RU2754340C1 - Test system and method for differential detection of antibodies to sars-cov-2 - Google Patents

Abstract

FIELD: biotechnology and medicine.SUBSTANCE: invention relates to biotechnology and medicine. The invention is a test system and a method for the differentiated (separate) detection of all classes of specific antibodies to the nucleocapsid and the receptor-binding domain of the spike protein of the coronavirus severe acute respiratory syndrome-2 (SARS-CoV-2) in order to assess the immune response to the transferred or current coronavirus COVID-19 infection, laboratory diagnosis of the current infection in combination with other studies, confirmation of positive or questionable test results, minimization of false positive test results. Includes an immunosorbent based on coronavirus antigens, which are the receptor-binding domain (RBD) and nucleoprotein (N) of the SARS-CoV-2 coronavirus, and detecting reagents, the above antigens are sorbed in different wells of plates for enzyme-linked immunosorbent assay, 96-well collapsible polystyrene tablets.EFFECT: invention allows determining a standardized relative amount of antibodies to different proteins of the virus due to the standard setting conditions and a single level of the threshold value (cutoff), provides increased sensitivity, simplification and ease of analysis, while in combination with other studies can confirm positive or questionable research results, minimizing false positive results (FPR).3 cl, 8 tbl, 4 ex

Description

The invention relates to the field of biotechnology and medicine. Usage: differentiated (separate) detection of all classes of specific antibodies to the nucleocapsid and the receptor-binding domain of the spike protein of the coronavirus severe acute respiratory syndrome-2 (SARS-CoV-2), in order to assess the immune response to past or current coronavirus infection COVID-19, laboratory diagnosis of the current infection in combination with other studies, confirmation of positive or questionable test results, minimization of false positive test results. Includes an immunosorbent based on coronavirus antigens, which are the receptor-binding domain (RBD) and nucleoprotein (N) of the SARS-CoV-2 coronavirus, and detecting reagents, the above antigens are sorbed in different wells of plates for enzyme-linked immunosorbent assay, 96-well collapsible polystyrene tablets.

The invention makes it possible to determine a standardized relative amount of antibodies to different proteins of the virus due to the standardization of the setting conditions and a single level of the threshold value (cutoff), provides increased sensitivity, simplification and convenience of the analysis.

Essence: obtaining an enzyme-linked immunosorbent assay for identifying a spectrum of antibodies of all classes to individual proteins of the SARS-CoV-2 coronavirus in human blood serum (plasma). The developed test system can be used to assess the immune response to a postponed or current coronavirus infection COVID-19, laboratory diagnostics of the current infection in combination with other studies, confirm positive or questionable test results, minimize false positive test results, separate natural (native) and artificial active acquired (post-vaccination) immunity (in the case of using spike-protein-based vaccines as an immunogen).

State of the art

The SARS-CoV-2 (2019-nCoV) coronavirus, which causes COVID-19 infection, is the seventh coronavirus capable of infecting humans and the third coronavirus to emerge over the past two decades, causing outbreaks with high disease severity and significant mortality across countries. The symptoms of COVID-19 are non-distinctive and overlap with those of other severe respiratory infections caused by the MERS-CoV and SARS-CoV coronaviruses.

Laboratory diagnostics of coronavirus infection COVID-19 is based on:

a) PCR diagnostics to detect nucleic acids of the SARS-CoV-2 virus;

b) detection of SARS-CoV-2 antigens;

c) detecting antibodies to SARS-CoV-2.

Serological diagnosis of new coronavirus infection is of great clinical importance. According to the "Interim Guidelines of the Ministry of Health of the Russian Federation (BMP of the Ministry of Health of the Russian Federation)" Prevention, Diagnosis and Treatment of New Coronavirus Infection (COVID-19) ", version 9 (26.10.2020), testing methods for antibodies to the SARS-CoV-2 virus are recommended to be used in the following cases:

“- as an additional method for diagnosing an acute infection (taking into account the seronegative period) or when it is impossible to study smears using the amplification of nucleic acids, including during hospitalization for somatic pathology;

- to identify individuals with an asymptomatic form of infection;

- to establish the fact of a previously transferred infection when examining risk groups and conducting a mass survey of the population to assess the level of population immunity;

- for the selection of potential donors of immunocompetent plasma ".

To detect antibodies in COVID-19, enzyme-linked immunosorbent assay (ELISA), immunochemiluminescence assay (ICLA) and rapid immunochromatographic (ICA) tests are used. ELISA is based on the immobilization of viral antigens on plates, to which antibodies from the sample bind. In the presence of specific antibodies of class G, M and A to SARS-CoV-2 antigens in the test samples, an antigen-antibody immune complex is formed, which is detected by a conjugate - a mixture of recombinant SARS-CoV-2 antigens or antibodies to human immunoglobulins labeled with horseradish peroxidase.

The main antigenic targets of the SARS-CoV-2 virus against which antibodies are detected are the spike protein (S-protein) and the nucleocapsid phosphoprotein (N-protein).

The S-protein present on the surface of SARS-CoV-2 is required for the virus to enter the cell; antibodies to this protein, in particular to its receptor binding domain (RBD), have neutralizing properties and prevent the binding of the virus to human cells, thereby providing an important role in determining the individual protective immune response to SARS-CoV-2 (Grigoryan L., Pulendran B. The immunology of SARS-CoV-2 infections and vaccines, Semin Immunol. 2020 Aug; 50: 101422).

N-protein is the most abundantly expressed immunodominant protein that interacts with RNA. The formation of antibodies to the nucleocapsid indicates the activation of cellular immunity (Ferretti A.R. et al. Unbiased screens show CD8 + T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein. Immunity. 2020 Nov 17; 53 (5): 1095-1107.e3). However, the presence of antibodies to the N-protein may indicate a previous infection with other seasonal human coronaviruses, because The nucleocapsid protein contains a number of conserved sequences in common (Kathleen M. McAndrews et al. Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity.JCI Insight. 2020 Sep 17; 5 (18): el42386 ). The dynamics of the formation of antibodies to different proteins of the coronavirus differs in each individual person - some individuals initially develop antibodies to N, others to RBD, and still others to both proteins simultaneously (Seow J. et al. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans.Nat Microbiol. 2020 Dec; 5 (12): 1598-1607; Kathleen M. McAndrews et al. Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity. JCI Insight. 2020 Sep 17; 5 (18): e142386.). The persistence of antibodies to different proteins also varies from person to person (Kathleen M. McAndrews et al. Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity. JCI Insight. 2020 Sep 17; 5 ( 18): e142386).

Since the sequence of the S-protein of SARS-CoV-2, especially RBD, is unique, the determination of antibodies to this protein is a very specific marker of the immune response to S ARS-CoV-2. This also has its negative aspects - due to the rapid variability of the virus, new strains of the pathogen appear with mutations in important epitopes, especially in epitopes associated with vital functions, for example, with the penetration of the virus into the cell. This can lead to undetectable SARS-CoV-2 mutant strains. The nucleocapsid sequence is more conserved. Therefore, some cross-reactivity with other seasonal coronaviruses is possible. But at the same time, the determination of antibodies to this protein, less vulnerable to mutations, ensures the stability of the results.

Taking into account that antibodies to the N and S proteins of SARS-CoV-2 may differ in the dynamics and duration of their presence in the blood, different levels of cross-reactivity with seasonal coronaviruses, the ability to participate in the formation of an immune response to the vaccine (depending on the type of vaccine used) , for an objective assessment of the immune status of the test sample, it is necessary to determine the presence of antibodies to 2 (these two) immunodominant proteins of the virus.

A separate problem is the possibility of DM, associated mainly with the presence of antibodies to seasonal coronaviruses.

To minimize the decision-maker of the BMP study of the Ministry of Health of the Russian Federation "Prevention, diagnosis and treatment of new coronavirus infection (COVID-19)", version 9 (26.10.2020), it is recommended to "introduce an algorithm for sequential testing of patients who have received initial positive results for antibodies of the IgM / IgA classes or IgG, using a different test. For this purpose, it is necessary to use a test system with maximum sensitivity and specificity, as well as with the simultaneous detection of antibodies of classes A, M, G, which will play the role of a reference (verification) test system. As such a test system, a test system can be used to detect the receptor-binding domain of the surface glycoprotein S SARS-CoV-2. The use of such an algorithm will enable reliable diagnostics. " There is international experience in using sequential testing for different SARS-CoV-2 antigens. A similar testing algorithm for antibodies to the causative agent COVID-19 is set out in the Interim Guidelines for the Center for Disease Prevention and Control (CDC, USA) dated 01.08. 2020 (https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines.html) and in a number of publications on the epidemiological assessment of the prevalence of specific antibodies in the population (Gudbjartsson DF et al. Spread of SARS-CoV-2 in the icelandic population.N Engl J Med. 2020 Jun 11; 382 (24): 2302-15; Ripperger TJ Orthogonal SARS-CoV-2 serological assays enable surveillance of low-prevalence communities and reveal durable humoral immunity Immunity 2020 Nov 17; 53 (5): 925-3.e4). The proposed solution fully complies with the described algorithms and allows you to confirm the obtained positive test results in accordance with Russian and international recommendations.

The approach for making a decision on screening in order to establish a current or past coronavirus infection is the previously described “Method of using recombinant SARS-COV-2 proteins as part of a test system for enzyme-linked immunosorbent assay with the determination of levels of antibodies of the IgM, IgG, IgA classes in serum / plasma blood of patients with COVID-19 "according to patent RU 2730897. However, the described method for determining antibodies to the sum of proteins sorbed together does not allow the use of such a test system to determine the spectrum of antibodies in order to minimize DM or to separate native and post-vaccination immunity.

Many tests have been developed to detect antibodies to SARS-CoV-2. All of them differ from each other by the type of diagnostic target, detected by the spectrum of antibodies, the volume of the tested sample, the setting procedure, and the level of the threshold value. Table 1 summarizes the characteristics of the main tests available on the market for the diagnosis of SARS-CoV-2.

The variety of designs and different analytical procedures lead to low convergence of test results, especially during the period of early seroconversion or when examining people with little or no symptoms of the disease. So in the work of Gudbjartsson D.F. et al. (2020) showed that the convergence of test results using 2 different tests using N and S proteins as a diagnostic target varies from 32.4 to 93.4%, depending on the group studied (Gudbjartsson DF et al. Spread of SARS -CoV-2 in the icelandic population. N Engl J Med. 2020 Jun 11; 382 (24): 2302-15).

One of the most important points, along with the diagnostic target and classes of antibodies, is the established threshold value (cut off or critical optical density, OD crit.). This value is determined by the test manufacturer based on statistical processing of the negative sample signals. The size of the sample of negative samples, their origin and the method of statistical processing vary from manufacturer to manufacturer during the creation and validation of the test system and, accordingly, determine the difference in the established threshold value, which is individual for each test system. It has been shown that the use of a single or normalized threshold value can increase the repeatability of the study results, as well as its sensitivity and specificity (National SARS-CoV-2 serology assay evaluation group. Performance characteristics of five immunoassays for SARS-CoV-2: a head-to -head benchmark comparison. Lancet Infect Dis. 2020 Dec; 20 (12): 1390-1400; Haselmann V. et al. Comparison of test performance of commercial anti-SARS-CoV-2 immunoassays in serum and plasma samples. Clin Chim Acta . 2020 Nov; 510: 73-78).

The use of the sum of the results of different-format tests, not standardized according to the conditions of the analysis, also does not allow us to draw objective conclusions about the presence of antibodies to individual SARS-CoV-2 proteins (especially about their relative amount), and, therefore, about the possibility of confirming positive or questionable research results. , minimization of the decision-maker of the study.

Disclosure of the essence of the invention

The present technical solution consists in creating a method for the differentiated detection of specific antibodies of classes A, M, G to the nucleocapsid and the receptor-binding domain of the spike protein of the coronavirus severe acute respiratory syndrome-2 (SARS-CoV-2) and an enzyme-linked immunosorbent assay for detecting the spectrum of antibodies to coronavirus SARS-CoV-2, which causes COVID 19, allowing for the differentiated (separate) detection of all classes of specific antibodies to the nucleocapsid and the receptor-binding domain of the SARS-CoV-2 spike protein. Includes an immunosorbent based on coronavirus antigens representing the receptor-binding domain (RBD) and nucleoprotein (N) SARS-CoV-2 and detection reagents, and the above antigens are sorbed in different wells of ELISA plates; for sorption, 96-well collapsible polystyrene plates are used.

The technical result consists in providing increased sensitivity, the possibility of confirming positive or dubious research results, minimizing the decision-maker of the study, separating native and post-vaccination immunity, simplifying and convenient analysis.

The advantage of the proposed test system is that it allows one to assess the immune status of an individual in relation to COVID 19 infection under standard conditions, while simultaneously detecting antibodies to both the adhesion and nucleocapsid of the SARS-CoV-2 virus. The proposed test is developed in the "sandwich" -format ELISA, allows you to detect total antibodies (classes G, M and A) to the specified proteins SARS-CoV-2. According to the setting procedure, a one-stage ELISA version (30 minutes at 37 ° C in a shaker at 500 rpm), followed by washing and the manifestation of the reaction by adding a chromogenic mixture. The reaction is recorded spectrophotometrically on an ELISA reader.

This technical solution differs from the known ones:

1) Separate determination of antibodies to N and S proteins of the SARS-CoV-2 virus in one set, sorbed in different wells of polystyrene plates;

2) Standard test conditions and common test reagents for both proteins to ensure the same kinetics of the immune and enzymatic reactions;

3) A single cut-off value for both proteins, determined using the same group of samples and the same statistical methods;

4) Ease of use, which consists in the provision in one set of all the necessary reagents for the determination of antibodies to 2 different antigens of the SARS-CoV-2 virus.

The proposed solution is characterized by:

- high sensitivity and specificity (tables 2, 3, 4, 5);

- the possibility of confirming positive or questionable research results and minimizing the decision maker (Tables 7, 8)

- the possibility of separating native and post-vaccination immunity (table 6, 8)

Implementation of the invention

A set of reagents for detecting the spectrum of antibodies of classes G, M, A to the coronavirus SARS-CoV-2 (COVID-19) by ELISA ("DS-ELISA-ANTI-8AK8-CoU-2-SPECTRUM") is intended for the separate detection of antibodies of classes G , M and A to the nucleocapsid and spike protein SARS-CoV-2, which causes coronavirus infection COVID-19, in human serum or plasma.

A set of reagents can be used to assess the immune response to a postponed or current coronavirus infection COVID-19, laboratory diagnostics of the current infection in combination with other studies, confirm positive or questionable results of the study, minimize the decision-maker of the study (in accordance with clause 4.2. Of the section "Laboratory diagnostics of COVID-19 "BMP of the Ministry of Health of the Russian Federation" Prevention, diagnosis and treatment of new coronavirus infection (COVID-19) ", version 9, dated 26.10.2020).

Means and methods

Reagent kit composition

ELISA

The results for anti-8AK8-CoV-2-nucleocapsid and aHTH-SARS-CoV-2 spike should be recorded separately (for Nc-labeled plate strips, for Sp-labeled plate strips).

Take into account the reaction if the optical density (OD) in the wells with K + is not less than 0.600, and the average OD value in the wells with K- is not more than 0.150.

OP crit. calculate by the formula:

OP crit. = OP cp.K- + A,

where A is the coefficient determined by the manufacturer by the method of statistical processing of the ELISA results, the value of which is indicated for each batch in the instructions for use and in the batch passport.

Calculate the coefficient of positivity KP using the formula:

CP = sample OD / OD crit.

Consider the test samples as positive: if CP ≥ 1.0 in at least one well.

The test samples are regarded as negative: if the CP is <1.0 in both wells.

Interpretation of results

The effectiveness of the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" is demonstrated by the following examples.

Example 1.

Diagnostic sensitivity

Diagnostic sensitivity based on the commercial AccuSet ™ SARS-CoV-2 Performance Panel 0820-0410, SeraCare. According to the data from the panel's passport, numbers 1-10 contain antibodies to SARS-CoV-2, number 11 does not contain antibodies to SARS-CoV-2. The test results are presented in table 2.

The sensitivity of the DS-IFA-ANTI-SARS-CoV-2-SPECTRUM according to the AccuSet ™ SARS-CoV-2 Performance Panel, 0820-0410 manufactured by SeraCare was 100%.

The diagnostic sensitivity of the set of reagents "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" 26) were evaluated in the study of 171 blood serum samples from 105 patients with a confirmed diagnosis of COVID-19, obtained at different periods of disease development (0-47 days after the onset of symptoms) provided by the Privolzhsky District Medical Center of the Federal Biomedical Agency (FBUZ POMC FMBA of Russia). In all patients, the diagnosis of COVID-19 was confirmed by laboratory PCR testing.

We examined 35 clinical samples of blood serum from patients with a confirmed diagnosis of COVID-19, obtained at different periods of the development of the disease, samples were provided by the FBUZ "Republican Clinical Infectious Diseases Hospital", Syktyvkar, Russia.

To assess the diagnostic sensitivity, commercial samples (n = 4) of blood serum from patients with a confirmed diagnosis of COVID-19 Positive SARS-CoV-2 sample, Cantor Bioconnect, Canada, lot 190188, lot 190679, lot 190680, lot 190778 were used.

All studied samples were divided into three groups according to the time of collection from patients from the moment of the onset of the disease (the onset of the first symptoms): for the early stage of the disease ≤1 week, for the current infection 1-2 weeks (8-14 days), and for the late stage> 2 weeks from the onset of symptoms of the disease (table 3).

Long Q.X. et al. (2020), in patients diagnosed with COVID-19, seroconversion of antibodies to SARS-CoV-2 occurs 17-19 days after the onset of the disease (Long QX et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020 Jun; 26 (6): 845-8). Based on the data presented in the study, a panel of 49 clinical samples was formed to assess the sensitivity of the DS-IFA-ANTI-SARS-CoV-2-SPECTRUM test system. The panel consists of 49 serum (plasma) blood samples from patients with a confirmed diagnosis of COVID-19 (positive results from PCR testing and chest CT). All samples were collected from 17 to 47 days from the onset of symptoms of the disease (after two and a half weeks) (table 4).

In "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" all samples 49/49 showed a positive result, the diagnostic sensitivity of the test system for detecting antibodies in this group of patients (> 2.5 weeks from the onset of the disease) was 100% (95% CI: 92.73-100.00%).

Example 2.

Panel testing of samples collected from day 1 to day 28 after onset of symptoms of COVID-19, in parallel with a comparison test

Tested simultaneously in "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" and SARS-CoV-2-CoronaPass comparison test, Biopalitra, 33 samples from patients with a clinically confirmed diagnosis of COVID-19 from FBUZ POMC FMBA of Russia. The results are shown in Table 5.

The table shows that separate testing of antibodies to each antigen is inferior in sensitivity to the option that takes into account simultaneously the immune response to both antigens (DS-ELISA-anti-SARS-CoV-2-SPECTRUM) - 90.9% and 93.9% against 100 , 0%. The sensitivity of DS-ELISA-anti-SARS-CoV-2-SPECTRUM is also higher than that of SARS-CoV-2-CoronaPass, Biopalitra, even in terms of testing antibodies to the SARS-CoV-2 spike protein, which is the basis of the test system production of Biopalitra (93.9% versus 78.8%).

Tested 54 serum samples from 54 laboratory-confirmed COVID-19 patients obtained during the convalescent period of the disease (59-127 days after the onset of symptoms). The diagnostic sensitivity was 100% (95% CI: 93.36 - 100.00%).

Example 3.

Testing serum samples from vaccinated individuals

27 samples of human blood serum obtained on the 21st day after vaccination with the second dose of the drug "Gam-COVID-Vac Combined vector vaccine for the prevention of coronavirus infection caused by the SARS-CoV-2 virus," N.F. Gamalei "of the Ministry of Health of Russia, which were provided by the State Healthcare Institution" Lipetsk Regional Clinical Infectious Diseases Hospital ", the State Healthcare Institution" Lipetsk Regional Center for the Prevention and Control of AIDS and IZ ", the State Healthcare Institution" Gryazinsky Central District Hospital ", the State Healthcare Institution" Lipetsk Emergency Hospital No. 1 ", GUZ "Eletsk city hospital №1 named after ON. Semashko ”, State Healthcare Institution“ City Hospital No. 4 Lipetsk-Med ”.

The samples were examined in "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" (series 243001 ex.-pr., Valid until 2021-11-26), the results are presented in table 6.

In the study in "DS-ELISA-ANTI-SARS-CoV-2-SPECTRUM" 27 blood serum samples obtained from persons after vaccination, antibodies to the nucleocapsid SARS-CoV-2 were not detected, since the vaccine "Gam-COVID-Vac Combined vector vaccine for the prevention of coronavirus infection caused by the SARS-CoV-2 virus ”is based on the SARS-CoV-2 spike protein sequence; antibodies to the spike protein contain 27 out of 27 samples. The sensitivity of "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" for this sample was 100% (95% CI: 87.54 - 100.00%).

Example 4.

Assessment of the specificity of the test system

A collection of human serum samples were examined that were collected before November 2019 and gave a false-positive reaction when detecting antibodies to SARS-CoV-2 (n = 22). This collection was tested in the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM", the results are presented in table 7.

The results of the study showed that 4/22 samples from the collection of false positives showed a positive result for aHTH-SARS-CoV-2-nucleocapsid, all samples from this collection showed a negative result for anti-SARS-CoV-2-spike.

Nucleocapsid alignment data for SARS-CoV-2 relative to the nucleocapsid sequences of seasonal human coronaviruses (HKU1, NL63, OC43, 229E) using the NCBI National Center for Biotechnology Information, BLAST Protein, showed that there are several short conserved regions. In contrast, the SARS-CoV-2 spike (RBD) is a unique region that does not have homologous regions with seasonal human coronaviruses.

Based on the foregoing, the determination of antibodies to the SARS-CoV-2 nucleocapsid can give DM in the presence of antibodies to the homologous epitopes of seasonal coronaviruses in the sample, and this phenomenon is not typical for the determination of anti-spike antibodies (RBD).

Diagnostic specificity

The diagnostic specificity of the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" was assessed in the study:

- 242 random samples of blood serum from donors collected before November 2019 amounted to 99.17% (95% CI: 97.04 - 99.77%);

- 48 blood serum samples from hospital patients with non-communicable diseases and amounted to 100% (95% CI: 92.59 - 100.00%).

Analytical specificity

The analytical specificity of the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" was assessed in the study:

- 48 serum samples of pregnant women. Specificity was 100%) (95% CI: 92.59 - 100.00%);

- 80 samples of blood serum (plasma) of patients with infectious diseases caused by hepatitis B, C viruses, human immunodeficiency virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus. The specificity was 100% (95% CI: 95.42 - 100.00%).

Analytical interference

The influence of potentially interfering substances on the results of studies in the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" was evaluated by testing:

- 50 samples of IN.VENT interfering substances panel. The specificity was 100.00% (95% CI: 92.86 - 100.00%). The absence of interference was found in the study of blood serum samples containing antibodies to Escherichia coli, HAMA, as well as blood serum samples of pregnant women. It was determined that the presence of rheumatoid factor in blood serum samples up to 77.4 U / ml, bilirubin up to 502 μmol / L (0.3 mg / ml), triglycerides up to 6.61 mg / ml, cholesterol up to 2.04 mg / ml , hemoglobin up to 2.65 mg / ml does not affect the quality of studies in the test system.

Cross reactivity

The influence of potentially cross-reacting substances on the operation of the test system "DS-IFA-ANTI-SARS-CoV-2-SPECTRUM" was evaluated in the study:

- 6 artificial samples of human blood serum (plasma) containing molecules of monoclonal / polyclonal antibodies to the pathogens of SARS and MERS and Fc fragments of human immunoglobulin. The specificity was 100.00% (95% CI: 60.97 - 100.00%);

- 8 human serum samples containing antibodies to the causative agent of malaria and influenza A (H1N1), A (H3N2) and B. The specificity was 100.00% (95% CI: 67.56 - 100.00%).

Reproducibility

The coefficient of variation for testing positive samples within the plate, between plates of the same series and between series of the DS-IFA-ANTI-SARS-CoV-2-SPECTRUM test system does not exceed 10%.

Equivalence of human serum and plasma samples

The study of positive (n = 25) and negative (n = 25) for SARS-CoV-2 paired samples of human serum and plasma containing various anticoagulants (EDTA, heparin, sodium citrate), showed their equivalence, which makes it possible to attribute the indicators of diagnostic sensitivity and specificity to both types of test samples.

Antibody Spectra and Interpretation of Results

The interpretation options for the results for different groups of the studied samples are shown in Table 8. It can be seen from the presented data that the S + N + spectrum is present in 58, 3-100% of patients with current or previous infection, depending on the duration of the disease, in none of the obviously negative donors. (donation until 2019), none of the vaccinated. The S-N + spectrum is characteristic of the early phase of the disease - from 2.4-3.3% (for periods of 0-14 days), as well as 0.8%) of obviously negative donors, indicating a previous infection caused by seasonal coronaviruses. The presence of antibodies only in the spike protein of the S + N- virus is also observed in the early (up to day 15) stages of infection in 4.5-6.7%) of cases, in 100% of persons vaccinated with a vaccine based on the spike protein of the virus, and is not observed in a group of known negative controls. The absence of antibodies to both S-N- proteins can be observed in the first 2 weeks of the disease - 3.3-33.3%.

The data presented demonstrate the high diagnostic efficiency of the DS-IFA-ANTI-SARS-CoV-2-SPECTRUM reagent kit. The test system according to the invention can be used to detect all classes of specific antibodies to the nucleocapsid and the receptor-binding domain of the spike protein of the SARS-CoV-2 coronavirus, in order to assess the immune response to a past or current coronavirus infection COVID-19, laboratory diagnosis of current infection in in combination with other studies, confirmation of positive or questionable research results, minimization of the decision-maker of the study.

In this case, the diagnostic efficiency of the test system and method is not associated with specific sequences of Nc and RBD Sp antigens, but with the separate determination of antibodies to N and S proteins of the S ARS-CoV-2 virus in one set, sorbed in different wells of polystyrene plates; with standard conditions and common test reagents for both proteins, allowing to ensure the same kinetics of the immune and enzymatic reactions; a single cut-off value for both proteins, determined using the same group of samples and the same statistical methods.

SEQ ID NO: 1

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

SEQ ID NO: 2

VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGATPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKPGEQKLISEEDLSAGGHHHHHHHHHH

Claims (18)

1. Test system for the detection of specific antibodies of classes A, M, G to the nucleocapsid (Nc) and the receptor-binding domain of the spike protein of the coronavirus of severe acute respiratory syndrome-2 (SARS-CoV-2), containing a set of reagents for the detection of specific antibodies of the classes A, M, G to nucleocapsid and receptor binding domain (RBD) spike protein (Sp) of SARS-CoV-2 coronavirus: a 96-well collapsible polystyrene plate to strips or to wells, in the wells of which the recombinant SARS-CoV-2 antigens - Nc and RBD Sp are separately sorbed, and the short names of the immobilized antigens (immunosorbent) are applied on the surface of the strips; a mixture of recombinant antigens Nc and RBD Sp SARS-CoV-2 labeled with horseradish peroxidase (conjugate); control positive sample, inactivated (K +); control negative sample, inactivated (K-); concentrate (× 25) phosphate buffered saline with tween (FSB-T); sulfuric acid solution 0.2 M; substrate buffer solution (SB) - citrate buffer containing hydrogen peroxide solution; a solution containing 3,3 ', 5,5'-tetramethylbenzidine - TMB (× 11). 2. The test system according to claim 1, where the SARS-CoV-2 antigens - Nc and RBD Sp have the sequences

3. A method for the differentiated detection of specific antibodies of classes A, M, G to the nucleocapsid (Nc) and the receptor-binding domain of the spike protein (Sp) of the SARS-CoV-2 coronavirus, including the use of the test system according to PP. 1, 2 in a one-step enzyme-linked immunosorbent assay (ELISA), the required number of strips with immobilized Nc and RBD Sp antigens are inserted into the frame; 80 μl of a mixture of recombinant antigens Nc and RBD Sp SARS-CoV-2 labeled with horseradish peroxidase is added to each well; for each antigen, 20 μl K + and 3 wells K- are added to 1 well; the plate is covered with a protective film and kept in a thermal shaker for 30 minutes at a temperature of 37.0 ± 1.0 ° C and shaking at 500 rpm; wash the plate 4 times with a concentrate (× 25) of phosphate-buffered saline with tween (FSB-T), carefully adding the specified concentrate to the wells of the plate to the edges, at least 350 μl per well, then remove the contents of the wells into a container with a disinfectant solution and develop reactions by adding a chromogenic mixture; in all wells of the plate add 100 μl of the substrate mixture (citrate buffer with hydrogen peroxide and TMB); the plate is kept for 20 minutes in a place protected from light at room temperature 18-25 ° C; the reaction is stopped by adding to all wells 150 μl of a 0.2 M sulfuric acid solution and after 1-2 minutes, but not later than 20 minutes, the optical density (OD) is measured at a wavelength of 450 / 620-680 nm; the results of the reaction are recorded if the optical density (OD) in the wells with (K +) is not less than 0.600, and the average OD value in the wells with (K-) is not more than 0.150, while the OD crit. calculated by the formula: OPcrit. = OPsr. (K -) + A, where A is the coefficient determined by the manufacturer by the method of statistical processing of the ELISA results, the value of which is indicated for each batch in the instructions for use and in the batch passport, the positivity coefficient (KP) is calculated by the formula: KP = sample OD / OD crit., the test samples are considered positive if CP ≥ 1.0 in at least one well, test samples are considered negative if the CP is <1.0 in both wells.

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