key: cord-0832815-fisdjxzy
authors: Slev, Patricia R.
title: SARS-CoV-2 Serology Testing – A Laboratory Primer
date: 2021-11-03
journal: Clin Lab Med
DOI: 10.1016/j.cll.2021.10.003
sha: f4d08e778df29678f6f718878077c9690c54e52f
doc_id: 832815
cord_uid: fisdjxzy

In 2019 an emerging coronavirus, SARS-COV-2 was first identified. In the months since, SARS-CoV-2 has become a global pandemic of unimaginable scale. In 2021, SARS-CoV-2 continues to be a huge public health burden and a dominating issue in healthcare. In addition, SARS-CoV-2 has placed a spotlight on laboratory medicine and its’s key role in infectious disease management. The SARS-CoV-2 antibody testing landscape is vast and consists of dozens of antibody tests that have received EUA. The laboratory is faced with choosing the right test, staying current with the rapidly evolving recommendations and updating test information for clients and clinicians. This review addresses what we know about the humoral response in SARS-CoV-2 infection and how this knowledge translates into appropriate serology test choice, utility and interpretation.

In 2019 a new coronavirus virus, SARS-CoV-2, emerged that would lead to a 94 worldwide pandemic and highlight the importance of laboratory medicine in infectious 95 disease management 1 . In 2021, SARS-CoV-2 remains a priority for laboratory testing. 96 Although diagnostic testing to determine who was infected with the virus was at the 97 forefront of the pandemic, as serology testing became available, public interest in 98 testing quickly rose and demanded that laboratories offer serology testing, even though 99 antibody testing utility was limited. In the early days of the pandemic, March and April 100 2020, serology testing was not recommended for clinical purposes and was deemed of 101 limited clinical value 2,3 . Therefore, the FDA did not see a need for strict regulations for 102 antibody testing. This led to a proliferation of SARS-CoV-2 antibody tests, dominated 103 early on by lateral flow assays (LFA) imported from various parts of the world. At the 104 time, the FDA only required that the manufacturer notify the FDA of their intent to bring 105 an antibody assay to market without any data requirements to support the performance 106 characteristics of the assay. The consequence was a rapid and unprecedented 107 proliferation of unvalidated, expensive assays quickly made available to anyone who 108 wanted access. In addition, many were confused about rapid tests and incorrectly 109 assumed that because of the ease of use that these rapid tests could be used in any 110 setting, such as physicians' offices, without laboratory oversight or validation. The 111 combination of public curiosity as to whether they had been infected with the virus and 112 the lack of validated antibody tests used indiscriminately in any setting was 113 accompanied by considerable amount of bad press because many of the assays were 114 J o u r n a l P r e -p r o o f inaccurate. This situation quickly escalated and highlighted the need for quality serology 115 tests, FDA oversight and the importance of the laboratory in validating serology assays. 116 In early May 2020, the FDA issued new guidance for Emergency Use Authorization 117 (EUA) claims for serology assays, that stated that, although manufacturers could notify 118 the FDA of their intent to bring a serology assay to market as a first step to obtaining 119 EUA, the manufacturer also had to provide supporting data to the FDA within 10 days of 120 the notification. In addition, the FDA instituted an umbrella protocol that allowed for 121 serology assays to be independently evaluated through NIH by agencies such as the performance characteristics (please refer to section on serology assay evaluation).

The pandemic and serology testing have rapidly evolved and today we have a 128 plethora of EUA serology assays available, and the list is still growing every day. There 129 have been 21 new serology assays approved just since January 1, 2021. The good 130 news is that many advances have been made and there are many high quality assays 131 but there is now increased confusion about test choice, test utility and test result 132 interpretation. The SARS-CoV-2 EUA serology testing landscape has been recently 133 reviewed by Ravi and colleagues 4 . Confusion is driven not just by the large number of 134 assay options but also by the rapidly evolving science about antibody kinetics, antibody While neutralizing antibody assays provide a functional indication of the immune system Due to the role of neutralizing antibodies, many studies have investigated 161 correlation between commercial serology assays that measure binding antibodies and 162 neutralization assay results. There is a general qualitative agreement and a positive 163 correlation between binding and neutralizing antibody assays. Studies also show that 164 not surprisingly, there is a higher degree of correlation between neutralizing antibody 165 assays and binding antibody assays that use the spike protein as a target 11, 12, 13 . Ongoing studies will further refine these findings. have become the most commonly utilized assays because antibody testing is not 265 recommended for diagnosis. Therefore, assays that detect IgG or total antibodies can 266 be used to determine exposure and are the most widely utilized.

Antibody isotype is just one of the SARS-CoV-2 serology assay attributes that a 268 laboratory must consider when choosing which SARS-CoV-2 assay to implement. there is only one assay that has received EUA that specifically detects neutralizing 290 antibodies. This is in part because developing a neutralizing antibody assay that can be 291 adapted to a clinical laboratory is difficult to achieve. The gold standard for measuring 292 neutralizing antibodies (Nab) is the plaque reduction neutralization test (PRNT). A 293 classic PRNT assay determines the serum dilution that inhibits viral growth (50% or 294 90% inhibition) in cell culture and can therefore provide a titer. However, these assays Clinicians and epidemiologists may want to determine who has been exposed to 401 infection and who has been vaccinated. Because the spike protein is the target of the 402 vaccines that have been approved to date in the US, it is reasonable to think that one 403 can distinguish between these two scenarios by testing for spike and nucleocapsid 

In addition, assay performance characteristics not only vary between assays, but 419 even small differences in specificity and sensitivity between assays can translate to 420 substantial differences in positive predictive value (PPV) and negative predictive value 421 (NPV) depending on disease prevalence. For example, an assay that has 98.1% 422 sensitivity and 99.6% specificity that translates into 99.9% NPV and only 92% PPV 423 when disease prevalence is 5.0%. If the disease prevalence is 10% the NPV only drops 424 to 99.8% but the PPV increases to 96.1%. It is understandable that PPV was of 425 particular concern during the early days of the pandemic, when disease prevalence was 426 low. Therefore, the CDC made the following recommendation in order to increase 427 positive predictive value (PPV): 1) test only individuals who have a high likelihood of 428 exposure to SARS-CoV-2, 2) test with an assay that has >99.5% specificity, and 3) if 429 not possible to test with an assay that has >99.5% specificity then implement an 430 orthogonal approach to testing 16 .

The orthogonal approach to testing is based on testing with one serology assay 432 and if the sample is positive by the first assay, then the sample is tested by a second 433 assay. Ideally, the assay with the highest specificity should be used first to minimize 434 discrepant results between the two assays used in an orthogonal testing approach.

Otherwise, the assays used in this type of algorithm can be same antigenic target but 436 different method (ELISA spike and CIA spike), or same method but different antigenic 

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