Immunoassay method for determining a soluble form of hla - dr antigens in the serum of a person

 

The invention relates to medicine, in particular to immunology. The essence of the invention is that of solid media are specific immobilization of antibodies on a solid medium, sequential incubation with the test serum and peroxidase conjugate, at the same time as the first antibodies used polyclonal antibodies against surface antigens of mononuclear cells as well as secondary antibodies used HLA-DR-specific monoclonal antibodies ICA-1, conjugated to horseradish peroxidase. 2 C.p. f-crystals, 1 tab., 1 Il.

The invention relates to medicine, in particular to immunology. The content of soluble HLA-DR molecules is an important indicator in transplant immunology. The content of soluble HLA-DR antigens in serum significantly increased in rheumatoid arthritis (Claus et al.//Cell Immunol, 2000, V. 206, no. 2, R. 85-100; Verbruggen et al.//Tissue Antigens, 2000, V. 56, No. 5, P. 436-440), autoimmune hepatitis (Hagihara et al.//Autoimmunity, 1999, V. 31, No. 2, RV 85-93). Notes prognostic significance of the level of molecules sHLA-DR in the serum of patients With hepatitis C treated with interferon alpha (Hosoi et al.//Tokai. J. Exp. Clin. Med., 2000, v.25, No. 3, p.117-124). Known correlation between si is, vortochny level of soluble HLA-DR antigens can be used as a prognostic indicator of current diseases of different nature.

Known some methods for determining the soluble forms of HLA-DR antigens:

1) a Method of inhibition of antibody-based microlymphocytotoxicity samples, which are as follows: to test the monospecific anticigarette add different analyzed for HLA-DR fluid samples, after incubation contribute lymphocytes containing HLA corresponding to the antibodies in the antisera. Then hold microlymphocytotoxicity test with the use of complement (see Terasaki P. J., Mc Glelland J. D. // Nature, 1964, v.204, p.998-1000; modification Rahimov A. A. // Immunology, 1982, No. 3, S. 34-36). However, this method has low sensitivity, because the assessment of the intensity of cytotoxic reactions produce by the percentage of cell death.

2) high-speed Method nephelometry, based on the dependence of the rate of interaction of HLA antigens with monoclonal antibodies on the concentration of HLA molecules (see Konenkov C. I., Naumov, Y. N., Prokofiev C. F.//Immunology, 1987, No. 6, S. 61-63). But this method requires expensive equipment for registration of the change of turbidity of the investigated solutions (turbidity meter).

The closest to p is nn S., Claus R., Buchwald, S., Kohler H., Hausmann, D., Falk, U., Wegener S.//J. Immunoassay, 1996, 17, 257-75). In this way the immunological tablets absorb HLA-DR-specific monoclonal antibody BL-Ia/5 if 4C, then incubated with serum samples and standards at 37With over 90 minutes the Next stage - sequential incubation with HLA-DR-specific monoclonal antibody L243 (1 h), streptavidin, conjugated to peroxidase (30 min) and o-phenylenediamine as a substrate (30 min). The reaction is stopped with sulfuric acid, measuring the optical density at 492 nm and transferred by means of a calibration curve (in ng. This method is characterized by high sensitivity and ease of production, but due to the use of monoclonal antibodies foreign production is quite expensive. However, an indirect connection of the second antibody and enzyme reduces the specificity of the reaction.

The aim of the invention is a cheaper method with high sensitivity and specificity.

The invention consists in that for the determination of soluble molecules HLA-DR as the first antibodies used polyclonal antibodies against surface antigens of mononuclear cells in humans, and in quality the om center of RAMS, conjugated directly with horseradish peroxidase.

The method of determination of soluble HLA-DR molecules by using enzyme-linked immunosorbent assay as follows.

Polyclonal antibodies against surface antigens of mononuclear cells secrete using ammonium sulfate and ion-exchange chromatography on DEAE-cellulose and absorb in a dilution of 1:1000 in 100 μl into the wells of polystyrene plates to assay for 16-18 h at 4-8C. After washing is not bound peroxidase antibodies in the wells contribute samples of the investigated sera. Tablets incubated at room temperature for 16-18 h with subsequent laundering is not bound peroxidase proteins. Then holes tablets handle working solution of purified monoclonal antibodies ICA ICA-1, conjugated to horseradish peroxidase (Immunological methods /edited, Primes, S. 438) at a dilution of 1:400 and incubated for 1 hour at 37C. After washing is not bound peroxidase labeled antibodies in the wells of tablets make the substrate: 10 ml citrate buffer 3 mg o-phenylenediamine, followed by incubation for 15-20 min at room temperature. The reaction is stopped LA-DR antigens appreciate translating units of optical density in arbitrary units (U/ml) calibration curve using standard representing stored at -60With the samples of blood serum containing soluble HLA-DR in the same concentrations.

The optimal concentration of polyclonal antibodies for the sorption of tablets was determined by measuring the optical density at different dilutions from 1:100 to 1:1500. The maximum difference between experimental and baseline values was observed at a thousand-fold dilution of polyclonal antibodies (see Fig. 1A). The most effective concentration of monoclonal antibodies conjugated with horseradish peroxidase, was also determined using the dilution of conjugate from 1:50 to 1:800. Optimal was the breeding of 400 times (drawing In).

The practical application

In the study the level of soluble form of HLA-DR molecules in the serum 198 healthy donors was shown that it is normally 115,914,61 U/ml. Serum samples were obtained from 22 patients with rheumatoid arthritis (18 women and 4 men). The average age of patients was 44,22.3 years, mean disease duration of 7.719 U/ml (p<0,01). There was a significant correlation between serum levels of sHLA-DR and parameters of disease activity patients: the erythrocyte sedimentation rate, rheumatoid factor, C-reactive protein (see table). Thus, determination of serum concentrations of sHLA-DR molecules allows to judge about the activity flow of rheumatoid arthritis.

The advantages of the developed method is easy to use, no complex equipment, expensive reagents, high specificity, the ability to determine the minimum concentration of a substance (nanogram/ml). Conjugation of the second antibody directly with the enzyme reduces the stage.

Due to the fact that the molecules of the major histocompatibility complex plays a Central role in the immune response, determining the severity of the reaction of the immune system of the individual for each antigenic exposure, the study level restson viral diseases, autoimmune and other wildlife, as well as in the transplantation of various organs.

The method developed and implemented in the laboratory of molecular immunology bacterial and viral infections, research Institute of epidemiology and Microbiology. Acad. I. N. Blokhina, D. N. Novgorod.

Claims

1. The method for determining a soluble form of HLA-DR antigens in the serum of a person, including immobilizing specific antibodies on a solid medium, sequential incubation with the test serum and peroxidase conjugate, characterized in that as the first antibodies used polyclonal antibodies against surface antigens of mononuclear cells in man in a dilution of from 1:100 to 1:1500, and as secondary antibodies used HLA-DR-specific monoclonal antibodies ICA-1, conjugated to horseradish peroxidase at a dilution of 1:50 to 1:800.

2. The method according to p. 1, characterized in that the optimal dilution of polyclonal antibodies against surface antigens of mononuclear cells in humans is 1:1000.

3. The method according to p. 1, characterized in that the optimal dilution of HLA-DR-specific monoclonal antibodies ICA-1, conjugated to peroxidase is

 

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