Current assays reflect non-neutralizing antibodies, finds study

By Dr. Liji Thomas, MDJul 14 2020

Several serologic tests have been developed to help diagnose the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), either currently or in the past. However, all of them are not equally effective.

A new study published on the preprint server medRxiv* in July 2020 examines the performance of two serologic tests (DiaSorin and Roche) on serum samples to try and correlate the antibody level with neutralizing antibody activity.  At the same time researchers looked for possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2.

Study: Poor correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects. Image Credit: Catalin Rusnac / Shutterstock

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

Several older studies have examined the development of antibodies to SARS-CoV-2, where IgM antibodies were detected in five days from the first symptom, and IgG within seven days. As of now, many serologic tests are available, but not all have been adequately evaluated. Such testing is necessary to confirm the best antigen to be used, and the antibody isotype to be tested for as well.

These will be crucial in evaluating the antibody response to the vaccine, selecting the blood samples to be used in clinical trials, and examine in detail the process of seroconversion. The current study aimed to analyze how well two commercially available tests performed in the diagnosis of COVID-19. The tests selected were produced by DiaSorin and Roche, respectively.

The study included 46 COVID-19 patients and 85 samples predating the pandemic. The results from both assays was then assessed side-by-side with the ability of the serum to neutralize a clinical sample that yielded an isolate of the virus.

The 46 positive samples were taken from symptomatic patients on the day of admission to hospital and after 15 days. The 85 earlier samples were collected over the period 2012 to 2018 and were tested for antibodies to the virus.

The DiaSorin assay detects IgG against recombinant S1 and S2 proteins of SARS-CoV-2, while the Roche assay detects IgG, IgM, and IgA antibodies against the N protein. The viral isolate was grown in Vero E6 cells and infected with eight dilutions of virus stock, each diluted tenfold the previous dilution. Plaque reduction assay and endpoint dilution assays were both performed to observe the reduction in plaques and the presence of cytopathic effects (CPE).

Study: Poor correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects. Image Credit: Corona Borealis Studio

Assays Fail to Detect Significant Numbers of Positives

The Roche assay picked up 46% of cases at the time of admission, and 100% after fifteen days. It was 100% specific. The DiaSorin assay was 20% sensitive on the day of admission, but it was 100% sensitive at day 15. It picked out 3 of 85 pre-pandemic samples as positive, giving a specificity of 97%.

Selective Examination of Antibody Activity of Five Samples

The T0 (day of admission samples) samples were non-neutralizing, as expected from the low antibody titers. On the 15^th day, only one of five samples showed neutralizing activity at a high dilution, and another two at very low dilution. Even then, they showed only 22% and 11% neutralizing activity. One sample showed potent neutralization on day 15 though it had a relatively low antibody titer.

Overall Neutralizing Capacity

All the rest of the sera were tested for neutralizing activity on day 15 because of the very low activity on T0, and all at low dilution of 1:200, both based on the earlier five serologic results. The researchers found there was no correlation between antibody detected and neutralizing activity. Neither of these was, in turn, had any link with ICU admission.

Implications

The researchers, therefore, concluded that both assays were very sensitive at 15 days from the presentation of illness. The specificity was slightly less with the DiaSorin assays on the pre-pandemic sera, perhaps because of cross-reactions as described by the manufacturer.

The much lower sensitivity of the DiaSorin assay was less than half that of the Roche assay on the day of presentation, mainly because the latter detects IgM antibodies as well. This could explain the negative findings in some patients who have a clinical picture corresponding to COVID-19 but whose nasopharyngeal swabs or bronchoalveolar fluid lavage samples test negative for the virus.

A third difference between the two assays is the type of recombinant antigen to which antibody is detected. The Roche assays detects antibodies against recombinant N protein, while the DiaSorin assay picks up IgGs targeting the S1 or S2 portion of the spike protein. The S protein is responsible for receptor binding and host cell entry by the virus via membrane fusion.

The researchers conclude: “Both tests are endowed with low sensitivity on the day of hospital admission, which 33 increased to 97.8 and 100% for samples collected after 15 days for DiaSorin and Roche tests, 34 respectively.”

Antibody levels and neutralizing activity

The researchers also wanted to understand how antibodies correlated with protection against the infection. The results showed that at all dilutions from 1:100 to 1:800 the antibody levels were not related to the neutralizing capability of the serum sample.

At the second level, the researchers tested the neutralization capacity against a viral isolate of SARS-CoV-2 from a patient sample. This confirmed the finding that there was a poor correlation. Thus, even though the DiaSorin assay brochure states that “the test can give a clue on the presence of neutralizing antibodies directed against SARS-CoV-2,” This does not agree with the current observations.

Even more vital is the knowledge that similar methods are used today to measure the protection against SARS-CoV-2 conferred by candidate vaccines based on the S protein.

Directions for Future Research

If the presence of these antibodies does not correlate with protection, other techniques must be worked on to help unravel both the epidemiology and the clinical diagnostics of this virus. The role played by neutralizing antibodies, and the ability to detect them with binding assays is another crucial area of research in developing effective vaccines against the virus.

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