RU2360253C2 - Way of revealing antibodies to antigens of hepatitis viruses - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely to diagnostics of infectious diseases and concerns a method of revealing antibodies to antigens of hepatitis viruses. The essence of the way consists in use of an immunochromatographic strip which consists from fiber glass non-sorbing matrix (6) on which the strips containing conjugates of painted particles with monoclonal antibodies to human immunoglobulins G and M (7,8) plastic substrates are placed and immobilised by means of drying, covered with an adhesin layer with low penetrating ability (1) on which a microporous nitrate cellulose membrane with evaporated on a side close to the substrate with a protective polymeric basis (2) is mounted, and admixtures of antigens of viruses of a hepatitis A, B, C (3, 4, 5) are placed perpendicularly to the long side of the strip on the membrane from the non-protected side. The strip is moistened with investigated blood serum and at detection of the painted strip around the strip of antigens of hepatitis A, antibodies to an antigen of a virus of hepatitis A are detected. At detection of the painted strip around the strip of antigens of hepatitis B, antibodies to an antigen of a virus of hepatitis B are detected. At detection of the painted strip around the strip of antigens of hepatitis C, antibodies to an antigen of a virus of hepatitis C are detected, which are formed at linkage of antibodies of blood serum with conjugates of antibodies to immunoglobulins G and M with the painted particles.

EFFECT: method allows to tap simultaneously antibodies to antigens of viruses of a hepatitis A, B, C and to define to what type they are specific to.

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Description

The invention relates to medicine, in particular to the diagnosis of infectious diseases. The method provides differential screening express diagnostics of hepatitis A, B and C. For this purpose, differential detection of antibodies to the hepatitis antigen is carried out, while the hepatitis A, B and C antigens are applied separately, forming a diagnostic area for each antigen within the same immunochromatographic strip. In the course of immunochromatography, a complex of antibodies to the corresponding antigens from the studied sera and conjugates of nanocolloidal particles with antibodies against human immunoglobulins, previously immobilized in an inert non-absorbent carrier in the sample application zone, migrate and bind in the application area of the corresponding antigen, visualizing the presence of antibodies to hepatitis A virus antigens, B and C, which allows us to simultaneously detect the presence of such antibodies and determine which of the viruses they are specific to.

Description of the invention

The invention relates to medicine, in particular to applied immunology and the diagnosis of infectious diseases.

Known enzyme-linked immunosorbent assay for detecting progressive titers of antibodies to hepatitis A virus in the prodromal period of the disease and the initial phase of the heat-up period (EP Shuvalova. Infectious diseases. M., 1990, p. 154). The disadvantage of this method is its duration and the detection of antibodies only to hepatitis A.

A known method for the diagnosis of hepatitis A using an enzyme-linked immunosorbent diagnostic test system for detecting hepatitis virus using magnosorbents (patent RU 2065164). The disadvantage of this method is the low diagnostic value due to the small amount of antigen in biological fluids. In addition, this method allows you to diagnose only hepatitis A.

There is a method of enzyme immunoassay for the determination of hepatitis C by detecting antibodies to a peptide composition that simulates the antigenic composition of the hepatitis C virus (patent RU 2071350). The disadvantage of this method is the duration of the analysis and the fact that the method allows you to determine only hepatitis C.

Known nanodiagnostic test system for the detection of hepatitis virus, including the use of a two-channel biosensor "resonant mirror" with a biochip consisting of a biosensor cell, at the base of which is a prism with a waveguide conjugated to it, formed from a layer of a medium with a high refractive index - "nel" and a quartz layer with a low refractive index "ncl" with thicknesses of the order of several hundred nanometers, depending on the material of the layer, to the sensitive surface of which is simultaneously the surface on cuvettes, probe molecules are immobilized, forming specific complexes in the biological fluid with macromolecular disease markers in the surface layer of a few hundred nanometers in size to the waveguide, by the interaction of antibody molecules (anti-HBs) and HBsAg (hepatitis B virus antigen), and / or interaction hepatitis C surface nucleocapsid antigen and antibodies to it, and / or hybridization of oligonucleotides with complementary regions to hepatitis B DNA, and / or hybridization of oligonucleotides with complementary asthma to hepatitis C virus RNA, with the subsequent determination of changes in the refractive index of light in the surface layer at the waveguide boundary (Application for invention RU 2004134192/15). The disadvantage of this system is the need for special devices for the detection and interpretation of the results.

Known immunochromatographic strips for detecting antibodies to hepatitis C virus antigens manufactured by VedaLab (France). The disadvantage of these tests is that they do not allow the diagnosis of other types of hepatitis. Thus, a universal rapid test that allows to differentially diagnose the presence of AT classes of IgG and IgM to hepatitis A, B and C is not currently proposed.

Known immunochromatographic strips for the detection of HBs antigen manufactured by Syntron (USA) (the closest analogue), however, these tests only diagnose patients with hepatitis B.

The challenge facing the authors was to create an express system for the complex differential diagnosis of hepatitis A, B and C by simultaneously determining antibodies of classes G (IgG) and M (IgM) to the antigens of these hepatitis in the blood serum of patients. For this purpose, a strip design for indirect immunochromatographic analysis was proposed, including the following elements (drawing):

1) a plastic substrate (1) coated with an adhesive layer (12) with low penetration;

2) a microporous cellulose nitrate membrane mounted on it (2) with a protective polymer base sprayed onto the side adjacent to the substrate, and on the membrane from the unprotected side they are applied perpendicular to the long side of the strip of the mixture of hepatitis A virus antigens (AHAG) (3), hepatitis B (AGHB) (4) hepatitis C (AHVHC) (5) so that the bands are parallel to each other;

3) a glass fiber non-absorbent matrix — PED (6), on which strips containing conjugates of colored nanocolloidal particles with monoclonal antibodies to human G-IgG (7) and M-IgM (8) human immunoglobulins were applied and immobilized by drying, moreover, the antibodies were applied perpendicular to the long side of the strip, while the stripes were parallel to each other;

4) cellulose filter suction (9);

5) lower protective dacron mask (10);

6) the upper protective mylar mask (11).

In the course of immunochromatography, the PED of the strip was moistened with the test serum (diluted or whole), presumably containing antibodies to the antigens of one of the hepatitis viruses. During lateral movement, the sample dissolved the conjugates in the PED, forming, in the case of the studied antigens, a complex conjugate against IgG (kg α IgG) or conjugate against IgM (kg α IgM) with antibodies (AT) of hepatitis A viruses (ATVGA), hepatitis B (ATVHV) and C (ATVGS), which, in turn, in the process of further lateral movement through test zones 3, 4 and 5 bind to the corresponding antigens, forming complexes of kg α IgA-ATVGA-AGHVA, kg α IgB-ATVHV- AGVGV, kg α IgC-ATVGS-AGVGS. Such complexes, immobilizing in the corresponding test zones, visualized the presence of the corresponding antibodies, forming colored bands. In the absence of AT, no band was stained for these AHs, indicating the absence of patient contact with the studied viruses. Thus, using one strip, it was possible to determine antibodies of the human IgG and IgM classes to AH of hepatitis A, B, and C viruses by the appearance of colored bands in the test zone of the strip, and the type of virus was differentiated by the location of the colored strip.

The concentration of the mixture of antigens in the experimental bands during application ranged from 0.1 to 1 mg / ml As a positive and negative control of the reaction used serum production GISK them. Tarasevich. Ten sera containing each of the tested ATs and ten donor sera not containing such antibodies were tested. The same sera were examined using enzyme-linked immunosorbent assay systems manufactured by Vector-Best CJSC. The coincidence of enzyme-linked immunosorbent assay and immunochromatographic analysis was 100%.

Example 1. On a block consisting of a plastic substrate (1) coated with an adhesive layer (12) and mounted on it with a nitrocellulose membrane with a pore diameter of 10 μm manufactured by MDI (India), a suction (9) was mounted so that it 1 mm overlapped the membrane, but did not go beyond the border of the substrate (1). A mixture of genetically engineered AGs from NPO LLC was applied to the membrane (2) at a concentration of 0.1 mg / ml in stripes 1 mm wide. Diagnostic systems (Russia) in the following order - AGVGS (5) 1.5 cm from the edge of the suction, AGVGV (4) - 2 cm, AGVGA (3) - 2.5 cm and dried in a laminar air flow at 37 ° C for 30 minutes. Mixtures of AG had the following composition:

moreover, in each mixture, the constituent AGs were taken in equal weight parts.

Conjugates of nanocolloidal particles were obtained by sorption of monoclonal antibodies against IgG and IgM on colloidal gold with a particle diameter of 30 nm from ImmunoGold (Great Britain) according to the copy attached to colloidal gold. After sorption, the conjugate was centrifuged at ten thousand rpm and a temperature of 4 ° C for 30 minutes and stabilized by adding an equal volume of a thirty-three percent solution of bovine serum albumin to the precipitate (SIGMA, USA). The conjugates were applied onto PED with a strip of 2-4 mm thickness and dried with two bands of kg IgG (7) and kg IgM (8) at 37 ° С. After drying, the PEM was mounted on the adhesive surface (12) of the substrate (1), opposite to that on which the suction (9) was mounted so that the edge of the PED was 1 mm above the edge of the membrane, and the opposite edge of the PED coincided with the edge of the block. Then, a mask was mounted on top of the PED so that it protruded 1 mm beyond the edge of the PED to nitrocellulose, and its opposite edge coincided with the edge of the block. The side of the mask adjacent to the PED was coated with an adhesive layer and was fixed on the PED and nitrocellulose. The free side of the mask was painted so as to differ from the mask covering the suction. Arrows were drawn on it, pointing to the edge of the PED opposite the edge of nitrocellulose. This edge comes into contact with the sample during the reaction, so the arrows on the mask are necessary to eliminate the wrong orientation of the strip during analysis. A mask was mounted on top of the suction so that it protrudes 1 mm above the edge of the suction on nitrocellulose. Its opposite edge coincided with the edge of the block. The adhesive layer of the inside of the mask stuck the mask to the suction and nitrocellulose. After the installation of the block was completed, it was cut into strips 5 mm wide and stored in sealed bags from an aluminized laminate with a desiccant at a temperature of 4-25 ° C.

Testing of the obtained strips was carried out as follows. Diluted in accordance with the instructions of the studied sera were placed in the wells of a 96-well plate (350 μl / well). The edge of the strips marked with arrows was placed in the same wells. The reaction was taken into account 10 minutes after the start of the formulation. We used 10 experimental sera containing antibodies to HAV, HBV, HCV, and 10 control sera. The coincidence of the results of immunochromatography with the data of GISK them. Tarasevich about these sera was 100%.

Example 2. Strips were prepared similarly to Example No. 1. In test zones 3, 4 and 5, respectively, AH at a concentration of 1 mg / ml was applied - AGHA (3) - 2.5 cm from the edge of the suction, AGHH (4) - 2 cm, AGHH (5) 1.5 see. We used 10 experimental sera containing antibodies to HAV, HBV, HCV, and 10 control sera. The coincidence of the results of immunochromatography with the GISK im.Tarasevich on these sera was 100%.

Claims (1)

A method for detecting antibodies to antigens of hepatitis A, B, C viruses, characterized in that antibodies of classes G and M to antigens of hepatitis A, B, C viruses are simultaneously determined using an immunochromatographic strip, which consists of a glass fiber non-absorbent matrix, onto which are applied and immobilized by drying, strips containing conjugates of colored particles with monoclonal antibodies to human immunoglobulins G and M, a plastic substrate coated with an adhesive layer with a low permeability, on which a microporous cellulose nitrate membrane was mounted with a protective polymer base sprayed onto the side adjacent to the substrate, and a mixture of hepatitis A, B, C virus antigens from solutions with a concentration of 0.1-1 mg / ml was applied perpendicular to the long side of the membrane on the membrane; the strip is wetted with the test blood serum and when a colored band is detected in the region of the hepatitis A antigen band, antibodies to the hepatitis A antigen are detected, when a colored band is found in the region of the hepatitis B antigen band, antibodies are detected for the hepatitis B virus antigen, if a colored band is detected in the region of the antigen band hepatitis C antibodies to hepatitis C virus antigen are detected, which are formed by binding of blood serum antibodies to conjugates of antibodies to immunoglobulins G and M with colored particles.