Quantifying antibodies made in response to SARS-CoV-2 vaccination: comparable outcomes from five commercial assays

By Mariana Bego

The evaluation of antibodies against SARS-CoV-2 is critical to the management of the COVID-19 pandemic. During our current vaccination efforts, assays detecting anti-SARS-CoV-2 antibodies are key to monitoring the success of our vaccination strategy. Early assays measuring SARS-CoV-2-specific antibodies were designed to distinguish between naïve and infected individuals. These were usually developed as qualitative rather than quantitative assays and were designed to achieve the highest possible specificity and sensitivity. In a pre-print, investigators from Medical University of Vienna characterized the immunization response after the first dose of an mRNA vaccine using five commercial quantitative assays, recently evaluated side-by-side, and found they generated very comparable results.

Of all the proteins made by the virus during a natural infection, commercial assays that detect SARS-CoV-2 antibodies focus in two: the viral spike (S) and nucleocapsid (N). Antibodies directed against SARS-CoV-2 N protein are induced early and strongly in most infected individuals. They are very specific as no reactivity is observed even with closely related viruses. They are often used as a marker of prior infection, but the physiological significance of these antibodies is unclear. On the other hand, many antibodies directed against SARS-CoV-2 S protein can neutralize the virus. The spike proteins of SARS-CoV-2 mediate viral entry in human cells. The region of the spike that binds to the cellular receptor, ACE2, to trigger viral entry is known as the Receptor Binding Domain (RBD).  Many neutralizing antibodies recognize the RBD and prevent its binding to ACE2. Numerous studies have shown a correlation between levels of antibodies that bind to spike and various neutralization assays. Hence, the evaluation of these neutralizing antibodies post-immunization is an important readout to validate the success of vaccination efforts. However, true neutralization assays are generally labor-intensive, require live virus and cannot be performed at high throughput. Alternatively, assays measuring anti-spike antibodies can be used instead, as long as they can consistently and reliably quantify specific antibody levels, and correlate with the presence of neutralizing antibodies.

In a pre-print, investigators from Medical University of Vienna characterized the immunization response after the first dose of an mRNA vaccine using five commercial quantitative anti-spike antibody assays in a head-to-head comparison. The authors eliminated samples from individuals with previous SARS-CoV-2 infection by excluding samples recognizing N (while antibodies recognizing the N and S proteins are made after a natural infection, mRNA vaccines only induce antibodies recognizing S protein). For the exclusion step, they used Roche Elecsys Anti-SARS-CoV-2 assay, which detects total antibodies to the viral nucleocapsid. They also estimated the sera neutralization capacity using a commercial surrogate virus neutralization test (sVNT, GenScript).

The features of each assay are described in Table 1. Most of the assays measured presence of IgG, except for the assay from Roche which measures total antibodies (tAb, IgA plus IgM plus IgG). The assay targets are also slightly different: one recognizes the full-length spike, one uses the cleaved S1 and S2 subunits of the spike, one uses spike on a trimeric form, and two use the RBD of the S1 subunit. For all the assays, the manufacturers-reported range of detection, clinical specificity, and sensitivity. The sensitivity was linked to the pre-determined manufacturer recommended cut-offs, and values above these cut-offs should be denoted as positive.

Table 1: Features of each assay used in the comparison.

In order to standardize readings (when applicable), the binding antibody units per milliliter (BAU/mL), traceable to the WHO International Standard for anti-SARS-CoV-2 immunoglobulin, were calculated by applying conversion factors as suggested by the manufacturers. Yet, these calculations did not make final readings comparable. The authors concluded that a direct comparison of the numerical outputs from different test systems was unlikely, even after recalculation attempts using WHO standards. They also observed differences in the measurement ranges and found that upper limits were exceeded in two out of five assays (DiaSorin TriS IgG and Serion S IgG).

Despite these differences, all five assays returned comparable outcomes when results were converted into tertiles (0–33.3%, 33.4–66.7%, 66.8–100%). For example, a sample detected within the lowest tertile of test A was also detected in the lowest tertile of test B. Among all 69 samples that were tested, only one individual, who had also undergone immunosuppressive therapy, consistently showed no detectable antibodies in all five assays used. Nevertheless, 10% of the individuals were negative in the surrogate neutralization assay used. Ultimately, the authors concluded that while positive results are diagnostically valuable, they are not indicative of sufficient neutralization capabilities. It is unclear at the moment what would be the threshold, and if it exists, in the amount of antibodies that would be directly correlated with neutralization.

Perkmann T, Perkmann-Nagele N, Koller T, Mucher P, Radakovics A, Marculescu R, Wolzt M, Wagner OF, Binder CJ, Haslacher H. Anti-Spike protein assays to determine post-vaccination antibody levels: a head-to-head comparison of five quantitative assays. MedRxiv preprint. 2021 Mar 8. DOI: 10.1101/2021.03.05.21252977.