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Viral Testing Summary underlying immune competence, such as specific
immunoglobulin deficiencies, will also affect whether they
Samples • Sputum, if available, then nasopharyngeal, are capable of mounting an efficient immune response
nasal, and lastly, oropharyngeal swabs. resulting in positive antibody titers.
Saliva testing is now also available and As immunoassays are developed, it becomes important
works best when collected from a deep to determine the most appropriate viral antigens to use.
cough after arising. There are many companies currently developing serology
• Take samples within seven days of testing, and many are not forthcoming as to the antigen
symptom onset. being used in their assay. Recent studies have looked at the
Methods • Ensure viral transport medium is not whole spike protein (S) or the S1, S2, or receptor-binding
expired and follow proper collection
techniques. domain (RBD) subunits of S, in addition to the nucleoprotein
• Send for RT-PCR at FDA, (N), envelope (E), and the membrane protein (M). The S
EUA-approved CLIA lab. and N proteins are the main immunogenic proteins, while
Timing • Viral shedding can begin 3-21 days after S1 and RBD correlate with neutralizing antibodies (NAbs),
exposure. since they are the proteins on the viral surface that facilitate
• Viral RNA shedding can occur for up to entry into the host cell. Neutralizing antibodies not only
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21 days after symptom resolution. bind to a viral protein but are capable of blocking a viral
• Testing evaluates viral RNA, not shedding infection by neutralizing or inhibiting its biological effect.
of intact virus This is opposed to binding Abs, which bind to a specific
(i.e., infectivity). antigen flagging them. The antibody-antigen complex can
• It appears that intact viral shedding is, in 6
most cases, complete 14 days after then trigger T cells to destroy the flagged antigen. NAbs
symptom onset—note that this differs can directly inhibit viral infection without the need for
from the time course of viral RNA further cellular immune support, and are thus key to
shedding, as measured by NAA. preventing subsequent infection. This explains why
therapeutic convalescent plasma is effective if high levels of
NAbs are present.
Antibody Testing The choice of viral antigen used for the immunoassay
Serological Testing is also important because other coronaviruses have similar
Serological testing is used for assessing an individual’s proteins, and an immune response to a previous coronavirus
immune response to SARS-CoV-2 exposure. The addition infection may cross-react, resulting in a false positive test
of serological testing to viral RNA testing dramatically (specificity). Not all individuals will mount an Ab response
increases the sensitivity of a COVID-19 diagnosis and to every viral antigen, making the choice of viral antigen
facilitates assessment of past exposure (especially in also important for increasing the sensitivity of the test.
asymptomatic cases). 4,8,17 Serological testing from blood, Thus, the level of sensitivity and specificity of each test will
serum, or plasma determines if one or more vary with the antigen used, the serotype of the antibody
immunoglobulin subtypes (IgM, IgA, or IgG) are present being tested, and the parameters of the assay itself. 1
to specific viral antigens. Despite immense variability, most individuals develop
The development of specific antibodies to different some type of seropositivity within seven to ten days
protein components of a virus occurs in a sequential following onset of symptoms. Testing prior to seven days
manner, with IgM and IgA arising initially, followed by post symptom onset is likely to yield a negative antibody
IgG, which confers long-term immunity. IgM tends to be result. In one study, the positive rate for IgG or IgM at less
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the more general, immediate response immunoglobulin, than seven days was 38%, while the rate at greater than
while subsequent isotype switching to IgG selects for seven days was 89%. Another study in 23 hospitalized
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antibodies with higher binding affinity. IgA is most often patients revealed that by day 14, rates of seropositivity
found at the mucous membranes. Current evidence were 94% for anti-NP IgG, 88% for anti-NP IgM, 100% for
supports this immunological paradigm in SARS-CoV-2 anti-RBD IgG, and 94% for anti-RBD IgM, indicating that
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infection as well. 3,5,10 While it appears that close to 100% of testing later in the time course of the disease is more
individuals who are infected (PCR positive) go on to sensitive and that there is variability in the immunoglobulin
develop some type of antibody response to the virus, there isotype and the viral antigens.
is tremendous individual variability in the time course of
antibody production, the amount of antibody produced, Types of Antibody Testing
and the specificity of the antibodies, i.e., which viral Laboratory Standard = ELISA Testing
proteins are recognized. Antibody levels also appear to Enzyme-linked immunosorbent assay (ELISA) is a
correlate with severity of disease and presence of plate-based technique that uses whole blood, plasma, or
comorbidities. More severe disease results in higher viral serum samples from patients for testing. The antigen of
load and also higher antibody titers. An individual’s interest (for example, the S protein) is coated on the
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48 Integrative Medicine • Vol. 19, No. S1 • Epub Ahead of Print Messier—Primer on SARS-CoV-2 Testing