RU2797874C2 - REAGENT KIT FOR ENZYME IMMUNOASSAY QUANTITATIVE DETERMINATION OF VIRUS-NEUTRALIZING ANTIBODIES TO SARS-CoV-2 - Google Patents

Abstract

FIELD: laboratory diagnostics.

SUBSTANCE: invention can be used for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to the receptor-binding domain — RBD of surface glycoprotein S of the SARS-CoV-2 virus in human serum or blood plasma and laboratory animals. The invention is a set of reagents for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to SARS-CoV-2, which implements a two-stage "competitive" variant of enzyme-linked immunosorbent assay.

EFFECT: obtaining a reagent kit for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to SARS-CoV-2.

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Description

The invention relates to the field of biotechnology and medicine, namely to laboratory diagnostics.

The invention can be used to quantify the humoral immune response to a current or past infection caused by the SARS-CoV-2 virus, to determine the level of post-vaccination immunity formed on the S-protein (including RBD) of the SARS-CoV-2 virus, and also to detect virus-neutralizing activity of human and animal blood sera, monoclonal antibodies of various species.

In the kit of reagents for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to SARS-CoV-2, a two-stage "competitive" variant of enzyme-linked immunosorbent assay is implemented. The kit of reagents is intended for the quantitative determination of virus-neutralizing antibodies to the receptor-binding domain - RBD surface glycoprotein S of the SARS-CoV-2 virus in human and laboratory animal blood serum (plasma) by enzyme-linked immunosorbent assay.

The results of the quantitative determination of antibodies to the SARS-CoV-2 virus have a correlation coefficient with a neutralization reaction of 0.84, unlike other reagent kits for the quantitative determination of class G immunoglobulins to SARS-CoV-2 surface glycoprotein S in human serum (plasma), for which it is 0.7.

The kit is intended for professional use in clinical laboratory diagnostics and research organizations.

The invention is new, since publicly available sources in the Russian Federation do not contain information about a set of reagents that allows for the quantitative analysis of virus-neutralizing antibodies by a two-stage "competitive" enzyme-linked immunosorbent assay, which includes calibration samples certified against the WHO International Standard First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human), NIBSC code: 20/136 IU - international units").

As a prototype of the proposed invention, a set of reagents for enzyme immunoassay quantitative determination of class G immunoglobulins to SARS-CoV-2 "SARS-CoV-2-IgG quantitative-ELISA-BEST" was chosen, intended for the quantitative determination of class G immunoglobulins to surface glycoprotein S of SARS-CoV -2 in human serum (plasma) by enzyme-linked immunosorbent assay.

The choice of the kit specified above is due to the similarity of the composition of the reagent kit, as well as the purpose of both kits for the quantitative determination of antibodies to the receptor-binding domain - RBD of the surface glycoprotein S of the SARS-CoV-2 virus in human serum (plasma) and laboratory animals by enzyme-linked immunosorbent assay

The features of the present invention, which distinguish it from its analogue, include:

- the proposed set of reagents is intended for the quantitative determination of virus-neutralizing antibodies, the comparison set is for the determination of class G immunoglobulins (IgG);

- in the proposed set, the determination method is based on a two-stage "competitive" variant of enzyme-linked immunosorbent assay, in the comparison set - on a two-stage "indirect" variant of enzyme-linked immunosorbent assay.

The proposed invention is industrially applicable, since typical biotechnological equipment, as well as reagents and materials widely used in the biotechnological industry, can be used to manufacture the kit.

The kit of reagents for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to SARS-CoV-2 "SARS-CoV-2-VHA-ELISA-RHBZ" includes:

1. A collapsible 96-well plate for carrying out a reaction with an immobilized recombinant extracellular domain of the human angiotensin-converting enzyme ACE-2 - 1 pc;

2. Calibration samples containing known amounts of IgG to SARS-CoV-2 - 0; 25; 100 IU/ml - 5 bottles of 1.5 ml;

3. Control sample (blood serum containing antibodies to SARS-CoV-2 at a concentration of 60-90 IU /) - 1 bottle of 1.5 ml;

4. Conjugate (conjugate of recombinant S-protein vipyca SARS-CoV-2 with horseradish peroxidase) - 1 vial 11 ml;

5. Concentrate of phosphate-buffered saline solution with tween - 1 bottle of 28 ml;

6. Tetramethylbenzidine solution - 1 vial 13 ml;

7. Stop reagent (sulfuric acid 4.9%) - 1 vial 12 ml;

8. 96-well collapsible plate for preliminary dilution of test samples - 1 pc.;

9. Films for gluing the tablet - 2 pcs.;

10. Tips for dispensers for 2-200 µl - 16 pcs.;

11. Tray for reagents - 1 pc.

Analysis using a set of reagents for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to SARS-CoV-2 "SARS-CoV-2-VHA-ELISA-RCBZ" is carried out according to the following scheme.

135 μl of the conjugate and 15 μl of the test sample are added to the wells of the fluorimeter for dilution and incubated.

100 μl of 10-fold diluted samples, including calibration samples 0; 25; 100 IU/ml and control sample.

Measurement of the results of the analysis is carried out at a wavelength of 450 nm.

The strips are sealed with a film and incubated for 60 minutes in a thermostatically controlled shaker at a temperature of (37±1)°C with a stirring intensity of 300 rpm.

At the end of the incubation, the sticky film is removed and placed in a vessel with a disinfectant solution. Using the wash device, wash the wells of the plate 5 times with the wash solution, alternating between aspiration and immediate filling of the wells of each strip. 350 μl of fluid is added to each well during each wash cycle. The time between filling and emptying the wells should be at least 30 seconds. At the end of washing, the remaining moisture from the wells is carefully removed.

100 μl of tetramethylbenzidine solution are added to all wells and incubated in a place protected from direct sunlight for 25 minutes at a temperature of (18-26)°C.

Add 100 µl of Stop Reagent to all wells.

Measurement of the results of the analysis is carried out at a wavelength of 450 nm and a reference wavelength in the range of 620-655 nm.

The time between stopping the reaction and measuring the optical density should not exceed 5 minutes.

The concentration of virus-neutralizing antibodies to SARS-CoV-2 (IU/ml) in the analyzed samples is determined according to the calibration curve (dependence of the optical density value on the concentration), built in linear coordinates based on the readings of the calibration samples (IU/ml).

Claims (1)

A set of reagents for enzyme immunoassay quantitative determination of virus-neutralizing antibodies to the receptor-binding domain - RBD surface glycoprotein S of the SARS-CoV-2 virus in human and laboratory animal serum or plasma, including calibration samples containing known amounts of IgG to SARS-CoV-2 - 0; 25; 100 IU/mL, negative control, conjugate, tween phosphate buffered saline concentrate, tetramethylbenzidine solution, stop reagent, 96-well collapsible reaction plate and pre-dilution plate for test samples, sealing films, tips for dispensers, a bath for reagents, characterized in that the recombinant extracellular domain of the human angiotensin-converting enzyme ACE-2 is immobilized in the wells of the plate for the reaction, the conjugate is the recombinant S-protein of the SARS-CoV-2 virus with horseradish peroxidase, the dilution of serum acts as a control sample blood containing antibodies to SARS-CoV-2 at a concentration of 60-90 IU / ml.