The likelihood that the COVID-19 pandemic will not subside anytime soon has sparked intensive research on its detection and treatment, to prevent onward transmission. A new paper by researchers at McMaster University and Hamilton Health Sciences, published on the preprint server medRxiv* in September 2020, reports on the development of a serologic assay that is suitable for sensitive high-throughput antibody testing for COVID-19.
This transmission electron microscope image shows SARS-CoV-2, the virus that causes COVID-19, isolated from a patient in the U.S. Virus particles are shown emerging from the surface of cells cultured in the lab. The spikes on the outer edge of the virus particles give coronaviruses their name, crown-like. Image captured and colorized at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, Montana. Credit: NIAID
Detecting Asymptomatic COVID-19
COVID-19 symptoms usually appear 2-14 days after being exposed to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but many people are asymptomatically infected – in fact, up to 80% of cases do not display symptoms. Since such cases mediate viral spread in half of all known infections, serologic testing becomes vital to pick up actual infection rates.
The diagnostic test used in COVID-19 is a real-time reverse transcription-polymerase chain reaction (RT PCR) in which the viral RNA genome is amplified to allow detection. This viral nucleic acid is obtained typically from nasal or throat swabs. The chances of getting an accurate positive result from such swabs are highest in the four days before symptoms usually start. In fact, starting from the first day of symptoms, RNA detection is possible, peaking in the first week of infection. However, by week 3, PCR tests begin to revert to negative, unless in severe cases. A caveat is that PCR positivity indicates only the presence of viral RNA and not viable or infective virus. This test is about 80% sensitive but 100% specific.
It is evident that SARS-CoV-2 testing is essential for the containment of viral transmission, calling for highly accurate testing methods. One such option is serology, to detect an immune response and thus help pick up those who are infected among contacts, identify potential convalescent plasma donors and evaluate vaccine efficacy.
Anti-SARS-CoV-2 antibodies indicate that the person has perhaps developed protective immunity directed against the large spike glycoprotein S, composed of two domains, S1 and S2. The first contains the receptor-binding domain (RBD), which is bound by the majority of neutralizing antibodies bind.
The S protein exists in nature as a homotrimer, binding to the human cell receptor angiotensin-converting enzyme 2 (ACE2). Subsequently, virus-cell fusion is triggered, leading to the entry of the virus into the host cell.
Earlier studies indicate lower titers of specific IgG antibodies in people with asymptomatic or mild infection but high levels in those who have recovered from severe COVID-19.
Establishing a High-Throughput Assay
The current study describes the results of a new testing method used on ten PCR-positive convalescent patients, 4 PCR-negative individuals, and 332 pre-COVID controls. All patient samples were obtained between day 23 and day 89 of infection. Of the controls, 26 were healthy, while 306 were thought to have low platelet counts, either immune in nature or due to heparin usage.
The investigators then developed an ELISA test for the full-length S and RBD protein. They established the optimal concentrations of RBD and S to be 2 µg/mL (Figure 1A) and S protein at 5 µg/mL, in terms of characterizing positive and negative values. Similarly, the optimal serum dilution for a cut-off between positive and negative was found to be 1/100.
Strongly Reactive and Reproducible ELISAs
Convalescent samples were uniformly strongly reactive to both antigens, more against the full-length S than the RBD. Pre-COVID-19 samples were mostly non-reactive. They found that anti-S and anti-RBD IgM were least specific and IgA for both antigens, most specific among all antibodies.
They also ensured the assay results were reproducible by repeating the test four or more times, with minimal variability between assays. Many methods are used to ensure safety while handling virus-contaminated samples, such as heat-treatment and Triton X-100. They found that heat treatment affected the detection rate for IgG and IgM. This could be due to denaturation and aggregation of the antigens on heating. IgM has heavy chain structures that make it more vulnerable to thermal instability.
Treatment with Triton X-100 had no significant effect on sample reactivity in the patient samples. Antibody titers in the pre-COVID-19 titers became higher after treatment by either of these methods.
Inhibition of IgG binding to RBD
The COVID-19 patient samples were uniformly found to show inhibition of anti-RBD binding by about 80%, as was one control who tested positive for anti-RBD IgG. Validation of the assays was done using commercially available assays.
The researchers say, “We describe a high throughput, reproducible, semi-quantitative serological method to detect antibodies against SARS-CoV-2.” The assay measures all three antibody types that are reactive to both S and RBD viral proteins.
From earlier studies, it is apparent that all antibody isotypes increase steadily, with both IgA and IgM peaks at 1-3 weeks after infection, and subsequently IgG. The ability to measure all three isotypes, therefore, allows a broader visualization of the immune response with a lower risk of false-negative results.
Moreover, the full-length S and RBD antigens were used in the current assay. The specificity of such tests depends on many factors, including the type of test and antigen, as well as other host autoantibodies such as rheumatoid factor and other heterophile antibodies.
Cross-Reactivity with Other Coronaviruses
Cross-reactivity with antigens from other coronaviruses is also a significant factor that has been identified in commercially available SARS-CoV-2 ELISA kits and in the current assay. IgAs appear to be the least specific among COVID-19 antibodies. Thus, the reason for positive test results in 19 pre-COVID-19 controls may be cross-reactivity. The researchers say, “The pre-COVID-19 controls that had anti-RBD antibody were not false positive per se because antibody binding could be inhibited by excess RBD.” This is most likely to be due to cross-reactive antibodies generated to other seasonal coronaviruses.
The specificity is also a variable of the control population selected. The investigators found that with the optimized ELISA used here, the specificity was 94% or more based on the selected antibody class and antigen. These results are also in accordance with those reported using two commercial assays with sensitivity and specificity of 100% and 90%, respectively. In some earlier papers, however, the latter has been reported to have significantly lower sensitivity and specificity of 65% and 74%, respectively.
An advantage of the current ELISA testing approach is that it is rapid and straightforward, without the need for any specialized instruments. It is capable of being adapted to standard diagnostic laboratories and is highly reproducible between different assays when the tests are repeated.
This sensitive, specific, and reproducible high-throughput ELISA is likely to be useful in detecting infection rates and the changes in immunological responses to the virus in longitudinal studies. It can also complement viral RNA testing to allow researchers to pick up recent infections and describe the antibody response to SARS-CoV-2 more accurately. It could also help screen convalescent plasma donors and healthcare workers, so as to select those at least risk to serve COVID-19 patients of different categories. The duration of immunity could also be better understood by longitudinal serological studies on both symptomatic and asymptomatic individuals.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.