COVID-19 neutralizing  antibodies (NAbs)  have been one of the big stories  of the coronavirus pandemic. Produced by immune system B cells, NAbs stop infections by incapacitating the invading pathogen. From the moment their discovery was announced in May 2020, coronavirus  NAbs were touted as possible treatments and diagnostic targets.

SARS-CoV-2 NAbs usually form around 1-2 weeks after  infection. Serologic tests thus expand the testing time window beyond the acute  infection phase, but they cannot diagnose acute infections. The assays also  help track infection chains and answer clinical, virological, and  epidemiological questions, including how many infected individuals remain  asymptomatic, and whether a robust antibody response has occurred.

Almost all coronavirus NAb assays carry the  research use-only (RUO) designation. RUO does not imply any level of adherence  to regulatory or quality directives, and products designated as such carry no  warrantee except for their contents. Documentation, validation, or certifications  of purity or quality are provided for product differentiation, not to satisfy  regulations.

By contrast in vitro diagnostics (IVDs) are  approved, highly regulated medical devices whose development follows ISO 13485  guidance as well as jurisdictional regulations like CE marking and the newer  European In Vitro Diagnostic Regulation.

An exercise in risk management

Patient variability always makes the journey from  RUO to IVD challenging, and coronavirus NAbs are no exception. "The  concentration of NAbs in blood varies greatly from person to person," says  Kai Fechner, Director of International Sales and Marketing for EUROIMMUN, a  PerkinElmer company.

A wide range of NAb titers means that correlations  between antibody levels and disease or immunity status becomes murkier, and in  the world of health regulation murkiness equals risk.

"Regulators of IVDs are concerned mainly with  risk, and that the device does what it promises, nothing more and nothing  less," says Marcus Manocha, Custom Services Manager at Absolute Antibody.  "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 toward a common goal. At its core IVD approval is a task in risk  management."

As with therapeutics based on coronavirus NAbs,  regulators have made exceptions to normal approval protocols for COVID-19  serologic (antibody) diagnostics, which the Agency approves through Emergency  Use Authorizations (EUAs).

FDA issues EUAs as needs arise, bypassing months or  years of development, but EUAs remain in effect only for the duration of the  "emergency." It remains to be seen whether regulators will apply  lessons learned during 2020 toward revising the risk-benefit algorithms they  apply to future drug and device approvals, perhaps leading to more streamlined  licensing of critical products under their purview.

PRNT and improved PRNT
Coronavirus NAbs are unique, among the various  immunoglobulins raised in response to infection, in that they block the  interaction between the coronavirus spike protein receptor binding domain and  its cognate receptor, ACE2. Or, they prevent viruses from transferring genes to  host cells. Either way, interpreting semi-quantitative NAb assays depends on  where the designated signal cutoff is set.

"Assays like ELISA, lateral flow, and  bead-based assays detect binding, not functional inhibition of infection,"  says Anis H. Khimani, Senior Strategy and Market Segment Leader at PerkinElmer.  "An ideal assay measures NAb levels as well as their activity in blocking  infection."

PerkinElmer's EUROIMMUN business unit recently released a test,  Anti-SARS-CoV-2 QuantiVac ELISA (IgG), which incorporates a recombinant S1  subunit of the SARS-CoV-2 spike protein. "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 a FDA EUA Neutralization Antibody Detection  Kit," Fechner says. "This assay has already been CE-marked and we plan to file a request for its emergency use  authorization with the FDA."

The gold standard for neutralization assays is the  plaque reduction neutralization test (PRNT), which uses virus suspension and  appropriate host cells to detect levels of infection or neutralization. PRNT is  cumbersome and time-consuming, and requires Biosafety Level 3 facilities, which  is why higher-throughput and more convenient ELISA-based NAb assay kits are  more generally used.

PRNT assays have been developed with automated  plate-imaging capabilities, which provide assay throughput while maintaining  test accuracy and consistency. "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," Khimani says. General accessibility and utility  of PRNT assays is enhanced as well.

ACROBiosystems has launched a ready-to-use  neutralizing antibody titer serologic  assay kit  that can sensitively and  specifically detect COVID-19 neutralizing antibodies in 2 hours, showing a high  correlation with FDA EUA approved ELISA kit.

Side-stepping

In addition to surveillance and  therapeutic uses, NAb assays are used during clinical trials to quantify  vaccine-induced or post-treatment protective immunity. "NAb assays can  also assess the effectiveness of vaccines in generating immunity against novel  strains," says Prajwal Paudel, Product Development Scientist at  ACROBiosystems. “NAb tests cannot, however, diagnose active infection.  ACROBiosystems has also received CE  Mark for our assay kit that measures NAb blocking of SARS-CoV-2 RBD binding to  ACE2 protein.

An EUA designation sidesteps the  lengthy 510k process for approving IVDs but still sets criteria on sample  qualification, collection and processing methods, assay performance and result  interpretation, plus requires disclosures of test principle, assay steps,  product manufacturing, as well as control(s) and calibration materials.  "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," Paudel  tells Biocompare. Tests must demonstrate sensitivity with 90% positive percent  agreement and 95% negative percent agreement with the comparator serum PRNT  assay, and assay cutoffs should be established using independent samples.

Cross-reactivity with 12 viral  infection samples that include influenza, common cold coronavirus, HIV, and  respiratory syncytial virus is encouraged to be assessed as well, Paudel says,  and recommend to include at least 10 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."

(Source: https://www.biocompare.com/Editorial-Articles/572420-Neutralizing-Antibodies-in-the-Fight-against-COVID-19/)