Desenvolvimento e Padronização de um Ensaio Imunoenzimático para Diagnosticar a COVID-19

Abstract

Severe Acute Respiratory Syndrome 2 coronavirus (SARS-CoV-2) is a new virus, which emerged in Wuhan, Hubei Province, China, in early December 2019, capable of causing mild, moderate and severe respiratory crises in humans. and, due to its high transmission capacity, it quickly became a pandemic disease, the Coronavirus disease 2019 (COVID-19). During the outbreak of the disease, it was observed that many infected patients, despite being asymptomatic, are able to transmit the virus to other people, contributing to the spread of the virus. Initially, while vaccines were not available for the entire population, the World Health Organization (WHO) proposed restriction and social isolation in controlling the transmission of SARS-CoV-2. As a diagnostic method, the RT-PCR test has been used, using samples from the upper respiratory tract to detect the presence of the SARS-CoV-2 viral genome. Alternatively, serological testing and rapid tests recognize the presence of specific antibodies against the virus, after at least 14 days after infection. In this perspective, the present study aimed to develop a low-cost, high-sensitivity and high-specificity diagnostic test, based on an ELISA (Enzyme-Linked Immunosorbent Assay) enzyme immunoassay. For this, through bioinformatics approaches, and heterologous expression, antigens from structural proteins of SARS-CoV-2 were screened to compose the serological test. After their production, the antigens were validated using a panel of samples from negative (RT-PCR negative) and positive (samples obtained from the 15th day after positive diagnosis by RT-PCR) volunteers, in which the presence of specific anti-SARS-CoV-2 IgA, IgM and IgG antibodies. Likewise, comparative tests were carried out against the diagnostic kits already marketed and the analysis of antibody reactivity after vaccination with the immunizers CoronaVac and AstraZeneca. In addition, a proof of concept was developed for an immunochromatographic test using the protein antigen. Among the results obtained, it is noteworthy that the identification of peptide antigens screened by bioinformatics showed excellent diagnostic performance by ELISA in the identification of IgA, IgM and IgG anti-Sars-Cov-2 antibodies. Similarly, Recombinant Antigen 1 demonstrated the reactivity of anti-Sars-Cov-2 IgA, IgM and IgG antibodies by ELISA, equivalent to commercial serological tests (EUROIMMUN, BIOLISA, Allserum, ErbaLisa and Labtest). In addition, Recombinant Protein Antigen 1 was able to detect the seroconversion of individuals immunized with CoronaVac. These results stimulated the making of the prototype of the rapid immunochromatographic test for the detection of IgG against Sars-Cov-2 using the Recombinant Protein Antigen 1, showing promising results. Given the above, the present study was able to develop and standardize a diagnostic test capable of identifying antibodies against SARS-CoV-2 infection, which is in the process of technological transfer to the private sector.