodies have allowed exceptions to the expected approval protocols for COVID-19 serologic (antibody) diagnostics as they have with therapeutics based on coronavirus NAbs. The Agency does this through the use of EUAs, or Emergency Use Authorizations.
EUAs are issued by the FDA as needed to bypass years or months of development but only remain active for the emergency’s duration.
Currently, it is unclear what lessons regulators will learn from 2020 and how they will be applied to the revision of the risk-benefit algorithms that will be applied to future device and drug approvals; it is possible one result will be more streamlined licensing of critical products that follow under their purview.
PRNT and improved PRNT
Among the various immunoglobulins raised in response to infection, coronavirus NAbs are unique. This is because they block the interaction between ACE2 (the cognate receptor) and the coronavirus spike protein receptor binding domain.
Or, they prevent the transferral of genes to host cells by viruses. The placement of the designated signal cutoff dictates the interpretation of semi-quantitative NAb assays either way.
Assays like ELISA, lateral flow, and bead-based assays detect binding, not functional inhibition of infection. Aan ideal assay measures NAb levels as well as their activity in blocking infection.”
Anis H. Khimani, Senior Strategy and Market Segment Leader, PerkinElmer
A test known as Anti-SARS-CoV-2 QuantiVac Elisa (IgG) was recently released by PerkinElmer’s EUROIMMUN business unit. This test incorporates a recombinant S1 subunit of the SARS-CoV-2 spike protein.
Fencher has said, “this assay detects and quantifies anti-S1 IgG antibodies in standardized, comparable units referenced to the World Health Organization's standard for Anti-SARS-CoV-2 IgG, and correlates well with an FDA EUA Neutralization Antibody Detection Kit.”
He added, "this assay has already been CE-marked and we plan to file a request for its emergency use authorization with the FDA."
PRNT, or the plaque reduction neutralization test, uses appropriate host cells and virus suspension to detect levels of neutralization or infection and is the gold standard for neutralization assays.
This process requires Biosafety Level 3 facilities and is time-consuming and cumbersome. For these reasons, there is more general use of the more convenient and higher-throughput ELISA-based NAb assay kits.
Automated plate-imaging capabilities have been developed for PRNT assays, which maintains test consistency and accuracy while providing assay throughput.
Khimani notes, “migrating assays from the conventional 24-well plate assay format to 96-well or higher formats, combined with imaging capabilities and algorithm-supported data analysis, significantly enhances the value of NAb detection.”
Additionally enhanced are the utility and general accessibility of PRNT.
Post-treatment or vaccine-induced protective immunity are quantified during clinical trials through the use of NAb assays in addition to their use for therapeutic and surveillance work.
Product Development Scientist at ACROBiosystems, Prajwal Paudel, comments “NAb assays can also assess the effectiveness of vaccines in generating immunity against novel strains.”
He has additionally stated, “NAb tests cannot, however, diagnose an active infection.” The CE mark has been awarded to ACROBiosystems for their assay kit for measuring the NAb blocking of SARS-CoV-2 RBD binding to ACE2 protein.
The lengthy 510k IVS approval process is sidestepped by EUAs. However, EUAs still require the disclosure of calibration and control material(s), product manufacturing, assay steps, and test principle, as well as setting criteria for result interpretation, assay performance, processing and collection methods, and sample qualification.
Paudel has told Biocompare that “particularly for COVID-19 serologic tests, regulators require evaluation in at least 30 unique SARS-CoV-2 RT-PCR positive patients as well as 75 negative samples to provide results with 95% confidence interval."
Assay cutoffs should be established through the use of independent samples, and tests must demonstrate sensitivity with 95% negative percent agreement and 90% positive percent agreement with the comparator serum PRNT assay.
Paudel says there is an assessment of cross-reactivity with 12 viral infection samples, including respiratory syncytial virus, HIV, common cold coronavirus, and influenza. It is also recommended that there are at least anti-HIV positive samples.
Currently, there is no requirement to provide mutant strain reactivity or inclusivity in the EUA application. Not yet, but how those variants affect the NAb protectivity in vaccinated and recovered population need to be closely monitored. ACROBiosystems have been quickly developing SARS-CoV-2 mutant proteins to support these kinds of studies, such as the UK B1.1.7 and South African variants."
Prajwal Paudel, Product Development Scientist, ACROBiosystems
ACROBiosystems is a leading manufacturer of recombinant proteins and other critical reagents to support the development of target therapeutics. The company employs an application-oriented development strategy, with a particular focus on product design, quality control, and solution-based support. Our products and services enable anyone in the field of drug development to have a more intuitive and streamlined process.
To respond to the coronavirus pandemic, ACROBiosystems has developed SARS-CoV-2 antigens specifically designed and optimized for serological test kits, including Spike-derived antigen S1, RBD, and Nucleocapsid protein. Proteins have been supplied to diagnostic companies in large quantities.
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