CONTINUING EDUCATION :: ANTIBODIES
antigen targets are known to be associ- ated with differing assay performance characteristics. Studies have shown that N and S1 ELISAs detect SARS-CoV-2 antibodies with high specificity, in comparison to the full length S protein due to the S2 subunit being highly con- served among coronaviruses compared to S1.19,27
been observed with N and other non-S targets compared to S1 targets.9
protein has shown high sensitivity and specificity for antibody detection.28
Increased cross reactivity has The RBD The
RBD antibody has a strong association with SARS-CoV-2 neutralizing antibod- ies (Figure 2).28
The region of the RBD is
poorly conserved among coronaviruses and is known to be a major target of human antibodies.29 A study published in Science Immunol-
Figure 3: IDSA Guidelines on the Diagnosis of COVID-19: Serologic Testing (
idsociety.org) Adapted from Hanson KE, et al. Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Serologic Testing. Infectious Diseases Society of America 2020; Version 1.0.0. https://www.
idsociety.org/practice-guideline/covid-19-guideline-serology/. Accessed October 10, 2021.
depending on their EUA intended use. These methods require either plasma or serum samples and are performed in a moderate- or high-complexity laboratory setting.
Neutralizing antibody (nAb) tests detect antibodies that bind to a specific part of the SARS-CoV-2 virus and have been observed to prevent infection by SARS-CoV-2 in vitro.21 Neutralizing an- tibody tests are required to be performed in a high complexity clinical laboratory. There are three types of nAb tests: Virus neutralization test (VNT), Pseudovirus neutralization tests (pVNT), and Com- petitive neutralization tests (cVNT).21,22 Currently, only one neutralizing antibody test (cVNT) has received an FDA EUA for the qualitative detection of nAbs.21 Additionally, the FDA requires SARS-
CoV-2 Ab test developers to continu- ally monitor for new and emerging viral mutations and variants, as well as their potential impact on test performance.23 For any viral mutations or variants that are identified as prevalent and/or clini- cally significant, test developers should assess whether the amino acid change(s) in the viral proteins are critical to their test design. If so, these mutations and variants should be evaluated using clinical speci- mens to assess the impact of the mutation or variant on test performance. The FDA states that the aggregate impact of the mutations should not reduce the clinical performance of a test by 5% or more or
10 DECEMBER 2021
MLO-ONLINE.COM
decrease the clinical performance point estimates for the test below the clinical performance recommendations.23
SAR-CoV-2 Ab assays target antigens Different SARS-CoV-2 Ab assays detect antibodies to different major viral anti- gens, such as the SARS-CoV-2 nucleo- capsid (N) protein, spike (S) protein (including subunits S1 and S2), and RBD (recep- tor binding domain) of the S protein.24
The
N protein, an im- munodominant protein, is highly conserved among the CoV genus and is found within the virus on the nucleocapsid.21,25 Facilitating viral entry into host cells, the S protein is located on the surface of virus, protruding from the envelope, and is comprised of three seg- ments: the ectodomain, single-pass transmembrane, and intracellular tail.26
ogy in June 2020 concluded that utilizing recombinant SARS-CoV-2 RBD antigen for the detection of antibodies induced by SARS-CoV-2 is highly sensitive and spe- cific.29
Antigen targets also influence the clinical information that can be rendered by a specific SARS-CoV-2 Ab test. For example, the majority of individuals that recover from infection with SARS-CoV-2 elicit antibody responses to N, S, RBD and other proteins. 6,21
In contrast, because all
current EUA COVID-19 vaccines utilize only mRNA sequences encoding for the SARS-CoV-2 S protein,14-16
vaccinated
individuals (that have not had a previous infection with SARS-CoV-2) have been observed to only have antibodies to the SARS-CoV-2 S and RBD proteins.21
SARS-CoV-2 Ab assays and isotype detection SARS-CoV-2 Ab tests collectively can detect a variety of immunoglobulin isotypes or isotype combinations. There
The ectodomain consists of the
S1 subunit, containing the RBD, and the S2 subunit.26
An antigen target(s) is an important factor when evaluating a test, as different
Photo Courtesy of LumiraDx
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