A serological assay to detect SARS-CoV-2 seroconversion in humans

Original Article: F. Amanat, T.H.O. Nguyen, V. Chromikova, S. Strohmeier, D. Stadlbauer, A. Javier, K. Jiang, G. Asthagiri Arunkumar, J. Polanco, M. Bermudez-Gonzalez, D. Caplivski, A. Cheng, K. Kedzierska, O. Vapalahti, J. M. Hepojoki, V. Simon, F. Krammer, A serological assay to detect SARS-CoV-2 seroconversion in humans, medRxiv (Pre-print, not peer-reviewed)

Author of summary: Alessia Villois; Reviewer: Bernadette Basilico

Date of Original Publication: March 19th, 2020

The article reports the development of a serological test for the diagnosis of COVID-19. It consists of an ELISA assay that uses recombinant antigens derived from SARS-CoV-2. 59 blood serum samples were analysed in the study, including 4 SARS-Cov-2 positive specimens. The method proves to be selective and specific. Based on an established and relatively simple technology, this technique represents a promising screening tool in populations affected by the virus.

SARS-CoV-2 infection is currently diagnosed with genetic, but not serological assays. In this article, SARS-CoV-2 seropositivity is determined by the ELISA method (enzyme-linked immunosorbent assay). The technique detects the presence of antibodies in the blood serum, which target a glycoprotein called spike protein (S protein), expressed by the virus to bind to the host cells.

The study was conducted on 59 serum samples, from people aged 20 to 65+, including four samples from three COVID-19 patients. Two of these samples were taken from the same patient 2 and 6 days after the symptom onset. In the other two cases, the samples were taken after 4 and 20 days respectively. Some participants, negative to SARS-CoV-2, presented previous viral infections (hantavirus, dengue, coronavirus NL63, chikungunya).

Two versions of the antigen were used in the study: the portion of S protein that binds to the receptor of the host cell (RBD) and the entire S protein. The two antigens were expressed in both insect and mammalian cells. In all the combinations of antigen and expression system, the difference in signal between COVID-19 patients and negative controls was significant. However, the reactivity of the COVID-19 samples was more pronounced towards the entire S protein, compared to the RBD fragment, probably because the S protein includes a higher number of epitopes.

Moreover, the four positive samples were subjected to a further ELISA assay to investigate which isotypes and subtypes of antibodies was more reactive. All samples showed a strong signal for IgG3, IgM and IgA. A signal for IgG1 was found in 3 samples out of 4. No signal was found for IgG4, while IgG2 was not examined. It should be noted that the IgG3 response was greater than that of IgG1, in contrast to the immune response to influenza.

In conclusion, the results show that SARS-CoV-2 infection causes a significant seroconversion that is easily to detect using this assay. Moreover, the antibodies from positive samples interact with both the S protein and the RBD, which represents the main target for neutralizing antibodies also in the case of other coronaviruses.

Finally, the researchers specifically commented on the results obtained from two samples:

  1. One of the four positive samples was tested in neutralization assays in another study and it was found to have a neutralizing titer of 1:160. Hence, seroconversion could lead to immunization, at least for some time.
  2. Testing the sample from a patient with a NL63 infection showed no signal, indicating that there may be no cross-reactivity between human coronaviruses and SARS-CoV-2. Even though this result is preliminary, it suggests that the SARS-CoV-2 is completely new to humans, which would explain the highly spreading.

Potentially, the method can be applied to find the actual attack rate and infection fatality rate of the disease and to determine the kinetics of the immune response to SARS-CoV-2.

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