WO2021246910A1 - Sars-cov-2 virus rbd recombinant protein and method for producing same - Google Patents

Abstract

The invention relates to biotechnology. Claimed are a genetic construct for the expression of SARS-CoV-2 virus RBD recombinant protein, and a Chinese hamster ovary cell strain CHO-S-RBD for producing recombinant protein of the receptor binding domain (RBD) of the SARS-CoV-2 virus which can be used for diagnostic purposes. Described is a method for producing a Chinese hamster ovary cell strain CHO-S-RBD for producing SARS-CoV-2 virus RBD recombinant protein, which contains a genetic construct including SEQ ID NO:1; the introduction of said genetic construct into cells by lipofection; and the selection of cells on the antibiotic Hygromycin В. Claimed is a Chinese hamster ovary cell strain CHO-S-RBD for producing SARS-CoV-2 virus RBD recombinant protein. Proposed is a method for producing SARS-CoV-2 virus RBD recombinant protein, which includes: culturing a Chinese hamster ovary cell strain CHO-S-RBD; chromatographically purifying SARS-CoV-2 virus RBD recombinant protein from the Chinese hamster ovary cell strain CHO-S-RBD culture medium; and confirming the production of SARS-CoV-2 virus RBD recombinant protein. Claimed is a SARS-CoV-2 virus RBD recombinant protein for detecting SARS-CoV-2 antibodies. Claimed is a test system for the enzyme-linked immunosorbent assay of human blood serum or plasma. Proposed is a method for the assay of COVID-19 convalescent blood serum or plasma in order to select the most promising samples for the treatment of patients infected with SARS-CoV-2. Also proposed is a method for the assay of human blood serum or plasma for determining SARS-CoV-2 antibody titre.

Description

RECOMBINANT PROTEIN RBD SARS-CoV-2 AND

The field of technology.

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 a while, 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 throughout the world, causing a pandemic affecting more than 200 countries. By May 1, 2020, the number of cases exceeded 3 million, 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 an increase in body temperature (in 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 registered. Mostly symptomatic treatment is performed. On April 1, 2020, the Moscow Department of Health issued an order "On the introduction of technology for the use of 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 has 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 administered to other patients, it 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% CO2 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 for 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 in various dilutions are added and at the same time PsV containing the luciferase gene is introduced. After 48 hours, evaluate luciferase activity 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. The publication (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 serum of patients, who have had 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). Substrate for HRP is then added and anti-RBD antibody titer is measured. 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 of analyzing the serum and plasma of COVID-19 convalescents to determine the most promising samples for therapy in 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 a quick 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 that are most promising for therapy for 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 through the development of a genetic construct for the expression of the recombinant RBD protein of the SARS-CoV-2 virus, including SEQ 10 NOT

Also, the technical result is achieved for the development of a Chinese hamster ovary cell strain CHO-S-RBD, a producer of the recombinant RBD protein of the SARS-CoV-2 virus, containing a genetic construct comprising SEQ ID NOT

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 including SEQ ID NOT;

- 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 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;

- substrate solution containing hydrogen peroxide solution;

- stop reagent.

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, including the use of a test system, where:

- in the wells of the plate for enzyme-linked immunosorbent assay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2, 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-fold wash buffer solution;

- add 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;

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

- add 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;

- 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 by developing a method for analyzing serum or plasma of human blood to determine 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 immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2 positive control, 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-fold wash buffer solution;

- add 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;

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

- add 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;

- 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 figure 1 - 4 presents the results of the study.

Implementation of the invention.

Brief description of the 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 the secretion of protein 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

The scheme of the plasmid vector pl-RBD-CoV-2, where CMV is the promoter / enhancer of early genes of human cytomegalovirus; genetic construct - 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 b-lactamase enzyme, which cleaves the b-lactam ring of ampicillin and some other antibiotics; ori- the sequence of the start of replication;

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

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

Hindlll, Xhol - recognition sites for the corresponding restriction enzymes.

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

In experimental studies, it was found 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 the 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 was 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 penetration of the virus into 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 the 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 is its small size, ease of synthesis and lack of influence on the function of the protein, its specific activity and structure.

In addition, as a result of this work, 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 on 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; substrate solution containing 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 pre-diluted test serum samples, or human blood plasma; incubation of these samples at a temperature 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-fold solution of wash buffer; 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-fold solution of wash buffer; 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 = A450 K- cf ± 3 x SD A450K-, where: Cut off - cutoff values of optical density,

A450 K-cf - the average value of optical density in the wells with negative control,

SD A450K- is the standard deviation of the optical density values of the negative control, while 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.

A method has also been developed for analyzing human serum or plasma to determine the titer of antibodies to the SARS-CoV-2 virus, including: adding to the wells of an enzyme-linked immunosorbent assay with SARS-CoV-2 virus RBD recombinant protein RBD adsorbed in the wells having SEQ ID NO : 2 positive control, negative control, as well as a series of 2-fold dilutions of the tested samples of human serum or plasma; incubation of these samples 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-fold solution of wash buffer; 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-fold solution of wash buffer; 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 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 = A450 K- cf + 3 x SD A450K-, where: Cut off - cutoff values of optical density,

A450 K-cf - the average value of optical density in the wells with negative control,

SD A450K- is the standard deviation of the optical density values of the negative control, while the antibody titer is determined as the highest dilution of a human serum or plasma sample, in the analysis of which the optical density at a wavelength of 450 nm is 2 times higher than the cut off 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 of 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 the affinity method. chromatography. Thus, the genetic construct SEQ GO NOT was developed.

The diagram of the genetic construct and is presented 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 Hindlll and Xhol into a plasmid vector for episomal expression in mammalian cell lines, and the resulting plasmid was named pl-RBD-CoV- 2 (Fig. 2). Then the resulting plasmid was produced in preparative amounts and purified using a commercial kit Plasmid Midiprep 2.0 (Evrogen / cat. N ° BC 124).

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

Example 2. Obtaining a strain of Chinese hamster ovary cells CHO-S-RBD, a 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 solid 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 is reached, 1 liter of suspension. Then the resulting suspension was transfected with the plasmid vector pl-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 pl-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, was obtained for the first time. SARS-CoV-2 virus. 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 virus 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 (Infers NT, 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, chromatographic purification of the protein from the clarified culture liquid was carried out using 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 liquid (100 ml) was diluted several times with a buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH = 7.4) and loaded onto a balanced 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. In this way, 10 purifications of 100 ml each were carried out. The fractions after purification were frozen at -20 ° C. The fractions after primary purification were thawed at room temperature and concentrated using Amicon Ultra -50, -10K centrifugal concentrators (Millipore, Ireland) to a protein concentration of 1–2 mg / ml. Protein concentration was measured using a Nanodrop 2000c spectrophotometer. Next, 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. A preparation with a concentration of 1–2 mg / ml was applied to the column through a syringe in a volume of 1 ml. Collected the first peak at UV280 corresponding to the target recombinant RBD protein. A total of 5-10 purification 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 a temperature of - 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 the culture medium (according to the results of three independent experiments). This is an indicator of the effectiveness of the developed production method, since the proteins of the coronavirus belong to the group that are 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 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 ° C for 5 min and added to the gel. Electrophoresis was performed in a Mini-Protean Tetra Cell (Bio-Rad, USA, cat. N ° 165-8000). Then the gel was stained with EZBlue ™ Gel Staining Reagent (Sigma-Aldrich, USA, cat. N ° 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). In the resulting reaction mixture was added sequentially 1 μl of 1M DTT and 10 μl of 110 mm iodoacetamide. After the reaction (56 ° С, 30 min in the dark), 4 μL of 1M DTT was added to the mixture to neutralize unbound iodoacetamide. Only trypsin was added to the remaining part, while 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 C 18), 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, Refl - 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). Smoothing according to the Savizky Golay algorithm (width 0.2 m / z, 1 cycle) and baseline subtraction according to the TopHat algorithm were applied to the mass spectra. 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 one and two 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-linked immunosorbent assay 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 CHO-S-RBD ovary cell strain of the Chinese hamster the RBD protein of the SARS-CoV-2 virus (examples 1-4), is a target for antibodies detected in the blood of COVID-19 convalescents.

The test system is a set of reagents that includes:

1) a plate for enzyme immunoassay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-22 virus 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) 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) 25x 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 the selection of samples of 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 treatment 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 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 convalescent plasma and serum samples from patients undergoing COVID-19 was carried out. Each sample was examined using a developed test system for enzyme-linked immunosorbent assay (described in example 5) and analyzed for the presence of 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, cannot be stored).

2. Analysis.

2.1 Add control and test serum samples Unpack the plate and add 100 μl positive control in 2 replicates 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 ° С 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 completely 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 an adhesive film and incubate for 1 hour at a temperature of +37 ° С with stirring at a speed of 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 ₅₀ ) within 10 minutes after stopping the reaction.

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

A _{45 °} K _Cp = (A 1 + A _{45o 45o} K A K 2) / 2 (1) wherein: - A ₄₅₀ K 1 - the value of the optical density of the sample 1 of the negative control,

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

A ₄₅₀ K ⁺ _ep = (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 _45O K ⁺ 2 - the value of the optical density of the sample 2 of the positive control. Calculate the average value A ₄₅₀ for each test sample (A ₄₅ oOP _cf. ) according to formula 3:

A ₄₅ oOP _av = (A _45O OP1 + A _45O OP2) / 2 (3) where:

- A ₄₅₀ OP1 - the value of the optical density of the sample 1,

- A ₄₅₀ OP2 - 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 the optical density in the wells with a negative control (A ₄₅₀ K-c _p ) 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 cut off values of the optical DENSITY 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 ₄₅ oOPav is 2.5 times higher than the Cut off value.

The analyzed serum or blood plasma sample is considered negative if A ₄₅ oOPav 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, 2x dilutions of serum or blood plasma were added, starting from 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 (conduction control) in 3 repetitions. 50 μl of control negative plasma (without virus neutralizing activity) at a dilution of 1/20, in 3 repetitions (plasma control).

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

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

Then, in all wells, except for the control of cells, 50 μl of vaccinated suspension with a concentration of 2000 TSW50 / ml was added.

As a control of viral activity (Control of the virus), dilutions of a vaccinated suspension were introduced into separate 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 2x10 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 action was suppressed in 2 out of 3 wells.

The 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 the developed test system and the presence of virus neutralizing antibodies (BHA) by direct virus neutralization of SARS-CoV-2 coronavirus on cell culture are presented in Table 1. The results of the dependence of the optical density (OD450) of the sample when diluted 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

Table 1.

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

Table 2.

Thus, as a result of the studies carried out, it was found that the diagnostic sensitivity of the developed test system for analyzing the serum or plasma of patients who underwent COVID-19 and have virus-neutralizing activity (titer 1/64 and higher) ranges 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. A 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 the analysis of serum or plasma of human blood to determine the titer of antibodies to the SARS-CoV-2 virus.

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

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, cannot be stored).

2. Analysis.

2.1 Add control and test serum samples

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

Into the remaining wells of the plate add a series of two-fold dilutions of the test samples (two replicates for each sample). Cover the tablet with foil and incubate for 1 hour at a temperature of +37 ° С 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 completely 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 an adhesive film and incubate for 1 hour at a temperature of +37 ° С with stirring at a speed of 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 ₅ oK) and positive (A ₄₅₀ K ⁺ ) controls according to formulas 1 and 2.

A _{45 °} K _cP ⁼ (A 1 + K ^~ _{45o 45o} A A K 2) / 2 (1) wherein:

- A _45O K 1 - the value of the optical density of the sample 1 negative control,

- A _45O K 2 - the value of the optical density of the sample 2 of the negative control.

А ₄ 5 _О К ⁺ _cf = (А ₄ 5 _О К ⁺ 1+ А _45О А К ⁺ 2) / 2 (2) where: - A ₄₅₀ K ⁺ 1 - the optical density of the sample 1 of the positive control,

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

Calculate the average value A ₄₅₀ for each test sample (A ₄₅ oOP _cf. ) according to formula 3:

A ₄₅ oOP _Sr = (A ₄₅₀ OPI + A _45O OP2) / 2 (3) where:

- А ₄₅₀ ОШ - the value of the optical density of the sample 1,

- A ₄₅₀ OP2 - 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:

2. Average value of optical density in wells with positive control (A _45O K ⁺ _CP ) - not less than 0.6;

3. The value of optical density in the wells with negative control (A ₄₅ oK ^_ _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 cut off values of the optical DENSITY A450 (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 A450OPav 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 from COVID-19 convalescents.

Table 3.

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 plasma with different variants of adsorbed coronavirus proteins by their ability to nonspecifically detect antibodies against seasonal human coronavirus OC43.

The purpose of this experiment was to compare several test systems for enzyme-linked immunosorbent assay 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 a temperature of + 4 ° C.

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

31 1) a 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) a plate for enzyme-linked immunosorbent assay with the full-length protein S of the SARS-CoV-2 virus adsorbed in the wells;

3) plate for enzyme-linked immunosorbent assay with SARS-CoV-2 virus adsorbed in the wells, inactivated by heating 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, cannot be stored).

Next, the following was applied to the wells of each of the 3 plates:

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 replicates of 100 μl each;

2) 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 repetitions of 100 μl each;

3) blood serum samples from patients who have suffered a disease caused by the seasonal coronavirus OS43, but did not suffer from 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 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 the filter paper folded in several layers. Then, 100 μl of a working solution of a 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 working buffer solution for plate washing.

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 a 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 (A450) within 10 minutes after stopping the reaction.

The arithmetic mean of the optical density of negative (A450CD and positive (A450I controls for each plate) was calculated according to formulas 1 and 2.

A450 K _cf = (A450 K 1+ A450A K 2) 12 (1) where:

- A450 KG1 - the value of the optical density of the sample 1 negative control,

- A450 K 2 - the value of the optical density of the sample 2 of the negative control.

A450 K ⁺ _Wed = (A450 K ⁺ 1 + A450A K ⁺ 2) / 2 (2) where:

- A450 K ⁺ 1 - the value of the optical density of the sample 1 of the positive control,

- A450 K ⁺ 2 - the value of the optical density of the sample 2 of the positive control.

We calculated the average A450 for each test sample (A45oOP _cf. ) according to formula 3:

A ₄₅ oOP _av = (A450OSh + A ₄₅ OOP2) / 2 (3) where:

- A450 OP1 - the value of the optical density of the sample 1,

- A450 OP2 - the value of the optical density of the sample 2.

To interpret the results, the cutoff values of absorbance A450 (Cut off) were calculated.

The Cut off value was calculated by the formula:

Cut off = A450 K- sr + 3 x SD A450K- where:

- SD A450K- is the standard deviation of the optical density values of the negative control.

The analyzed serum or blood plasma sample was considered positive if A450OPav higher than the Cut off value by 2.5 times.

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

The average optical density in the positive control wells (A450K + cf) was 1.960.

The average optical density in the wells with negative control (A450K-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 blood plasma sample was considered positive if A450OPav was 2.5 times higher than the Cut off value (2.5 * 0.052038 = 0.13).

The analyzed serum or blood plasma sample is considered negative if A450OPav 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 (A450K + cf) was 2.008.

The average optical density in the wells with negative control (A450K-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 blood plasma sample was considered positive if A450OPav was 2.5 times higher than the Cut off value (2.5 * 0.064542 = 0.16). The analyzed serum or blood plasma sample is considered negative if A450OPav 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 the wells, inactivated by heat and destroyed by ultrasound:

Average absorbance in positive control wells

(A450K + sr) was 2.081

Average absorbance in negative control wells

(A450K-sr) 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 blood plasma sample was considered positive if A450OPav was 2.5 times higher than the Cut off value (2.5 * 0.0613975 = 0.15).

The analyzed serum or blood plasma sample is considered negative if A450OPav 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 according to their ability to nonspecifically detect antibodies against seasonal human coronavirus OC43.

Table 4.

It was unexpectedly found that the use of the developed test system for enzyme immunoassay 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, while 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 serum and plasma enzyme immunoassay test system 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 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 titer antibodies 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 N ° l. The study was carried out according to the protocol described in example 7 (control).

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

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

Experiment N ° 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 N ° 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 N ° 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 N ° 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 N ° 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 N ° 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 N ° 1 - 11. Table 5.

As can be seen from the data presented, 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. Experiments were carried out according to the protocol described in example 6, with the exception of 1 parameter, which was changed.

Experiment Ml 2. The study was carried out according to the protocol described in example 6 (control).

Experiment M13. 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 Ml 4. 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 l 5. 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 l 6. The study was carried out according to the protocol described in example 6, except for the incubation temperature with the conjugate, which was + 38 ° C.

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

Experiment Ml 8. 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 Ml 9. 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 N ° 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 N ° 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 N ° 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 (A45 ₀ OD _cf ) at a wavelength of 450 nm, obtained in a series of experiments N ° 12-22.

Table 6.

As can be seen from the data presented, 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 you to quickly and efficiently perform a test to determine the most promising serum or plasma samples of COVID-19 convalescents for the treatment of patients infected with SARS-CoV-2.

Claims

Claim. 1. Genetic construct for the 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 protein RBD of the SARS-CoV-2 virus, containing a genetic construct comprising SEQ ID NO: E 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 for producing a recombinant RBD protein of the SARS-CoV-2 virus according to claim 4, comprising: - 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 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; - substrate solution containing hydrogen peroxide solution; - stop reagent. 7. A method for analyzing serum or blood plasma of COVID-19 convalescents for sampling the most promising patients for therapy, infected with SARS-CoV-2, including the use of a test system according to claim 6, where: - in the wells of the plate for enzyme-linked immunosorbent assay with adsorbed in the wells of the recombinant RBD protein of the SARS-CoV-2 virus having SEQ ID NO: 2, 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-fold wash buffer solution; - add 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; - wash the wells at least 3 times with 1-fold wash buffer solution; - add 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; - 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 for determining the titer of antibodies to the SARS-CoV-2 virus, comprising the use of a test system according to claim 6, in accordance with which: - positive control, negative control are introduced 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, and a series of 2-fold dilutions of the tested samples of human serum or plasma is added; - incubation of these samples is carried out 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-fold 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-fold 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 ° С to ± 25 ° С; - 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 for the interpretation of 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 = A450 K- cf ± 3 x SD A450K-, where: Cut off - cutoff values of optical density, A450 K-cf - the average value of optical density in the wells with negative control, SD A450K- - the standard deviation of the optical density values of the negative control, in this case, 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 value of the optical density Cut off, which is calculated by the formula Cut off = A450 K- cf ± 3 x SD A450K-, where: Cut off - cutoff values of optical density, A450 K-cf - the average value of optical density in the wells with negative control, SD A450K- is 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 cut off values of the optical density Cut off.