Alexandra Rak, Victoria Matyushenko, Polina Prokopenko, Arina Kostromitina, Dmitry Polyakov, Alexey Sokolov, Larisa Rudenko, Irina Isakova-Sivak.
Abstract
Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients.
Introduction
COVID-19 is an acute respiratory infection caused by the coronavirus SARS-CoV-2, characterized by high contagiousness and mortality of the infected population, which determines its high socio-economic significance. The global COVID-19 pandemic, which began in late 2019, has affected more than 704 million people and took the lives of 7 million victims. Despite to date the pandemic is finished, the disease cases continue to emerge in the form of successive waves of the worldwide spread of new SARS-CoV-2variants, which is associated with the high antigenic variability of the virus.
Materials and Methods:
The following SARS-CoV-2 viruses belonging to different lineages were obtained from the Smorodintsev Research Institute of Influenza:
African green monkey kidney Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and routinely maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic–antimycotic (AA) (all from Capricorn Scientific, Ebsdorfergrund, Germany).
The viruses were propagated on the Vero E6 cells using DMEM supplemented with 2% FBS, 10 mM of HEPES and 1× antibiotic-antimycotic (all from Capricorn Scientific, Ebsdorfergrund, Germany) at 37°C and 5% CO2. All experiments with live SARS-CoV-2 were performed in a biosafety-level-3 laboratory (BSL-3).
NCL5 antibodies against the N protein of SARS-CoV-2 (B.1), specifically binding N proteins of B.1.351, P.1, B.1.617.2 and B.1.1.529 VOCs were previously obtained using standard hybridoma approach, isotyped as IgG2a, dialyzed against 20 mM phosphate buffer, pH 7.4 (PBS), aliquoted and stored at -20°C.
Discussion
The neutralizing antibodies are detected in approximately 40%-70% of SARS-CoV-2-infected individuals, correlate with COVID-19 severity and are reported as key players in preventing viral entry into the host cell, so MN titer may be considered as an indicator of antiviral protection. It is well known that serum IgG antibody levels, similarly with the serum neutralizing activity gradually decrease in COVID-19 convalescents, but don’t disappear completely for a long time.
Acknowledgments:
We thank Dr. Ekaterina Stepanova for her advices on SARS-CoV-2 propagation and technical assistance, as well as all blood donors who participated in the study.
Citation: Rak A, Matyushenko V, Prokopenko P, Kostromitina A, Polyakov D, Sokolov A, et al. (2024) A novel immunofluorescent test system for SARS-CoV-2 detection in infected cells. PLoS ONE 19(5): e0304534. https://doi.org/10.1371/journal.pone.0304534
Editor: Boyan Grigorov, CRCL: Centre de Recherche en Cancerologie de Lyon, FRANCE
Received: February 26, 2024; Accepted: May 14, 2024; Published: May 31, 2024.
Copyright: © 2024 Rak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The minimal data cannot be shared publicly because of its confidentiality. The dataset used to prepare the article represents optical density and FFU values in the plate wells, so uploading them to public repositories without descriptions of groups, samples and dilutions (including location of samples within the plates) seems to be inappropriate. We uploaded a raw dataset related to our manuscript to the repository of the Local Ethics Committee of the Institute of Experimental Medicine (email: [email protected]), located on Yandex Disk at: https://disk.yandex.ru/client/disk/Datasets%20for%20External%20Requests/Rak%20et%20al.%202024%20PLoS%20One%20(FFU%20test%20system)%20raw%20data%20set Access to this repository is provided by the Local Ethics Committee staff using the login [email protected], and data may be stored there indefinitely. The minimal data are available for researchers who meet the criteria for access to confidential data upon request to the following member of the IEM Local Ethics Committee who is responsible for ensuring data access: Dr. Olga V. Kirik, email: [email protected], mob: +7 (951) 654-94-23.
Funding: This research was funded by the RSCF grant 21-75-30003. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
