High-performance ELISA for serology in mild-moderate non-hospitalized COVID-19

By Dr. Liji Thomas, MDDec 7 2020

Epidemiologic surveillance of COVID-19 patients is an essential part of pandemic monitoring. Not only does this provide a measure of exposure at the population level, but it helps understand the antibody correlates of protection against reinfection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In turn, such understanding will help to shape public health policy.  

Precision of the human anti-IgGAM SARS CoV-2 ELISA. Precision was measured using clinical and lab standards guidelines EP12-A2 in 40 replicates and assessed at clinical cut-offs C5, C50 and C95. The mean and coefficient of variation (%) was calculated for each clinical cut-off.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Low Performance at Low Antibody Levels

A continuing challenge in the surveillance of COVID-19 is the large proportion of mild or moderate COVID-19 cases. The relative insensitivity of many commercially available assays makes their serologic detection, based on anti-SARS-CoV-2 antibodies, difficult.

A new study published on the preprint server medRxiv* in December 2020 describes an ELISA assay that combines the detection of IgM, IgA, and IgG antibodies against the SARS−CoV−2 trimeric spike glycoprotein, with high sensitivity and specificity.

High-Performing ELISA Validated

The researchers describe the validation of a combined ELISA developed with the use of recombinant SARS-CoV-2 spike protein in adults with mild or moderate disease who were not hospitalized with COVID-19. They used the serum and dried blood spots (DBS) from adults with confirmed infection, that is, with positive polymerase chain reaction (PCR) tests. All were 14 or more days from symptom onset. They also collected the same samples from COVID-19-negative individuals.

All serum samples were stored at -20°C and thawed before testing. A pilot study was carried out using 624 and 62 COVID-19 negative and PCR-confirmed samples from non-hospitalized, mild or moderate COVID-19 patients. The researchers observed the sensitivity of the test to be 98.5% and the specificity to be 97.6%. They found that at least 167 PCR-positive samples from such patients would be required to validate the sensitivity of the ELISA in clinical samples.

Next, the manufacturing process was validated in 359 COVID-19 negative samples and 73 PCR-confirmed non-hospitalized, mild, or moderate COVID-19 samples, with the assay threshold being 1. This showed the sensitivity and specificity to be 98.6% and 98.3%, respectively.

Advantages of the Current ELISA

The assay was tested and validated for its precision, and it was shown to be free of interference from hemoglobin, bilirubin, or triglycerides at high concentrations. Either dried blood spots or serum could be used, and the results showed a strong correlation.

The ELISA was also proved to be non-reactive against eight other human viruses including coronaviruses. This indicates its high specificity, since alpha- and beta-coronaviruses are responsible for almost a third of common colds.

The kit was also found to retain its activity for 15 days after opening it, and for one day under extreme temperature conditions.

Next, the researchers validated the assay using clinical samples, with a group of 426 COVID-19 negatives and 226 PCR-positive patients. The other criteria were as before. They found the sensitivity to be 94.7% and the specificity 98.4%, for anti-IgGAM SARS-CoV-2.

Sensitive and Specific at Low Titers

An earlier study showed that the RBD, S1 subunit, and nucleocapsid antigens were all good targets for serologic assays in patients with severe COVID-19, but their effectiveness was limited at lower antibody titers. One reason is that current serologic tests have been developed and validated on samples from hospitalized patients.

In choosing to develop and test the performance characteristics of the current ELISA in samples from non-hospitalized adults who were 2 or more weeks from symptom onset, the researchers ensured the test would recognize antibodies at lower concentrations.

Broad Spectrum of Recognition

This assay recognizes epitopes over almost the whole of the spike trimer, extending its range over a much greater number of sites. Recent studies suggest that neutralizing anti-SARS-CoV-2 antibodies target not just the receptor-binding domain (RBD) but many other epitopes across the spike glycoprotein with high affinity and cross-reactivity.

The ability to detect IgA, IgG, and IgM simultaneously is important because of the uncoordinated development of these different antibody classes. For instance, some studies show that IgM may be detected in a few patients with the asymptomatic infection without IgG, while others indicate seroconversion for both classes occurs in synchrony.

However, yet others show that IgGAM occurs together in only half the patients. About 10% develop one of these antibodies first, 10% develop IgG and IgM together, while 3% each develop IgA with either IgG or IgM simultaneously.

In adults, the duration of the immune response is still unclear but may last for about 50 days, for IgA and IgM, with IgG being detected at 75 days or even longer. Recently, antibodies have been found to persist at 6 months.

Advantages Associated with DBS

The fact that DBS or serum may be used also makes the test ideal for remote samples, which has multiple advantages. DBS requires very little blood, reduces the risk of bacterial contamination, is non-invasive, can be easily posted to a testing center, remains stable for long periods, are cheap, and provide excellent results, while avoiding the need for patients and testing personnel to have close personal contact, reducing the risk of infection.

This risk is significant, with researchers reporting multiple clusters of infection among hospital patients in one center, and the tracing of 15% of COVID-19 among hospitalized patients to viral spread within the hospital. It also provides safe testing for high-risk segments of the population.

Conclusion

We demonstrate the human anti-IgGAM SARS-CoV-2 ELISA to be a reliable assay for future seroepidemiological studies of the general population, the majority of whom will not require hospitalization following infection with SARS-CoV-2.”

Both accurate and sensitive in its detection of antibodies targeting the virus in adults with mild-to-moderate disease, this test is also highly suitable for widespread and safe screening, with high accessibility for all segments of the population.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources