RU2723008C9 - Method for producing chinese hamster ovary cell strain, producer of sars-cov-2 virus recombinant rbd protein, chinese hamster ovary cell strain, producer of recombinant rbd protein of sars-cov-2 virus, method of producing recombinant rbd protein of sars-cov-2 virus, a test system for enzyme-linked immunosorbent assay of human blood serum or plasma and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: genetic construct for expression of recombinant protein RBD of virus SARS-CoV-2, strain of cells of Chinese hamster ovary CHO-S-RBD producing recombinant protein – receptor-binding domain (RBD) of SARS-CoV-2 virus, which can be used for diagnostic purposes. Described is a method of producing a Chinese hamster ovary cell strain CHO-S-RBD from a producer of recombinant RBD protein SARS-CoV-2 containing a genetic construct comprising SEQ ID NO: 1; introducing said genetic construct into cells by lipofection; selection of cells on antibiotic Hygromycin B. A Chinese hamster ovary cell strain CHO-S-RBD, a producer of recombinant RBD protein SARS-CoV-2 virus, is created. Method of producing recombinant protein RBD of virus SARS-CoV-2 including: cultivation of Chinese hamster ovary cell strain CHO-S-RBD; chromatographic purification of recombinant protein RBD of SARS-CoV-2 virus from culture medium of Chinese hamster ovary cell strain CHO-S-RBD; confirmation of obtaining of recombinant RBD virus SARS-CoV-2 virus. A recombinant RBD protein SARS-CoV-2 for detecting antibodies to SARS-CoV-2 is created. Test system is created for enzyme immunoassay of human serum or plasma. There is developed a method for serum or blood plasma analysis of convalescent COVID-19 for sampling the most promising for therapy of patients infected with SARS-CoV-2. A method of analyzing blood serum or human blood plasma for determining titre of antibodies to virus SARS-CoV-2 is developed.

EFFECT: invention provides a fast and effective test system for determining the most promising serum or blood plasma samples of convalescent COVID-19 for therapy of patients infected with SARS-CoV-2; invention enables to reduce the time and simplifies the procedure for analyzing serum or blood plasma of convalescent COVID-19 for sampling the most promising for therapy of patients infected with SARS-CoV-2; invention enables high and stable expression of recombinant RBD protein SARS-CoV-2.

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Description

Technology area

The invention relates to biotechnology and relates to the creation of a new strain of Chinese hamster ovary cells CHO-S-RBD, producing a recombinant protein - receptor-binding domain (RBD) of the SARS-CoV-2 virus, which can be used for diagnostic purposes.

State of the art

At the end of 2019, an outbreak of a new infection occurred in the People's Republic of China (PRC) with an epicenter in Wuhan city (Hubei province). After some time, the causative agent of this infection was discovered, which turned out to be a previously unknown coronavirus, called SARS-CoV-2. Within a few months, SARS-CoV-2 spread around the world, causing a pandemic affecting more than 200 countries. By May 1, 2020, the number of cases exceeded 3 million people, and the death toll was 220 thousand people. At the same time, a further increase in morbidity and mortality is predicted, since in most countries the epidemic has not yet reached its peak.

SARS-CoV-2 causes a potentially severe acute respiratory infection called COVID-19. This disease is characterized by the presence of such clinical symptoms as fever (more than 90% of cases); cough - dry or with a small amount of sputum (more than 80% of cases); shortness of breath (in 55% of cases); fatigue (in 44% of cases); a feeling of congestion in the chest (more than 20% of cases). The most severe complications of COVID-19 are pneumonia, acute respiratory distress syndrome, acute respiratory failure, acute heart failure, acute renal failure, septic shock, cardiomyopathies, etc.

Currently, there are no specific drugs for the treatment and prevention of COVID-19. Mostly symptomatic treatment is performed. On April 1, 2020, the Moscow Department of Health issued an order "On the introduction of the technology for using fresh frozen plasma from COVID-19 convalescent donors" for the treatment of patients with COVID-19 in the terminal stage of the disease.

Reconvalescents are people whose body successfully coped with the disease, and they recovered without complications. This happened, in part, because their immune system produced antibodies that neutralize the virus. If convalescent blood plasma containing such antibodies is injected into other patients, this can mitigate the course of their illness.

However, not all convalescent plasma samples contain the same amount of virus neutralizing antibodies. Therefore, an urgent issue is the development of a method that would allow large-scale screening of plasma and blood serum of COVID-19 convalescent donors in order to select the most promising samples for therapy.

There is a known method for assessing the serum of convalescents by determining the titer of neutralizing antibodies (Ch. Shen et al. Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma. JAMA. 2020; 323 (16): 1582-1589. Doi: 10.1001). This method is based on the fact that blood serum samples in various dilutions are added to Vero cells (kidney cells of the African green monkey) and at the same time they are infected with the SARS-CoV-2 coronavirus. Then the cells are incubated at 37 ° C, 5% CO ₂ for 5 days, then the cytopathic effect of the virus is assessed. The result is presented as the titer of neutralizing antibodies (the largest serum dilution that showed the inhibitory activity of SARS-CoV-2). The disadvantage of this method is the long term of the analysis (5 days) and the need to adhere to the rules for working with pathogenic biological agents (PBA) of the II pathogenicity group. All this entails low productivity and high cost of this analysis.

In addition, there is a known method for assessing the plasma of convalescents by determining the titer of neutralizing antibodies, in which pseudotyped lentiviral particles (PsV) are used instead of coronavirus (F.Wu Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications, medRxiv 2020.03.30.20047365). This method includes culturing 293T / ACE2 cells, to which blood serum samples are added in various dilutions and at the same time PsV containing the luciferase gene is introduced. After 48 hours, the luciferase activity is assessed with a standard kit (Promega). The result is presented as the titer of neutralizing antibodies (the largest serum dilution, when added, the luciferase activity decreases by 50%). The disadvantages of this method are the duration of the process (48 hours), labor intensity, the need for expensive equipment and highly qualified personnel.

It is known from the literature that the receptor-binding domain (RBD) of the S protein of the SARS-CoV coronavirus is the main target for neutralizing antibodies. In a publication by Z. Cao et al. Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients. Virol J 7, 299 (2010)) describes a method for detecting antibodies to RBD in the blood serum of patients with SARS-CoV infection. The method is implemented as follows: a series of dilutions of patient serum samples and control samples are added to the RBD-His adsorbed in the plate wells. Antibodies bound to RBD are detected using secondary antibodies conjugated to horseradish peroxidase (HRP). Then add substrate for HRP and estimate the titer of antibodies to RBD. This method, as the closest to the claimed one, was chosen as a prototype. The disadvantage of this method is that it does not allow the detection of antibodies to the SARS-CoV-2 coronavirus in the blood plasma of COVID-19 convalescents.

Thus, there is currently a need to develop a rapid method for analyzing the serum and plasma of COVID-19 convalescents to determine the most promising samples for therapy of patients with COVID-19.

Disclosure of the invention.

The objective of the claimed invention is to create an effective test system and method for rapid analysis of serum and blood plasma of COVID-19 convalescents to determine the most promising serum and plasma samples for the therapy of patients with COVID-19, as well as a method for rapid analysis of human serum and plasma to determine the titer of antibodies to the SARS-CoV-2 virus.

The technical result is to obtain a fast and effective test system for determining the most promising serum or plasma samples of COVID-19 convalescents for the treatment of patients infected with SARS-CoV-2.

The technical result consists in reducing the time and simplifying the procedure for analyzing the serum or blood plasma of COVID-19 convalescents for the selection of samples of the most promising for therapy of patients infected with SARS-CoV-2.

The technical result also represents a high and stable expression of the recombinant RBD protein of the SARS-CoV-2 virus.

In addition, the technical result consists in an unexpectedly manifested effect for the analysis of human serum and plasma for the presence of antibodies to the SARS-CoV-2 virus, the absence of false positive results in patients who have undergone the disease.

The technical result is achieved by developing a genetic construct for the expression of the recombinant RBD protein of the SARS-CoV-2 virus, including SEQ ID NO: 1

Also, the technical result is achieved for the development of the Chinese hamster ovary cell strain CHO-S-RBD, the producer of the recombinant RBD protein of the SARS-CoV-2 virus, containing the genetic construct comprising SEQ ID NO: 1

Also, the technical result is achieved through the development of a method for obtaining a strain of cells of the ovary of the Chinese hamster CHO-S-RBD, including:

- obtaining a genetic construct comprising SEQ ID NO: 1;

- introduction of the specified genetic construct into cells by lipofection;

- cell selection for the antibiotic Hygromycin B.

Also, the technical result is achieved through the development of a recombinant RBD protein of the SARS-CoV-2 virus for detecting antibodies to SARS-CoV-2, having SEQ ID NO: 2, which is the product of a Chinese hamster ovary cell strain.

Also, the technical result is achieved through the development of a method for producing the recombinant RBD protein of the SARS-CoV-2 virus, including:

- cultivation of the Chinese hamster ovary cell strain CHO-S-RBD;

- chromatographic purification of the recombinant RBD protein of the SARS-CoV-2 virus from the culture medium of the Chinese hamster ovary cell strain CHO-S-RBD;

- confirmation of the receipt of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2.

Also, the technical result is achieved through the development of a test system for the enzyme immunoassay of human serum or plasma, which is a set of reagents, including:

- plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2;

- a positive control comprising an inactivated human serum sample containing IgG to the RBD protein of the SARS-CoV-2 virus;

- negative control, which is an inactivated human serum sample that does not contain IgG to the RBD protein of the SARS-CoV-2 virus;

- a conjugate containing a 20-fold concentrate of monoclonal mouse antibodies to human IgG, conjugated with horseradish peroxidase;

- 25x Wash Buffer Concentrate containing PBS concentrate with Tween 20 for washing plates;

- buffer for dilution of serum or plasma and the specified conjugate;

- a chromogen solution containing a tetramethylbenzidine solution;

- a substrate solution containing a solution of hydrogen peroxide;

- stop reagent.

Also, the technical result is achieved by developing a method for analyzing serum or blood plasma of COVID-19 convalescents for sampling the most promising patients for therapy with SARS-CoV-2 infection, including the use of a test system, where:

- in the wells of the plate for enzyme-linked immunosorbent assay with the recombinant RBD protein of the SARS-CoV-2 virus, which has SEQ ID NO: 2, adsorbed in the wells, add a positive control, a negative control, and also add previously diluted test samples of human serum or plasma;

- incubation of these samples is carried out at a temperature from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour;

- wash the wells at least 3 times with 1 x wash buffer solution;

- 1-fold conjugate solution is introduced, followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour;

- wash the wells at least 3 times with 1 x wash buffer solution;

- introduce a chromogen-substrate solution into each well with incubation for 15 minutes in a dark place at a temperature of + 15 ° C to + 25 ° C;

- stop the peroxidase reaction by adding a stop reagent to each well;

- measure the optical density on a spectrophotometer in each well at a wavelength of 450 nm;

- carry out the interpretation of the results.

Also, the technical result is achieved through the development of a method for analyzing serum or plasma of human blood for determining the titer of antibodies to the SARS-CoV-2 virus, including the use of a test system, in accordance with which:

- in the wells of the plate for enzyme-linked immunosorbent assay with the recombinant RBD protein of the SARS-CoV-2 virus, which has SEQ ID NO: 2, adsorbed in the wells, make a positive control, a negative control, and also make a series of 2-fold dilutions of the tested samples of human serum or plasma;

- incubation of these samples is carried out at a temperature from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour;

- wash the wells at least 3 times with 1 x wash buffer solution;

- 1-fold conjugate solution is introduced, followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour;

- wash the wells at least 3 times with 1 x wash buffer solution;

- introduce a chromogen-substrate solution into each well with incubation for 15 minutes in a dark place at a temperature of + 15 ° C to + 25 ° C;

- stop the peroxidase reaction by adding a stop reagent to each well;

- measure the optical density on a spectrophotometer in each well at a wavelength of 450 nm;

- carry out the interpretation of the results.

Also, the technical result is achieved through the development of a method for analyzing the serum or blood plasma of COVID-19 convalescents for sampling the most promising for therapy of patients infected with SARS-CoV-2 (option 1).

Also, the technical result is achieved by developing a method for analyzing human serum or plasma to determine the titer of antibodies to the SARS virus - CoV-2 (option 2).

The essence of the claimed group of inventions is illustrated by drawings, where Fig. 1-4 show the results of the study.

Implementation of the invention

Brief description of figures

FIG. 1 is a schematic representation of a genetic construct for the expression of the receptor binding domain (RBD) of the SARS-CoV2 virus, where

SP is a signal peptide that ensures protein secretion into the culture fluid;

RBD - receptor-binding domain RBD virus SARS-CoV2;

His-tag is a histidine tag that provides protein purification by affinity chromatography.

FIG. 2 shows a diagram of the plasmid vector p1-RBD-CoV-2, where

CMV - promoter / enhancer of early genes of human cytomegalovirus;

genetic construct - the developed genetic construct SEQ ID NO: 1, expressing the receptor-binding domain (RBD) of the SARS-CoV2 virus;

poly (A) - polyadenylation signal;

EBV ori - sequence of the beginning of replication of the Epstein-Barr virus;

AmpR promoter - Ampicillin resistance gene promoter;

Amp - ampicillin resistance gene, which encodes the β-lactamase enzyme, which cleaves the β-lactam ring of ampicillin and some other antibiotics;

ori - replication start sequence;

TK promoter - human herpes simplex virus thymidine kinase gene promoter;

HygroB is a gene for resistance to Hygromicin B, which encodes the enzyme phosphatase, which inactivates this antibiotic by phosphorylation.

HindIII, XhoI - recognition sites for the corresponding restriction enzymes.

FIG. 3 shows an electropherogram of the RBD protein preparation after affinity purification, where

M - molecular weight marker;

1.- sample of RBD protein 0.75 μg / μl;

2.- sample of RBD protein 1 μg / μl;

3.- sample of RBD protein 0.5 μg / μl;

4.- sample of RBD protein 0.25 μg / μl.

FIG. 4 shows the results of mass spectral analysis of the RBD protein preparation after affinity purification, where

[M] + - distribution of molecular weights of a singly charged ion;

[M] 2 + - distribution of molecular weights of a doubly charged ion.

Experimental studies have shown that most antibodies with virus-neutralizing activity against the type of coronavirus (MERS, SARS, etc.) specifically bind to various epitopes of the receptor-binding domain of protein S (spike) (Hofmann H. et al. S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients.Journal of Virology. 2004, 78 (12): 6134-42; He Y. Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies. Journal of Immunology. 2005, 174 (8): 4908-15; He Y. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine Biochem Biophys Res Commun 2004, 324 (2): 773-81). It has been found that antibodies that prevent the binding of the SARS-CoV-2 virus S protein to the angiotensin converting enzyme 2 (ACE2) receptor on human cells have neutralizing activity. The receptor binding domain (RBD) is a structural domain of protein S, which forms a binding site for the ACE2 receptor; therefore, antibodies capable of binding to the RBD protein can block contact with the receptor and, as a result, prevent the virus from entering human cells. Therefore, the administration of serum or plasma from COVID-19 convalescent donors, containing antibodies to the RBD protein of the SARS-CoV-2 virus, to patients with COVID-19 is a promising therapy.

Thus, the analysis of the serum or blood plasma of COVID-19 convalescent donors for the presence of antibodies to the RBD protein of the SARS-CoV-2 virus will make it possible to select the most promising serum or plasma samples that have a therapeutic effect.

As a result of this work, a genetic construct was created for the expression of the recombinant RBD protein of the SARS-CoV-2 virus, including SEQ ID NO: 1.

This construct comprises a human immunoglobulin G heavy chain leader peptide fused to the RBD gene of the SARS-CoV-2 virus and a histidine tag.

Leader peptides are signal sequences that play a central role in targeting and translocation of almost all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The secretion of the recombinant protein directly depends on the choice of the signal peptide. The use of the leader peptide of the heavy chain of human immunoglobulin G allows for high levels of production of the recombinant RBD protein into the culture medium.

A histidine tag is a nucleotide sequence that encodes 6 amino acid residues of histidine. This label is required for subsequent protein purification on immobilized metal-affinity sorbents. This method exploits the ability of histidine to bind to a chelated transition metal ion such as nickel (Ni2 +). The main advantages of using a histidine tag are its small size, ease of synthesis and no effect on the function of the protein, its specific activity and structure.

In addition, as a result of the work carried out, a strain of Chinese hamster ovary cells CHO-S-RBD was created, a producer of the recombinant RBD protein of the SARS-CoV-2 virus, containing a genetic construct including SEQ ID NO: 1 and a method for its production was developed, including: obtaining a genetic construct comprising SEQ ID NO: 1; introduction of the specified genetic construct into cells by lipofection; cell selection for the antibiotic Hygromycin B.

Also, a recombinant RBD protein of the SARS-CoV-2 virus was created for detecting antibodies to SARS-CoV-2, having SEQ ID NO: 2 and a method for its production, including: culturing a Chinese hamster ovary cell strain CHO-S-RBD; chromatographic purification of the recombinant RBD protein of the SARS-CoV-2 virus from the culture medium of the Chinese hamster ovary cell strain CHO-S-RBD; confirmation of the receipt of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2.

In addition, a test system was created for the enzyme immunoassay of human serum or blood plasma, which is a set of reagents, including: a plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2; a positive control comprising an inactivated human serum sample containing IgG to the RBD protein of the SARS-CoV-2 virus; negative control, which is an inactivated human serum sample that does not contain IgG to the RBD protein of the SARS-CoV-2 virus; a conjugate containing a 20-fold concentrate of monoclonal mouse antibodies to human IgG, conjugated with horseradish peroxidase; 25x wash buffer concentrate containing PBS concentrate with Tween 20 to wash plates; buffer for dilution of serum or plasma and the specified conjugate; a chromogen solution containing a tetramethylbenzidine solution; a substrate solution containing a hydrogen peroxide solution; stop reagent.

A method was also developed for the analysis of serum or blood plasma of COVID-19 convalescents for the selection of samples of the most promising for therapy of patients infected with SARS-CoV-2, including: introducing into the wells of an enzyme-linked immunosorbent assay plate with the recombinant RBD protein of the SARS-CoV virus adsorbed in the wells -2, having SEQ ID NO: 2 positive control, negative control, and previously diluted test samples of human serum or plasma; incubation of these samples at temperatures from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; washing the wells at least 3 times with 1 x wash buffer solution; introduction of a 1-fold conjugate solution followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; washing the wells at least 3 times with 1 x wash buffer solution; adding a chromogen-substrate solution to each well with incubation for 15 minutes in a dark place at a temperature of + 15 ° C to + 25 ° C; stopping the peroxidase reaction by adding a stop reagent to each well; measuring optical density on a spectrophotometer in each well at a wavelength of 450 nm; interpretation of results.

A feature of this method for the analysis of serum or blood plasma of COVID-19 convalescents is that, to interpret the results, the analyzed human serum or plasma sample is compared with the cutoff value of the optical density Cut off, which is calculated by the formula:

Cut off = A ₄₅₀ K ^- _Wed +3 x SD A ₄₅₀ K ^- ,

where Cut off - cutoff values of optical density,

A ₄₅₀ K ^- _cf. - the average value of optical density in the wells with negative control,

SD А ₄₅₀ К ^- - the standard deviation of the optical density values of the negative control,

the sample is considered positive if, after the analysis, the optical density in the wells of the plate with the experimental sample at a wavelength of 450 nm is 2.5 times higher than the cutoff values of the optical density Cut off.

Also, a method was developed for the analysis of human serum or plasma to determine the titer of antibodies to the SARS-CoV-2 virus, including: adding to the wells of the plate for enzyme-linked immunosorbent assay with the recombinant RBD protein of SARS-CoV-2 virus adsorbed in the wells having SEQ ID NO : 2 positive control, negative control, and a series of 2-fold dilutions of the tested samples of human serum or plasma; incubation of these samples at temperatures from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; washing the wells at least 3 times with 1 x wash buffer solution; introduction of a 1-fold conjugate solution followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; washing the wells at least 3 times with 1 x wash buffer solution; adding a chromogen-substrate solution to each well with incubation for 15 minutes in a dark place at a temperature from + 15 ° С to + 25 ° С; stopping the peroxidase reaction by adding a stop reagent to each well; measuring optical density on a spectrophotometer in each well at a wavelength of 450 nm; interpretation of results.

A feature of this method for the analysis of human serum or plasma is that, when interpreting the results, the analyzed sample of human serum or plasma is compared with the value of the optical density Cut off, which is calculated by the formula

Cut off = A ₄₅₀ K ^- _Wed +3 x SD A ₄₅₀ K ^- ,

where Cut off - cutoff values of optical density,

A ₄₅₀ K ^- _cf. - the average value of optical density in the wells with negative control,

SD А ₄₅₀ К ^- - the standard deviation of the optical density values of the negative control,

the titer of antibodies is determined as the highest dilution of a sample of human serum or plasma, in the analysis of which the optical density at a wavelength of 450 nm is 2 times higher than the cutoff values of the optical density Cut off.

The implementation of the invention is confirmed by the following examples.

Example 1. Development of the design of a genetic construct containing the leader peptide of the heavy chain of human immunoglobulin G, the RBD gene of the SARS-CoV-2 virus and a histidine tag

At the first stage of the work, the authors developed a design of a genetic construct that allows efficient expression of the recombinant RBD protein of the SARS-CoV-2 virus in mammalian cells and its chromatographic purification.

For this, the gene for the receptor-binding domain (RBD) of the protein S of the SARS-CoV-2 coronavirus was selected, fused with the signal peptide of the heavy chain of human immunoglobulin G, which ensures the secretion of the protein into the culture fluid and a histidine tag for purification of the recombinant protein by affinity chromatography. Thus, the genetic construct SEQ ID NO: 1 was developed.

A schematic of the genetic construct is shown in FIG. 1. The developed genetic construct was synthesized at ZAO Evrogen. Thus, the pAL-TA-RBD-CoV-2 construct was obtained.

Then, using genetic engineering methods, the developed genetic construct from pAL-TA-RBD-CoV-2 was cloned using restriction endonucleases HindIII and XhoI into a plasmid vector for episomal expression in mammalian cell lines, and the resulting plasmid was named p1-RBD-CoV- 2 (Fig. 2). Then the resulting plasmid was produced in preparative quantities and purified using a commercial kit Plasmid Midiprep 2.0 (Evrogen / cat. # BC124).

Thus, as a result of this work, the p1-RBD-CoV-2 plasmid was obtained, which provides expression of the RBD gene of the SARS-CoV-2 virus.

Example 2. Obtaining a strain of Chinese hamster ovary cells CHO-S-RBD, producer of the recombinant RBD protein of the SARS-CoV-2 virus

The Chinese hamster ovary cell strain CHO-S-RBD, the producer of the recombinant RBD protein of the SARS-CoV-2 virus, was obtained on the basis of the CHO-S cell line. This is a commercial cell line based on Chinese hamster ovary cells CHO, adapted for cultivation in dense suspension in a special culture medium for increased protein expression.

CHO-S cells were cultured in an incubator shaker at 5% CO2 content, 80% humidity, and a rocking speed of 120 rpm in HyClone HyCell TransFx-C transfection media (GE Healthcare, USA) supplemented with 8 mM glutamine (PanEco , Russia), until a density of 2 * 10 ⁶ cells / ml 1 liter of suspension is reached. Next, the resulting suspension was transfected with the plasmid vector p1-RBD-CoV-2 containing the genetic construct SEQ ID NO: 1 using 1 mg / ml Polyethylenimine reagent (Polyscience, USA) according to the manufacturer's instructions. Then, for 2 weeks, the transfected cells were selected for the antibiotic hygromycin B.This antibiotic was chosen because the plasmid vector p1-RBD-CoV-2 contains the gene for resistance to the antibiotic Hygromycin B.

Thus, as a result of this work, a strain of Chinese hamster ovary cells CHO-S-RBD, a producer of the recombinant RBD protein of the SARS-CoV-2 virus, was obtained for the first time. In this case, the production of the specified protein ranges from 5 mg / l to 10 mg / l.

Example 3. A method of obtaining a recombinant RBD protein of the SARS-CoV-2 virus using the created producer strain

The developed method for producing the recombinant RBD protein of the SARS-CoV-2 SEQ ID NO: 2 includes the following steps:

1) cultivation of the strain-producer of the recombinant RBD protein of the SARS-CoV-2 virus;

2) selection of the culture medium;

3) chromatographic purification of the recombinant RBD protein of the SARS-CoV-2 virus;

4) confirmation of the authenticity of the obtained recombinant RBD protein of the SARS-CoV-2 virus.

The cultivation of the cells of the producer strain was carried out for 7 days in a Multitron shaker-incubator (Infors HT, Switzerland) at 5% CO2 content, 80% humidity and a rocking speed of 120 rpm. The viability of cells in suspension was determined daily using a TC20 Automated Cell Counter (Bio-Rad, USA). After 7 days of cultivation, the cell suspension was precipitated by centrifugation at 5000 rpm. The culture fluid was clarified by filtration through Millipore Express (PES) HP 0.45 μm syringe filters (Merckmillipore, Germany).

Then, the protein was chromatographed from the clarified culture liquid on an AKTA start device (GE Healthcare, USA). A HisTag HP protein purification column (5 ml) was connected to the instrument and equilibrated with a buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH = 7.4). Then, the clarified culture fluid (100 ml) was diluted several times with a buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH = 7.4) and applied to the equilibrated column at a rate of 5 ml / min. After the end of the application, the column was washed with 100 ml of buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH = 7.4) at a rate of 5 ml / min. After the end of washing, the bound protein was eluted from the column with a buffer (20 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, pH = 7.4) at a rate of 1 ml / min. So 10 purifications of 100 ml were carried out. After purification, the fractions were frozen at -20 ° C. Fractions after primary purification were thawed at room temperature and concentrated using Amicon Ultra -50, -10K centrifuge concentrators (Millipore, Ireland) to a protein concentration of 1-2 mg / ml. Protein concentration was measured using a Nanodrop 2000c spectrophotometer. Then the HiTrap desalting column was equilibrated with a buffer (20 mM sodium phosphate, 150 mM sodium chloride) at a rate of 5 ml / min. After equilibration, the flow rate was reduced to 1 ml / min. The drug with a concentration of 1-2 mg / ml was applied to the column through a syringe in a volume of 1 ml. Collected a first peak at UV280 corresponding to the target recombinant RBD protein. A total of 5-10 cleaning cycles were carried out. The drug fractions obtained after the second stage of purification were combined, and the protein concentration was measured on a Nanodrop 2000c spectrophotometer. Then the preparation was adjusted with a buffer (20 mM sodium phosphate, 150 mM sodium chloride) to a concentration of 1 mg / ml, poured in 1 ml each and stored at -20 ° C.

The yield of the recombinant RBD protein of the SARS-CoV-2 virus after chromatographic purification was from 5 mg to 10 mg per liter of culture medium (according to the results of three independent experiments). This is an indicator of the effectiveness of the developed production method, since coronavirus proteins belong to the group of difficult to obtain in traditional bacterial expression systems.

Confirmation of the authenticity of the obtained recombinant RBD protein of the SARS-CoV-2 virus was carried out using protein polyacrylamide gel electrophoresis, as well as mass spectrometric analysis.

For the protein electrophoresis, a 12% separating polyacrylamide gel and a 6% focusing polyacrylamide gel were prepared. Samples of the purified recombinant RBD protein were diluted to various concentrations (1 μg / μl, 0.75 μg / ml, 0.5 μg / μl, 0.25 μg / ml) with sample buffer containing B-mercaptoethanol. Then the samples were heated at a temperature of 98 ° С for 5 min and added to the gel. Electrophoresis was carried out in a Mini-Protean Tetra Cell (Bio-Rad, USA, cat # 165-8000). Next, the gel was stained with EZBlue ™ Gel Staining Reagent (Sigma-Aldrich, USA, cat # G1041) and developed using the Gel Doc EZ system (Bio-Rad, USA). The results of the electrophoretogram are shown in FIG. 3.

According to the results of electropherogram, the mobility of the recombinant RBD protein under denaturing conditions corresponded to ≈30 kDa, which is due to its glycosylation (the mass of the amino acid sequence without taking into account glycosylation is about 25 kDa). Thus, during the initial analysis of the drug, its expected molecular weight was confirmed.

A more detailed confirmation of the authenticity of the drug was carried out using mass spectrometric analysis according to the following scheme:

1. Protein hydrolysis with trypsin in solution

25 μg of recombinant RBD protein was dissolved in 20 mM ammonium bicarbonate and added to 100 μl of 25 mM ammonium bicarbonate (pH 8.3). To the resulting reaction mixture was added sequentially 1 μl of 1 M DTT and 10 μl of 110 mM iodoacetamide. After the reaction (56 ° С, 30 min in the dark), 4 μL of 1 M DTT was added to the mixture to neutralize unbound iodoacetamide. Only trypsin was added to the remainder; in both cases, the protein: enzyme ratio was 25: 1. The reaction was carried out at 37 ° C for 17 h. The resulting reaction mixture was dried to dryness using a vacuum concentrator (Savant SPD121P, Thermo Scientific, USA ) and dissolved in 20 μl of 0.1% formic acid. The sample was desalted on columns containing a reversed phase membrane (analogue C18), the sample was eluted with a 90% solution of acetonitrile in water. For application, 4 μl of a sample in 90% acetonitrile was taken and mixed with 1 μl of a saturated solution of 2,5-dihydroxybenzoic acid in a 30% acetonitrile solution containing 0.5% glacial acetic acid by volume on a steel target, which was then dried in air at room temperature.

2. MALDI mass spectrometric analysis

Peptides were analyzed on an Ultraflextreme time-of-flight MALDI mass spectrometer (Bruker Daltonics, Germany). Positive ions were detected in the reflectron mode at voltages: at the IS1 ion source - 20.12 kV, IS2 - 17.82 kV; on lenses - 7.47 kV, Ref1 - 21.07 kV, Ref2 - 10.80 kV. Ions were detected in the m / z range from 650 to 5000 Th. Peaks of autolytic fragments of trypsin and keratin, which were excluded from the final lists of detectable masses, were used as an internal standard.

3. Analysis of mass spectrometry data

Mass spectra were processed using Flex Analysis 3.4 software (Bruker Daltonik GMBH, Germany). The mass spectra were smoothed according to the Savizky Golay algorithm (width 0.2 m / z, 1 cycle), and baseline subtraction according to the TopHat algorithm. The following peak detection parameters were used: the peak detection algorithm was SNAP2, the signal-to-noise ratio was 3, the maximum number of peaks in the spectrum was 500). The search and identification of proteins by the “peptide mass fingerprint” method was carried out using the Proteinscape software package (version 4, “Bruker Daltonik GMBH, Germany), the following search parameters were used: accuracy of mass determination - 50 ppm; possible post-translational modifications - oxidation of methionine, histidine, tryptophan.

As a result of mass spectral analysis, it was demonstrated that the recombinant RBD protein of the SARS-CoV-2 virus is present in solution in the form of single and double charged ions, and has a molecular weight of 31 kDa, which clearly correlates with the data of electrophoresis (Fig. 4).

The results of the study of the recombinant RBD protein of the SARS-CoV-2 virus using protein electrophoresis in polyacrylamide gel, as well as mass spectrometric analysis, have convincingly proved its authenticity.

Thus, as a result of this work, the recombinant RBD protein of the SARS-CoV-2 virus SEQ ID NO: 2 was obtained for the first time. This protein forms a stable folded structure.

Example 5. Test system for enzyme immunoassay of human serum or plasma using the recombinant RBD protein of the SARS-CoV-2 virus

The authors were the first to create a test system based on an enzyme-linked immunosorbent assay of human serum or plasma, in which the recombinant RBD protein of the SARS-CoV-2 virus SEQ ID NO: 2, obtained using the Chinese hamster ovary cell strain CHO-S-RBD, a producer of recombinant the RBD protein of the SARS-CoV-2 virus (examples 1-3) is a target for antibodies detected in the blood of COVID-19 convalescents.

The test system is a set of reagents that includes:

1) plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus 2 SEQ ID NO: 2;

2) a positive control comprising an inactivated human serum sample containing IgG to the RBD protein of the SARS-CoV-2 coronavirus;

3) a negative control, which is an inactivated human serum sample that does not contain IgG to the RBD protein of the SARS-CoV-2 coronavirus;

4) a conjugate containing a 20-fold concentrate of monoclonal mouse antibodies to human IgG, conjugated with horseradish peroxidase;

5) 25-fold wash buffer concentrate containing PBS concentrate with Tween 20 for washing plates;

6) a dilution buffer containing a buffer solution for diluting serum or plasma and the conjugate;

7) a chromogen solution containing a tetramethylbenzidine solution;

8) a substrate solution containing a solution of hydrogen peroxide;

9) stop reagent containing 1 M sulfuric acid solution.

Example 6. A method of using a test system for an enzyme-linked immunosorbent assay for the analysis of serum or blood plasma of COVID-19 convalescents for sampling the most promising for therapy of patients infected with SARS-CoV-2

This example describes a method of using the developed test system for enzyme-linked immunosorbent assay (described in example 5) for the analysis of serum or blood plasma of COVID-19 convalescents to select samples that are most promising for the therapy of patients infected with SARS-CoV-2.

The most promising for therapy are serum or blood plasma samples that contain virus-neutralizing antibodies. It is known from the literature that the RBD protein of coronaviruses is one of the main targets for virus neutralizing antibodies Z. Cao et al. Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients. Virol J 7, 299 (2010)).

In this study, a comparative analysis of 218 samples of convalescent plasma and blood serum from patients undergoing COVID-19 was carried out. Each sample was examined using the developed test system for enzyme-linked immunosorbent assay (described in example 5) and analyzed for the presence of virus-neutralizing antibodies (BHA) by direct virus neutralization of the SARS-CoV-2 virus in cell culture.

The enzyme-linked immunosorbent assay using the developed test system was carried out according to the following protocol.

1. Preparation of working solutions

To prepare the working buffer solution for plate washing, dilute the wash buffer concentrate 25 times with distilled water.

To prepare a working solution of the conjugate, dilute the conjugate 20 times with dilution buffer and mix thoroughly by pipetting (the solution was prepared immediately before use).

To prepare a working chromogen-substrate solution, add 9.0 ml of a substrate solution to 1.0 ml of a chromogen solution and mix thoroughly (the solution must be prepared immediately before use, it cannot be stored).

1. Analysis.

1.1 Add control and test serum samples.

Unpack the plate and add 100 μl positive control in 2 repetitions and 100 μl negative control in 2 replicates.

Add 90 μl of dilution buffer to the remaining wells of the plate, and then add 10 μl of the test samples previously diluted 20 times (two wells for each sample).

Cover the tablet with foil and incubate for 1 hour at a temperature of + 37 ° C with stirring at a speed of 300 rpm.

2.2 Washing.

After the end of incubation, remove the liquid from the wells and wash the plate 4 times with a working solution of buffer for plate washing, on an automatic washing device or manually, filling the wells to the top (300 µl / well). Then finally remove the liquid and dry the plate by tapping on the filter paper folded in several layers.

2. 3 Introduction of the conjugate.

Add 100 μl of the working solution of the conjugate of monoclonal antibodies to each well, cover the plate with adhesive tape and incubate for 1 hour at + 37 ° C with stirring at 300 rpm.

2.4 Rinsing.

After the end of incubation, wash the plate 4 times with working buffer solution for plate washing.

2.5 Adding chromogen-substrate solution.

Add 100 μl of chromogen-substrate solution to each well and incubate for 15 minutes in a dark place at a temperature of + 20 ° C.

2.6 Stopping the peroxidase reaction.

Stop the reaction by adding 50 μl of stop reagent (1 M sulfuric acid solution) to each well and record the results on a spectrophotometer within 10 minutes after stopping the reaction.

2.8. Registration of results

Measure the optical density of the substrate mixture on a spectrophotometer with a vertical beam at a wavelength of 450 nm (A ₄₅₀ ) within 10 minutes after stopping the reaction.

Calculate the arithmetic mean of the optical density of the negative (A ₄₅₀ K ^- ) and positive (A ₄₅₀ K ⁺ ) controls according to formulas 1 and 2.

A ₄₅₀ K ^- _cf = (A ₄₅₀ K ^- ₁ + A ₄₅₀ A K ^- ₂ ) / 2 (1),

Where

- A ₄₅₀ K ^- ₁ - the optical density of the sample 1 negative control;

- A ₄₅₀ K ^- ₂ - the value of the optical density of the sample 2 of the negative control,

A ₄₅₀ K ⁺ _sr = (A ₄₅₀ K ⁺ ₁ + A ₄₅₀ A K ⁺ ₂ ) / 2 (2)

Where

- A ₄₅₀ K ⁺ ₁ - the optical density of the sample 1 positive control;

- A ₄₅₀ K ⁺ ₂ - the optical density of the sample 2 of the positive control.

Calculate the average A ₄₅₀ for each test sample (A ₄₅₀ OP _cf ) according to formula 3:

A ₄₅₀ OP _av = (A ₄₅₀ OP ₁ + A ₄₅₀ OP ₂ ) / 2 (3)

Where

- A ₄₅₀ OP ₁ - the value of the optical density of the sample 1;

- A ₄₅₀ OP ₂ - the value of the optical density of the sample 2.

In this case, the measurement results obtained on control samples must meet the following requirements:

1. The average value of optical density in the wells with a positive control (A ₄₅₀ K ⁺ _cf ) - not less than 0.6;

2. The value of optical density in the wells with negative control (A ₄₅₀ K ^- _sr ) is less than 0.2.

If other indicators are obtained, the study must be repeated.

2.9 Interpretation of results.

To interpret the results, calculate the cutoff values of the absorbance A ₄₅₀ (Cut off).

The Cut off value is calculated by the formula:

Cut off = A ₄₅₀ K ^- _Wed +3 x SD A ₄₅₀ K ^- ,

Where

- SD А ₄₅₀ К ^- is the standard deviation of the optical density values of the negative control.

The analyzed serum or blood plasma sample is considered positive if A ₄₅₀ OD _cp is 2.5 times higher than the Cut off value.

The analyzed serum or blood plasma sample is considered negative if A ₄₅₀ OD _cf does not exceed the Cut off value by 2.5 times.

The virus neutralization reaction was set in the variant: constant dose of the virus - dilution of serum or blood plasma.

In the wells of the plate, 2-fold dilutions of serum or blood plasma were made, starting with a dilution of 1/20 to 1/1280 in a volume of 50 μl / well in 3 repetitions.

Control samples are added to individual wells:

- dilutions of control plasma with a known BHA titer at 50 μl / well (control of conduction) in 3 repetitions.

- 50 μl of control negative plasma (not possessing virus neutralizing activity) in a dilution of 1/20, in 3 repetitions (control plasma).

- 100 μl of growth medium in 3 repetitions (RS) (control of cells).

- 50 μl of PC and 50 μl of vaccinated suspension with a concentration of 2000 TCID (tissue culture infective dose 50 / ml (control of the virus dose) in 3 repetitions.

Then, 50 μl of vaccinated suspension with a concentration of 2000 TCID50 / ml was added to all wells, except for the control of cells.

As a control of viral activity (Control of the virus), dilutions of a vaccinated suspension were introduced into individual wells of the plate (internal control of the introduced doses of the virus when studying the neutralizing activity of plasma).

Then the plate was incubated in an atmosphere of 5% CO2 at 37 ° C for 60 minutes.

Then, 20,000 Vero E6 cells were inoculated into all wells of the plate in a volume of 100 μl / well (cell concentration 2 × 10 ⁵ cells / ml) in freshly prepared PC. Plates with cells were incubated in an atmosphere of 5% CO2 at 37 ° C for 96 hours until the full manifestation of the cytopathic effect of the virus in the viral control in the expected range.

Evaluation of viral production by cytopathic action (CPE) was carried out on the basis of analysis of cell viability using microscopy, in order to visually determine the border of viral cell damage.

Virus neutralizing activity of plasma / serum was assessed visually under a microscope 96 hours after infection by inhibition of the CPP virus in a Vero E6 cell culture. Each of the plasma / serum dilutions was tested in three parallel wells. The result of the study was the conclusion about the neutralization of the cytopathic effect of the virus in the culture of Vero E6 cells when exposed to blood plasma / serum. The BHA titer of the studied plasma / serum was taken as its highest dilution, at which the cytopathic effect was suppressed in 2 out of 3 wells.

Results of a comparative analysis of serum or plasma samples of COVID-19 convalescents using two methods: enzyme immunoassay for antibodies to the recombinant RBD protein using a developed test system and for the presence of neutralizing antibodies (VNA) by direct virus neutralization of SARS-CoV-2 coronavirus in culture cells are presented in Table 1. The results of the dependence of the optical density (OD450) of the sample at a dilution of 200 times on the BHA titer were distributed as follows (Table 2).

Table 1. Results of a comparative analysis of plasma and serum samples of COVID-19 convalescents

No. Virus neutralizing antibody titer, 1 / Optical density of the sample at 200 times dilution (OD450) one 320 2.87 2 1280 2.85 3 320 2.78 4 1280 2.75 five 640 2.72 6 640 2.70 7 128 2.34 8 1024 2.26 9 40 2.02 ten 128 1.93 eleven 512 1.88 12 640 1.87 thirteen 40 1.83 14 1024 1.80 fifteen 320 1.76 16 80 1.75 17 320 1.74 eighteen 320 1.74 19 128 1.69 20 128 1.56 21 160 1.56 22 128 1.51 23 320 1.49 24 160 1.47 25 1024 1.46 26 80 1.45 27 40 1.43 28 80 1.43 29 80 1.42 thirty 1024 1.42 31 160 1.34 32 256 1.39 33 1280 1.38 34 64 1.33 35 160 1.32 36 320 1.32 37 160 1.30 38 80 1.17 39 160 1.16 40 256 1.15 41 128 1.14 42 128 1.13 43 160 1.12 44 160 1.10 45 16 1.09 46 256 1.09 47 160 1.08 48 256 1.08 49 640 1.06 fifty 256 1.06 51 320 1.05 52 80 1.05 53 256 1.04 54 160 1.03 55 128 1.02 56 128 1.00 57 64 0.99 58 40 0.98 59 20 0.97 60 64 0.97 61 40 0.97 62 512 0.95 63 256 0.94 64 32 0.93 65 40 0.91 66 40 0.89 67 20 0.85 68 20 0.84 69 80 0.84 70 128 0.83 71 16 0.82 72 64 0.82 73 64 0.81 74 256 0.81 75 320 0.80 76 40 0.78 77 128 0.78 78 20 0.78 79 16 0.78 80 20 0.75 81 256 0.74 82 256 0.74 83 16 0.72 84 20 0.72 85 20 0.72 86 64 0.72 87 20 0.71 88 20 0.71 89 16 0.70 90 40 0.70 91 20 0.68 92 1024 0.68 93 32 0.68 94 80 0.68 95 20 0.65 96 16 0.65 97 20 0.64 98 64 0.64 99 20 0.64 one hundred 20 0.63 101 16 0.63 102 40 0.63 103 32 0.62 104 20 0.62 105 20 0.61 106 64 0.61 107 40 0.60 108 32 0.60 109 640 0.59 110 64 0.59 111 40 0.57 112 20 0.57 113 20 0.57 114 20 0.56 115 128 0.55 116 32 0.55 117 320 0.54 118 64 0.52 119 16 0.51 120 256 0.51 121 20 0.51 122 20 0.51 123 64 0.50 124 16 0.50 125 20 0.50 126 64 0.49 127 80 0.49 128 20 0.48 129 32 0.48 130 20 0.46 131 16 0.45 132 20 0.45 133 640 0.45 134 20 0.44 135 20 0.44 136 80 0.44 137 20 0.43 138 64 0.43 139 20 0.42 140 20 0.42 141 20 0.42 142 20 0.41 143 20 0.41 144 80 0.41 145 20 0.39 146 160 0.39 147 20 0.38 148 16 0.38 149 160 0.35 150 32 0.35 151 20 0.35 152 32 0.35 153 20 0.34 154 16 0.33 155 20 0.32 156 16 0.32 157 16 0.32 158 32 0.32 159 16 0.32 160 16 0.31 161 16 0.31 162 20 0.30 163 20 0.30 164 32 0.30 165 16 0.30 166 32 0.29 167 32 0.27 168 128 0.27 169 32 0.27 170 20 0.26 171 16 0.26 172 64 0.25 173 20 0.24 174 32 0.24 175 32 0.23 176 16 0.23 177 16 0.22 178 16 0.21 179 16 0.21 180 32 0.21 181 16 0.21 182 16 0.21 183 20 0.21 184 20 0.20 185 16 0.20 186 32 0.20 187 16 0.19 188 1024 0.19 189 16 0.19 190 40 0.18 191 16 0.18 192 20 0.18 193 16 0.18 194 16 0.18 195 16 0.17 196 16 0.16 197 32 0.16 198 16 0.15 199 32 0.14 200 16 0.13 201 16 0.13 202 32 0.13 203 32 0.13 204 0 0.13 205 16 0.12 206 32 0.12 207 64 0.12 208 32 0.11 209 16 0.11 210 0 0.10 211 16 0.08 212 16 0.08 213 16 0.08 214 32 0.07 215 160 0.07 216 128 0.07 217 16 0.07 218 0 0.06

Table 2. Distribution of the dependence of the optical density (OD450) of the sample when diluted 200 times on the BHA titer.

Optical density of the sample at 200 times dilution (OD450) VNA titer from 1/64 and above BHA titer below 1/64 1.1 - 2.8 94% 6% 0.72 - 1 65% 35% 0.3-0.7 29% 71% Up to 0.2 3.8% 96.2

Thus, as a result of the conducted studies, it was found that the diagnostic sensitivity of the developed test system for the analysis of serum or blood plasma of patients who have undergone COVID-19 and have a neutralizing activity (titer 1/64 and higher) is from 96.8%. A correlation was established between the level of optical density (OD450) of the sample when diluted 200 times from the BHA titer, presented in Table 2. And the analysis time is 2.5 hours.

Example 7. Method of using the developed test system for the analysis of human serum or plasma to determine the titer of antibodies to the SARS-CoV-2 virus

This example describes a method of using the developed test system (example 5) for analyzing human serum or plasma to determine the titer of antibodies to the SARS-CoV-2 virus.

For the study, 10 samples of plasma and serum of COVID-19 convalescents were taken.

Immunoassay using the developed test system was carried out according to the following protocol.

1. Preparation of working solutions.

To prepare the working buffer solution for plate washing, dilute the wash buffer concentrate 25 times with distilled water.

To prepare a working solution of the conjugate, dilute the conjugate 20 times with dilution buffer and mix thoroughly by pipetting (the solution was prepared immediately before use).

To prepare a working chromogen-substrate solution, add 9.0 ml of a substrate solution to 1.0 ml of a chromogen solution and mix thoroughly. (the solution must be prepared immediately before use; it cannot be stored).

2. Analysis.

2.1 Add control and test serum samples

Unpack the plate and add 100 μl positive control in 2 repetitions and 100 μl negative control in 2 replicates.

Add a series of two-fold dilutions of the test samples to the rest of the plate wells (two replicates for each sample).

Cover the tablet with foil and incubate for 1 hour at a temperature of + 37 ° C with stirring at a speed of 300 rpm.

2.2 Washing.

After the end of incubation, remove the liquid from the wells and wash the plate 4 times with a working solution of buffer for plate washing, on an automatic washing device or manually, filling the wells to the top (300 µl / well). Then finally remove the liquid and dry the plate by tapping on the filter paper folded in several layers.

2. 3 Introduction of the conjugate.

Add 100 μl of the working solution of the conjugate of monoclonal antibodies to each well, cover the plate with adhesive tape and incubate for 1 hour at + 37 ° C with stirring at 300 rpm.

2.4 Rinsing.

After the end of incubation, wash the plate 4 times with working buffer solution for plate washing.

2.5 Adding chromogen-substrate solution.

Add 100 μl of chromogen-substrate solution to each well and incubate for 15 minutes in a dark place at a temperature of + 20 ° C.

2.6 Stopping the peroxidase reaction.

Stop the reaction by adding 50 μl of stop reagent (1M sulfuric acid solution) to each well and record the results on a spectrophotometer within 10 minutes after stopping the reaction.

2.8. Registration of results

Measure the optical density of the substrate mixture on a spectrophotometer with a vertical beam at a wavelength of 450 nm (A ₄₅₀ ) within 10 minutes after stopping the reaction.

Calculate the arithmetic mean of the optical density of the negative (A ₄₅₀ K ^- ) and positive (A ₄₅₀ K ⁺ ) controls according to formulas 1 and 2.

A ₄₅₀ K ^- _cf = (A ₄₅₀ K ^- ₁ + A ₄₅₀ A K ^- ₂ ) / 2 (1)

Where

- A ₄₅₀ K ^- ₁ - the optical density of the sample 1 negative control,

- A ₄₅₀ K ^- ₂ - the value of the optical density of the sample 2 of the negative control.

A ₄₅₀ K ⁺ _sr = (A ₄₅₀ K ⁺ ₁ + A ₄₅₀ A K ⁺ ₂ ) / 2 (2)

Where

- A ₄₅₀ K ⁺ ₁ - the optical density of the sample 1 of the positive control,

- A ₄₅₀ K ⁺ ₂ - the optical density of the sample 2 of the positive control.

Calculate the average A ₄₅₀ for each test sample (A ₄₅₀ OP _cf ) according to formula 3:

A ₄₅₀ OP _av = (A ₄₅₀ OP ₁ + A ₄₅₀ OP ₂ ) / 2 (3)

Where

- A ₄₅₀ OP ₁ - the value of the optical density of the sample 1,

- A ₄₅₀ OP ₂ - the value of the optical density of the sample 2.

In this case, the measurement results obtained on control samples must meet the following requirements:

1. The average value of optical density in the wells with a positive control (A ₄₅₀ K ⁺ _cf ) - not less than 0.6;

2. The average value of optical density in the wells with negative control (A ₄₅₀ K ^- _cf. ) is less than 0.2.

If other indicators are obtained, the study must be repeated.

2.9 Interpretation of results.

To interpret the results, calculate the cutoff values of the absorbance A ₄₅₀ (Cut off).

The Cut off value is calculated by the formula:

Cut off = A ₄₅₀ K ^- _Wed +3 x SD A ₄₅₀ K ^- ,

Where

- SD А ₄₅₀ К ^- is the standard deviation of the optical density values of the negative control.

The antibody titer was calculated as the last dilution of the sample, in which A ₄₅₀ OD _cp is 2 times higher than the Cut off value

The results of the study are presented in Table 3.

Table 3. Antibody titers to RBD in plasma and serum samples of COVID-19 convalescents

Patient number Antibody titer to RBD one 1: 512 2 1: 256 3 1:64 4 1:32 five 1: 256 6 1: 128 7 1: 512 8 1: 256 9 1: 128 ten 1: 256

Thus, the results of the study prove that the developed test system can be used for the analysis of human serum or plasma to determine the titer of antibodies to the SARS-CoV-2 virus.

An average specialist understands that this test system can also be used to detect antibodies to the RBD protein of the SARS-CoV-2 virus in serum or plasma of humans, to assess the strength of the immune response after administration of a vaccine containing the RBD receptor-binding domain of the SARS virus -CoV-2 or its gene.

Example 8 Comparison of test systems for enzyme-linked immunosorbent assay of human serum or blood plasma with different variants of adsorbed coronavirus proteins according to their ability to nonspecifically detect antibodies against seasonal human coronavirus OC43.

The purpose of this experiment was to compare several test systems for enzyme immunoassay based on the use of the recombinant RBD protein of the SARS-CoV-2 virus (developed test system), or the recombinant full-length protein S of the SARS-CoV-2 virus, or the destroyed SARS virus. CoV-2, by their ability to nonspecifically bind to antibodies to the seasonal OS43 coronavirus, which circulates in the human population.

At the first stage, the full-length protein S of the SARS-CoV-2 virus, or the SARS-CoV-2 virus, inactivated by heating at 56 ° C for 30 minutes and destroyed by ultrasound (within 1 minute on an ultrasonic homogenizer soniprep 150 Plus, MSE) was applied to the wells of plates for enzyme-linked immunosorbent assay at a concentration of 10 μg / ml in 100 μl and adsorbed overnight at + 4 ° C.

Thus, 3 plates were prepared by the beginning of the experiment:

1. plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus 2 SEQ ID NO: 2, which is part of the developed test system (example 5);

2. plate for enzyme immunoassay with adsorbed in the wells full-length protein S of the SARS-CoV-2 virus;

3. plate for enzyme-linked immunosorbent assay with SARS-CoV-2 virus adsorbed in the wells, inactivated by heat and destroyed by ultrasound.

Then we prepared working solutions required for analysis (test system example 5).

To prepare the working buffer solution for plate washing, dilute the wash buffer concentrate 25 times with distilled water.

To prepare a working solution of the conjugate, dilute the conjugate 20 times with dilution buffer and mix thoroughly by pipetting (the solution was prepared immediately before use).

To prepare a working chromogen-substrate solution, add 9.0 ml of a substrate solution to 1.0 ml of a chromogen solution and mix thoroughly (the solution must be prepared immediately before use, it cannot be stored).

Further, in the wells of each of the 3 plates were applied:

1) a positive control, including an inactivated human serum sample containing IgG to the RBD protein of the SARS-CoV-2 coronavirus (test system, example 5) in 2 repetitions of 100 μl;

2) a negative control, which is an inactivated human serum sample that does not contain IgG to the RBD protein of the SARS-CoV-2 coronavirus (test system, example 5) in 2 replicates of 100 μl each;

3) blood serum samples from patients who have suffered a disease caused by the seasonal coronavirus OS43, but did not have COVID-19 (10 samples), diluted 200 times in 2 repetitions of 100 μl.

Then all the plates were sealed with a film and kept for 1 hour at a temperature of + 37 ° C with stirring at a speed of 300 rpm.

After the end of the incubation, the liquid was removed from the wells and the plates were washed 4 times with working buffer solution for plate washing. Then the liquid was finally removed and the plates were dried by tapping on filter paper folded in several layers.

Then, 100 μl of the working solution of the conjugate of monoclonal antibodies was added to each well, the plates were covered with an adhesive film and incubated for 1 hour at a temperature of + 37 ° C with stirring at a speed of 300 rpm.

After the end of the incubation, the plates were washed 4 times with the working solution of the plate washing buffer.

Then, 100 μl of chromogen-substrate solution was added to each well of all 3 plates and incubated for 15 minutes in a dark place at a temperature of 20 ° C

After that, the reaction was stopped by adding 50 μl of stop reagent (1M sulfuric acid solution) to each well, and the results were recorded on a spectrophotometer within 10 minutes after stopping the reaction.

Next, the optical density of the substrate mixture was measured on a spectrophotometer with a vertical beam at a wavelength of 450 nm (A ₄₅₀ ) within 10 minutes after stopping the reaction.

Calculated the arithmetic mean of the optical density of the negative (A ₄₅₀ K ^- ) and positive (A ₄₅₀ K ⁺ ) controls for each plate according to formulas 1 and 2.

A ₄₅₀ K ^- _cf = (A ₄₅₀ K ^- ₁ + A ₄₅₀ A K ^- ₂ ) / 2 (1)

Where

- A ₄₅₀ K ^- ₁ - the optical density of the sample 1 negative control,

- A ₄₅₀ K ^- ₂ - the value of the optical density of the sample 2 of the negative control.

A ₄₅₀ K ⁺ _sr = (A ₄₅₀ K ⁺ ₁ + A ₄₅₀ A K ⁺ ₂ ) / 2 (2)

Where

- A ₄₅₀ K ⁺ ₁ - the optical density of the sample 1 of the positive control,

- A ₄₅₀ K ⁺ ₂ - the optical density of the sample 2 of the positive control.

Calculated the average A ₄₅₀ for each test sample (A ₄₅₀ OP _cf ) according to the formula 3:

A ₄₅₀ OP _av = (A ₄₅₀ OP ₁ + A ₄₅₀ OP ₂ ) / 2 (3)

Where

- A ₄₅₀ OP ₁ - the value of the optical density of the sample 1,

- A ₄₅₀ OP ₂ - the value of the optical density of the sample 2.

To interpret the results, the cutoff values of the absorbance A ₄₅₀ (Cut off) were calculated.

The Cut off value was calculated by the formula:

Cut off = A ₄₅₀ K ^- _Wed +3 x SD A ₄₅₀ K ^- ,

Where

- SD А ₄₅₀ К ^- is the standard deviation of the optical density values of the negative control.

The analyzed serum or blood plasma sample was considered positive if A ₄₅₀ OD _cp is 2.5 times higher than the Cut off value.

For the plate with the recombinant RBD protein of the SARS-CoV-2 virus 2 SEQ ID NO: 2 adsorbed in the wells:

The mean optical density in the positive control wells (A ₄₅₀ K ⁺ _cf ) was 1.960.

The average optical density in the wells with negative control (A ₄₅₀ K ^- _cf. ) was 0.052.

The average values of optical density allow us to say that the study was successful and its results are reliable.

Cut off value is:

Cut off = 0.052 + 3 x 0.0000125 = 0.052038

In this test, the studied serum or plasma sample was considered positive if A ₄₅₀ OD _cp was 2.5 times higher than the Cut off value (2.5 * 0.052038 = 0.13).

A test serum or blood plasma sample is considered negative if A ₄₅₀ OD _cf did not exceed the Cut off value by 2.5 times (2.5 * 0.052038 = 0.13).

For a plate with SARS-CoV-2 full-length protein S adsorbed in the wells:

The mean optical density in the positive control wells (A ₄₅₀ K ⁺ _cf ) was 2.008.

The average optical density in the wells with negative control (A ₄₅₀ K ^- _cf. ) was 0.064.

The average values of optical density allow us to say that the study was successful and its results are reliable.

Cut off value is:

Cut off = 0.064 + 3 x 0.0001805 = 0.064542

In this test, the studied serum or plasma sample was considered positive if A ₄₅₀ OD _cp was 2.5 times higher than the Cut off value (2.5 * 0.064542 = 0.16).

The test sample of serum or blood plasma is considered negative if A ₄₅₀ OD _cf did not exceed the Cut off value by 2.5 times (2.5 * 0.064542 = 0.16).

For a plate with SARS-CoV-2 virus adsorbed in wells, inactivated by heat and destroyed by ultrasound:

The average optical density in the wells with a positive control (A ₄₅₀ K ⁺ _cf ) was 2.081

The average optical density in the wells with negative control (A ₄₅₀ K ^- _cf. ) was 0.061.

The average values of optical density allow us to say that the study was successful and its results are reliable.

Cut off value is:

Cut off = 0.061 + 3 x 0.0001325 = 0.0613975.

In this test, the studied serum or plasma sample was considered positive if A ₄₅₀ OD _cf was 2.5 times higher than the Cut off value (2.5 * 0.0613975 = 0.15).

The test serum or blood plasma sample is considered negative if A ₄₅₀ OD _cf did not exceed the Cut off value by 2.5 times (2.5 * 0.0613975 = 0.15).

The results are shown in Table 4.

Table 4. Average optical density of experimental and control samples obtained as a result of comparative analysis of test systems for enzyme immunoassay of human serum or plasma with different variants of adsorbed coronavirus proteins by their ability to nonspecifically detect antibodies against seasonal human coronavirus OC43.

Table 4

Test sample RBD Full-length protein S SARS-CoV-2 virus one 0.055 0.159 0.132 2 0.060 0.493 0.519 3 0.053 0.152 0.414 4 0.054 0.142 0.117 five 0.055 0.268 0.436 6 0.055 0.143 0.134 7 0.048 0.065 0.136 8 0.048 0.351 0.581 9 0.049 0.130 0.090 ten 0.049 0.159 0.281 K- 0.052 0.064 0.061 K + 1,960 2.008 2.081

It was unexpectedly found that the use of the developed test system for enzyme-linked immunosorbent assay with the recombinant RBD protein of the SARS-CoV-2 virus SEQ ID NO: 2 (test system according to example 5) adsorbed in the wells for the analysis of human serum and plasma for the presence of antibodies to the SARS-CoV-2 virus does not give false positive results in patients who have undergone a disease caused by the seasonal human coronavirus OC43, whereas when using a test system for enzyme immunoassay of human serum and plasma with full-size protein S adsorbed in the wells, 30% of false-positive results were observed, and when using a test system for enzyme-linked immunosorbent assay of human serum and blood plasma with SARS-CoV-2 virus adsorbed in the wells, inactivated by heating and destroyed by ultrasound, 50% of false positive results were observed.

The results of the study can be explained by the fact that the recombinant RBD protein of the SARS-CoV-2 virus SEQ ID NO: 2 obtained by the authors has a low percentage of homology with the virus-binding domain of the OC43 virus S protein. Thus, the use of the recombinant RBD protein of the SARS-CoV-2 virus SEQ ID NO: 2 (compared to the full-length protein S of the SARS-CoV-2 virus or the destroyed SARS-CoV-2 virus) in the test system for enzyme immunoassay of serum and plasma human blood for the determination of antibodies to the SARS-CoV-2 virus will lead to more accurate and reliable results.

Example 9. Evaluation of the results obtained when using the test system for enzyme immunoassay of human serum or plasma in various experimental conditions.

In this study, we used a test system for enzyme-linked immunosorbent assay of human serum or plasma (example 5); as a test sample, we chose human blood plasma with a known titer of antibodies against the RBD protein of the SARS-CoV-2 virus (1: 512). A series of experiments was carried out in which the analysis of the antibody titer against the RBD protein of the SARS-CoV-2 virus was carried out according to the protocol described in example 7, with the exception of 1 parameter, which was changed.

Experiment # 1. The study was carried out according to the protocol described in example 7 (control).

Experiment # 2. The study was carried out according to the protocol described in example 7, except for the incubation temperature of the test samples, which was + 36 ° C.

Experiment # 3. The study was carried out according to the protocol described in example 7, except for the incubation temperature of the test samples, which was + 38 ° C.

Experiment # 4. The study was performed according to the protocol described in example 7, except for the incubation temperature with the conjugate, which was + 36 ° C.

Experiment # 5. The study was performed according to the protocol described in example 7, except for the incubation temperature with the conjugate, which was + 38 ° C.

Experiment # 6. The study was carried out according to the protocol described in example 7, except for the incubation temperature with the chromogen, which was + 15 ° C.

Experiment # 7. The study was carried out according to the protocol described in example 7, except for the incubation temperature of the test samples, which was + 25 ° C.

Experiment # 8. The study was carried out according to the protocol described in example 7, except for the stirring speed during incubation of the test samples, which was 250 rpm.

Experiment # 9. The study was carried out according to the protocol described in example 7, except for the stirring speed during incubation of the test samples, which was 350 rpm.

Experiment # 10. The study was carried out according to the protocol described in example 7, except for the stirring speed during incubation with the conjugate, which was 250 rpm.

Experiment 11. The study was carried out according to the protocol described in example 7, except for the stirring speed during incubation with the conjugate, which was 350 rpm.

The results are shown in Table 5.

Table 5. The titer of antibodies to the RBD protein of the SARS-CoV-2 virus, obtained in experiments No. 1-11

Experiment # one 2 3 4 five 6 7 8 9 ten eleven Titer
IgG to
squirrel
RBD 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512 1: 512

As can be seen from the presented data, a change in temperature, stirring rate during incubation with the test samples and antibodies, as well as temperature during incubation with a chromogen in the specified range of values, does not affect the result obtained.

In addition, a series of experiments was carried out in which the developed test system (example 5) was used to analyze the serum or blood plasma of COVID-19 convalescents to select samples that are most promising for the therapy of patients infected with SARS-CoV-2. The experiments were carried out according to the protocol described in example 6, with the exception of 1 parameter, which was changed.

Experiment # 12. The study was carried out according to the protocol described in example 6 (control).

Experiment 13. The study was carried out according to the protocol described in example 6, except for the incubation temperature of the test samples, which was + 36 ° C.

Experiment # 14. The study was carried out according to the protocol described in example 6, except for the incubation temperature of the test samples, which was + 38 ° C.

Experiment 15. The study was carried out according to the protocol described in example 6, except for the incubation temperature with the conjugate, which was + 36 ° C.

Experiment # 16. The study was performed according to the protocol described in example 6, except for the incubation temperature with the conjugate, which was + 38 ° C.

Experiment # 17. The study was performed according to the protocol described in example 6, except for the incubation temperature with the chromogen, which was + 15 ° C.

Experiment # 18. The study was carried out according to the protocol described in example 6, except for the incubation temperature of the test samples, which was + 25 ° C.

Experiment # 19. The study was carried out according to the protocol described in example 6, except for the stirring speed during incubation of the test samples, which was 250 rpm.

Experiment # 20. The study was carried out according to the protocol described in example 6, except for the stirring speed during incubation of the test samples, which was 350 rpm.

Experiment # 21. The study was carried out according to the protocol described in example 6, except for the stirring speed during incubation with the conjugate, which was 250 rpm.

Experiment # 22. The study was carried out according to the protocol described in example 6, except for the stirring speed during incubation with the conjugate, which was 350 rpm.

The results are shown in Table 6.

Table 6. The average value of the optical density of the test sample (A ₄₅₀ OD _av ) at a wavelength of 450 nm, obtained in a series of experiments No. 12-22

Experiment # 12 thirteen 14 fifteen 16 17 eighteen 19 20 21 22 A ₄₅₀ 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69

As can be seen from the presented data, a change in temperature, stirring rate during incubation with the test samples and antibodies, as well as temperature during incubation with a chromogen in the specified range of values, does not affect the result obtained. And the analysis time is 2.5 hours.

Thus, a genetic construct has been developed for the expression of the recombinant RBD protein of the SARS-CoV-2 virus, the Chinese hamster ovary cell strain CHO-S-RBD, the producer of the recombinant RBD protein of the SARS-CoV-2 virus and a method for its production, as well as a test system for an enzyme-linked immunosorbent assay for human serum or plasma, which allows a quick and effective test to determine the most promising serum or plasma samples of COVID-19 convalescents for the treatment of patients infected with SARS-CoV-2.

--->

Sequence listing

SEQ ID NO: 1

<110> FGBU "NITsEM named after N.F. Gamaleya" of the Ministry of Health of Russia

<120> Method for producing a Chinese hamster ovary cell strain, producer

recombinant protein RBD of SARS-CoV-2 virus, ovarian cell strain

Chinese hamster, producer of the recombinant RBD protein of the SARS-CoV-2 virus,

method for producing recombinant RBD protein of SARS-CoV-2 virus, test system

for enzyme immunoassay of human serum or plasma and its

application

<160> 1

<170> BISSAP1.3.6

<210> 1

<211> 765

<212> DNA

<213> SARS-CoV-2

<400> 1

atgggctgga gcttgatcct tctgttcctg gttgctgtgg ccaccagagt gctgtctcgg 60

gtgcagccca ccgaatccat cgtgcggttc cccaatatca ccaatctgtg ccccttcggc 120

gaggtgttca atgccaccag attcgcctct gtgtacgcct ggaaccggaa gcggatcagc 180

aattgcgtgg ccgactactc cgtgctgtac aactccgcca gcttcagcac cttcaagtgc 240

tacggcgtgt cccctaccaa gctgaacgac ctgtgcttca caaacgtgta cgccgacagc 300

ttcgtgatcc ggggagatga agtgcggcag attgcccctg gacagacagg caagatcgcc 360

gactacaact acaagctgcc cgacgacttc accggctgtg tgattgcctg gaacagcaac 420

aacctggact ccaaagtcgg cggcaactac aattacctgt accggctgtt ccggaagtcc 480

aatctgaagc ccttcgagcg ggacatctcc accgagatct atcaggccgg cagcacccct 540

tgtaacggcg tggaaggctt caactgctac ttcccactgc agtcctacgg ctttcagccc 600

acaaatggcg tgggctatca gccctacaga gtggtggtgc tgagcttcga actgctgcat 660

gcccctgcca cagtgtgcgg ccctaagaaa agcaccaatc tcgtgaagaa caaatgcgtg 720

aacttcgcgg ccgcacatca ccaccatcac catcatcatt gataa 765

SEQ ID NO: 2

<110> FGBU "NITsEM named after N.F. Gamaleya" of the Ministry of Health of Russia

<120> Method for producing a Chinese hamster ovary cell strain, producer

recombinant protein RBD of SARS-CoV-2 virus, ovarian cell strain

Chinese hamster, producer of the recombinant RBD protein of the SARS-CoV-2 virus,

method for producing recombinant RBD protein of SARS-CoV-2 virus, test system

for enzyme immunoassay of human serum or plasma and its

application

<160> 1

<170> BISSAP1.3.6

<210> 2

<211> 229

<212> PRT

<213> SARS-CoV-2

<400> 2

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe His

210 215 220

His His His His His

225

<---

Claims (55)

1. Genetic construct for expression of the recombinant RBD protein of the SARS-CoV-2 virus, comprising SEQ ID NO: 1. 2. The strain of cells of the ovary of the Chinese hamster CHO-S-RBD, the producer of the recombinant RBD protein of the SARS-CoV-2 virus, containing the genetic construct comprising SEQ ID NO: 1. 3. A method of obtaining a Chinese hamster ovary cell strain CHO-S-RBD according to claim 2, including: - obtaining a genetic construct comprising SEQ ID NO: 1; - introduction of the specified genetic construct into cells by lipofection; - cell selection for the antibiotic Hygromycin B. 4. Recombinant RBD protein of the SARS-CoV-2 virus for detecting antibodies to SARS-CoV-2, having SEQ ID NO: 2, which is the product of the strain according to claim 2. 5. A method of obtaining a recombinant RBD protein of the SARS-CoV-2 virus according to claim 4, including: - cultivation of the Chinese hamster ovary cell strain CHO-S-RBD according to claim 2; - chromatographic purification of the recombinant RBD protein of the SARS-CoV-2 virus from the culture medium of the Chinese hamster ovary cell strain CHO-S-RBD; - confirmation of the receipt of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2. 6. Test system for enzyme-linked immunosorbent assay of human serum or plasma, which is a set of reagents, including: - plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2; - a positive control comprising an inactivated human serum sample containing IgG to the RBD protein of the SARS-CoV-2 virus; - negative control, which is an inactivated human serum sample that does not contain IgG to the RBD protein of the SARS-CoV-2 virus; - a conjugate containing a 20-fold concentrate of monoclonal mouse antibodies to human IgG, conjugated with horseradish peroxidase; - 25x Wash Buffer Concentrate containing PBS concentrate with Tween 20 for washing plates; - buffer for dilution of serum or plasma and the specified conjugate; - a chromogen solution containing a tetramethylbenzidine solution; - a substrate solution containing a solution of hydrogen peroxide; - stop reagent. 7. A method for analyzing serum or blood plasma of COVID-19 convalescents for sampling the most promising for therapy of patients infected with SARS-CoV-2, including the use of a test system according to claim 6, where: - into the wells of the plate for enzyme-linked immunosorbent assay with the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2 adsorbed in the wells, make a positive control, a negative control, and also add previously diluted test samples of human serum or plasma; - incubation of these samples is carried out at a temperature from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; - wash the wells at least 3 times with 1 x wash buffer solution; - 1-fold conjugate solution is introduced, followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; - wash the wells at least 3 times with 1 x wash buffer solution; - introduce a chromogen-substrate solution into each well with incubation for 15 minutes in a dark place at a temperature of + 15 ° C to + 25 ° C; - stop the peroxidase reaction by adding a stop reagent to each well; - measure the optical density on a spectrophotometer in each well at a wavelength of 450 nm; - carry out the interpretation of the results. 8. A method for analyzing serum or plasma of human blood to determine the titer of antibodies to the SARS-CoV-2 virus, comprising the use of the test system according to claim 6, in accordance with which: - in the wells of the plate for enzyme-linked immunosorbent assay with the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2 adsorbed in the wells, make a positive control, a negative control, and also make a series of 2-fold dilutions of the tested samples of human serum or plasma ; - incubation of these samples is carried out at a temperature from + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; - wash the wells at least 3 times with 1 x wash buffer solution; - 1-fold conjugate solution is introduced, followed by incubation at a temperature of + 36 ° C to + 38 ° C with stirring at a speed of 300 (± 50) rpm for at least 1 hour; - wash the wells at least 3 times with 1 x wash buffer solution; - add a chromogen-substrate solution to each well with incubation for 15 minutes in a dark place at a temperature from + 15 ° C to + 25 ° C; - stop the peroxidase reaction by adding a stop reagent to each well; - measure the optical density on a spectrophotometer in each well at a wavelength of 450 nm; - carry out the interpretation of the results. 9. The method according to claim 7, in which to interpret the results, the analyzed human serum or plasma sample is compared with the cutoff value of the optical density Cut off, which is calculated by the formula Cut off = A ₄₅₀ K ^- _Wed + 3 x SD A ₄₅₀ K ^- , Where: Cut off - cutoff values of optical density, A ₄₅₀ K ^- _cf. - the average value of optical density in the wells with negative control, SD А ₄₅₀ К ^- - the standard deviation of the optical density values of the negative control, the sample is considered positive if, after the analysis, the optical density in the wells of the plate with the experimental sample at a wavelength of 450 nm is 2.5 times higher than the cutoff values of the optical density Cut off. 10. The method according to claim 8, in which, when interpreting the results, the analyzed human serum or plasma sample is compared with the optical density value Cut off, which is calculated by the formula Cut off = A ₄₅₀ K ^- _Wed + 3 x SD A ₄₅₀ K ^- , Where: Cut off - cutoff values of optical density, A ₄₅₀ K ^- _cf. - the average value of optical density in the wells with negative control, SD А ₄₅₀ К ^- - the standard deviation of the optical density values of the negative control, the titer of antibodies is determined as the highest dilution of a sample of human serum or plasma, in the analysis of which the optical density is obtained at a wavelength of 450 nm, which is 2 times higher than the cut off values of the optical density Cut off.

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