B-085 Sars-cov-2 nt-chip: A Multiplex eia Platform for Detect Humoral Response and Neutralizing Antibodies Against Sars-cov-2
Authors: Siqueira J R , Moreira M L , Carvalho E M , Moruzzi F , Luche D D , Kintrup M 
Published: September 2023

Abstract

Background
COVID-19 pandemic is a current health crisis that began in late 2019 and is caused by the pathogen SARS-CoV-2. This virus causes respiratory illness and has led to numerous infections and deaths worldwide. SARS-CoV-2 has an essential protein, the spike protein (S) containing a receptor-binding domain (RBD) binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Studies have shown that mutations in the RBD can affect its ability to bind to the ACE2 receptor, and some of these mutations have been associated with increased transmissibility or disease severity. During infection, the host immune response can generate a variety of antibodies against SARS-CoV-2 proteins, including neutralizing antibodies that can target RBD. Serological tests could detect the presence of these antibodies in blood samples and are being used to determine whether a person has been infected with SARS-CoV-2 in the past or has developed immunity against the virus after vaccination. These tests also can help to better understand the spreading of new VOCs and epidemiological profiles of COVID-19. However, it is essential to note that the presence of antibodies does not guarantee immunity.

Methods
SARS-CoV-2 VOC Virachip IgG test was designed for the multiplex detection of binding antibodies to multiple SARS-CoV-2 proteins, including nucleocapsid (N), spike fragment S1 (S1), spike fragment S2 (S2), and the RBDs of Wuhan strain (RBD-w), Delta VOC (RBD-d), and Omicron VOC (RBD-o). This test was modified and further developed to also measure the neutralization effectiveness of antibodies binding against different RBDs. The percentage of inhibition of neutralizing antibodies against Wuhan and Omicron RBDs was determined. To achieve this, a purified ACE-2 Alkaline-Phosphatase conjugate was incorporated into the assay, and its ability to bind RBD spots in the microarray wells was measured after preincubation with sera or plasma. The presence of neutralizing antibodies was assessed for its capability of preventing such interaction, hence allowing us to detect neutralizing antibodies indirectly. Furthermore, it was also possible to generally detect binding antibodies against RBDs and the N protein. Assessing past infections in people immunized with mRNA vaccines, and in the same assay, detecting the subset of antibodies that promote functional neutralization against SARS-CoV-2 infection was feasible.

Results
A cohort containing 50 pre-pandemic and 61 post-infection sera the SARS-CoV-2 NT-CHIP yielded 100% specificity and 96.7% sensitivity regarding neutralization antibodies against RBD-w. In case of RBD-o analysis of 111 serum samples from patients before the emergence of Omicron and 69 serum samples from patients after infection resulted in 99.1% specifictiy and 95.7% sensitivity. These tests were performed in comparison with PRNT, the gold standard test for measuring neutralization.

Conclusion
This innovative microarray platform allows for quick upgrades to incorporate new RBDs from currently important VOCs. Given the status of the pandemic, the emergence of new variant strains, and vaccine adjustments, the SARS-COV-2 NT-CHIP could be a very valuable tool for assessing the humoral immunity generated against future VOCs. Thus, it contributes to the decision-making processes in the public health care system and is well suited for new vaccine studies.