RU2293335C1 - Method for evaluating autoimmunity to human hepatic antigen - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with evaluating autoimmunity to ganglioside of human hepatocytes. In blood serum due to immunoenzymatic assay one should detect the content of autoantibodies IgM and IgG to ganglioside of hepatic tissue, calculate the ratio of the content of autoantibodies in tested serum against their content in negative control, and at its value being above 1 it is possible to detect the presence of autoimmune process in human liver. The innovation enables to fulfill diagnostics of autoimmune process in hepatic tissue at higher accuracy and specificity.

EFFECT: increased accuracy and efficiency of evaluation.

Description

The invention relates to medicine, namely to immunological and biochemical methods for studying the parameters of biological substrates in order to evaluate the autoimmune process in the human liver.

A known method for the determination of serum LKM-1 (autoantibodies to liver and kidney microsomes) (Yamamoto A.M. et al., 1993; K. Choudhuri et al., 1998). As a result, LKM-1 autoantibodies to liver and kidney microsomes are determined by enzyme-linked immunosorbent assay, but by this method, LKM-1 autoantibodies are detected only with type II autoimmune hepatitis. In addition, the production of anti-LKM-1 autoantibodies is induced not only in autoimmune pathology, but also in case of poisoning by a number of chemical and medicinal substances (phenobarbital, carbamazepine, etc.) and is also noted in viral hepatitis C and D.

There is also a method of determining antinuclear autoantibodies (ANA) (Feltkamp T.E. W. 1996, Alvarez F. et al., 1999). According to this method, in the diagnosis of autoimmune type I hepatitis in blood serum, autoantibodies to antigens of cell nucleus structures (ANA) are determined by enzyme-linked immunosorbent assay. The disadvantage of this method is that with type I autoimmune hepatitis, for the differential diagnosis of which the definition of antinuclear autoantibodies is mainly used, they are detected only in 70-80% of cases. In addition, ANA are not specific autoantibodies only for type I autoimmune hepatitis and are found in a wide range of autoimmune diseases of various localization, especially rheumatological pathology (systemic lupus erythematosus, Sjogren's syndrome, systemic scleroderma, Sharpe syndrome), as well as a number of other disorders autoimmune nature.

The closest to achieve the goal is the method according to Application No. 2003115886/15, G 01 N 33/564 dated 05/27/2003, "Method for the diagnosis of autoimmune liver lesions in patients with chronic hepatitis." This method determines antimitochondrial antibodies (AMA), immunoglobulins A, M, G and γ-globulins. Antigliadin antibodies (AGA), circulating immune complexes (CICs), antimitochondrial antibodies (with a value index of J> 1.1), antigliadin antibodies (more than 25 units / ml), circulating immune complexes (more than 120 units) are diagnosed, which also diagnose autoimmune lesions. Thus, the disadvantage of this method of diagnosing autoimmune liver damage is the need to use 5 or more tests, which are also not hepatospecific.

The aim of the proposed method is to increase the efficiency, specificity and reliability of the diagnosis of autoimmune liver damage.

We propose the identification of the presence of autoantibodies of various classes to the ganglioside of human hepatocytes, which are the object of aggression in autoimmune liver lesions. To achieve this goal, an enzyme-linked immunosorbent assay is performed on the blood serum of individuals to be examined.

Immunological plates are used, sorbed with an alcohol solution of highly purified ganglioside obtained in situ from human liver tissue. The content of autoantibodies of IgM and IgG classes to ganglioside of liver tissue is determined, the ratio of the content of autoantibodies in the test serum is calculated in relation to their content in the negative control, and if it is greater than unity, the presence of an autoimmune process in the human liver is determined.

The method is as follows:

A solution of highly purified ganglioside of liver tissue with a concentration of 1-10 μg / ml in ethanol is added to the wells of the immunological tablet of 0.02-0.05 ml and left to dry.

0.1 ml of test serum diluted 100 times diluted with phosphate-buffered saline (FSB, pH 7.2) containing 0.05% BSA is added to the wells of 2 tablets (or strips) sorbed with a solution of highly purified human hepatocyte ganglioside (or other solution with similar properties).

0.1 ml of FSB-BSA (background control) is added to the first well of the plates (strips). 0.1 ml diluted 100 times FSB-BSA, healthy donor blood serum (negative control) is added to the second well of plates (strips). In the third well of plates (strips), 0.1 ml diluted 100 times of blood serum with a high level of specific autoantibodies is added (it is possible to use the serum of patients with autoimmune hepatitis).

The tablets are closed with lids and incubated for 1 h at a temperature of (37 ± 0.5) ° C, after which they are washed 3 times with FSB-BSA.

Then, 0.1 ml diluted in the working concentration of FSB-BSA, IgM conjugate is added to each well of the first tablet (strip), and 0.1 ml of IgG conjugate is added to each well of the second tablet (strip). The tablets are closed with lids and incubated for 1 h at a temperature of (37 ± 0.5) ° C. Then washed 3 times with FSB-BSA. Then, 0.1 ml of substrate-indicator solution is added to all wells of the plates (strips), kept for 30 minutes in a dark place at a temperature of 18 to 20 ° C, after which 0.05 ml of stop solution is added to all wells reagent (2N sulfuric acid) and immediately record the results of the study using a photometric device.

Based on the obtained optical density (OD) data, the presence of specific autoantibodies of IgM and IgG to liver ganglioside is determined in arbitrary units. For this, the OD of the test serum is divided into the OD of the negative control serum and compared with the same indicators obtained for the positive control serum.

An example of a specific implementation of the method

In patients “A” and “B”, a venous or capillary blood is taken into a clean, dry tube without adding an anticoagulant, from which serum is subsequently obtained by the generally accepted method. The resulting serum is used for research.

In the first two wells of each of the two tablets (or strips), sorbed with a solution of highly purified ganglioside of liver cells, 0.1 ml diluted 100 times FSB-BSA, the blood serum of patients "A" and "B". 0.1 ml of FSB-BSA (background control) is added to other wells, 0.1 ml diluted 100 times in the following wells, healthy donor blood serum (negative control), 0.1 ml are added to the remaining wells, 100 times diluted blood serum of a patient with autoimmune hepatitis (positive control).

Tablets (strips) are closed with lids and incubated for 1 h at a temperature of (37 ± 0.5) ° С. Then washed 3 times with FSB-BSA. Then, 0.1 ml of diluted FSB-BSA, the IgM conjugate at a working concentration is added to each well of the first tablet (strip), and 0.1 ml of the IgG conjugate at a working concentration is added to each well of the second tablet (strip). The plates covered with covers are incubated for 1 h at a temperature of (37 ± 0.5) ° С. Then washed 3 times with FSB-BSA. Then, 0.1 ml of substrate-indicator solution is added to all wells of the plates (strips), kept for 30 minutes, in a dark place at a temperature of 18 to 20 ° C, after which 0.05 is added to all wells ml stop reagent (2N sulfuric acid) and immediately record the results of the study using a photometric device.

The following values of optical density (OD) were obtained

IgM autoantibodies:

- Background control (FSB-BSA) - 0.05

- Negative control (healthy donor serum) - 0.15

- Positive control (serum of a patient with autoimmune hepatitis) - 1.78

- Serum of the patient "A" - 0.36

- Serum of the patient "B" - 1.54

The level (index) of hepatocyte ganglioside IgM autoantibodies for a positive control serum (the ratio of the OD value of the positive control serum to the OD of the negative control serum) is 1.78: 0.15 = 11.9.

Then determine the index of the level of autoantibodies in the blood serum of the patient "A": 0.36: 0.15 = 2.4.

Similarly, determine the index of antibodies in the blood serum of the patient "B": 1.54: 0.15 = 10.3.

IgG autoantibodies:

- Background control (FSB-BSA) - 0.06

- Negative control (healthy donor serum) - 0.12

- Positive control (serum of a patient with autoimmune hepatitis) - 1.92

- Serum of the patient "A" - 0.28

- Serum of the patient "B" - 1.99

The level (index) of hepatocyte ganglioside autoantibodies IgG for a positive control serum (the ratio of the OD value of the positive control serum to the OD of the negative control serum) is 1.92: 0.12 = 16.0.

Then determine the index of the level of autoantibodies in the blood serum of the patient "A": 0.28: 0.12 = 2.33.

Similarly, determine the antibody index in the blood serum of patient "B": 1.99: 0.12 = 16.6.

Thus, the content of autoantibodies to human hepatocyte ganglioside in the blood serum of patient “B” turned out to be high both in the IgM class and in the IgG class, comparable with their concentration in the positive control serum. A higher content of IgG class autoantibodies is due to the chronicity of the process in patient “B”. In the blood serum of patient “A”, the content of autoantibodies to the human hepatocyte ganglioside was low.

Thus, the proposed method allows a more objective assessment of the specific autoimmune component in hepatitis, as it is based on the determination of autoantibodies to ganglioside of human hepatocytes. The proposed ganglioside is localized on the outer side of the hepatocyte membranes, is the biochemical basis of the structures of a number of receptor formations and is available for autoaggression without cytolysis of hepatocytes, which can determine the primacy of autoimmune damage to liver tissue.

The study of the level of autoantibodies to a specific ganglioside of human hepatocytes provides high accuracy, reliability and specificity of the diagnosis of autoimmune process in the liver tissue.

The use of an enzyme immunoassay for this purpose provides high methodological sensitivity, accuracy, reproducibility and speed of diagnosis.

The possibility of wide practical application is provided by the use of standard equipment for enzyme immunoassay, which is widely used in practical healthcare institutions. High performance with sufficient accuracy, along with complex automation, makes it possible to effectively use the proposed method in mass clinical examinations of individuals infected with hepatitis A, B, C, D viruses, especially with suspected development of an autoimmune process in the liver.

The method was developed and tested in the laboratory of immunochemistry of the Nizhny Novgorod Research Institute of Epidemiology and Microbiology. Academician I.N. Blokhina.

Bibliography

1. Yamamoto AM, Crestiel D., Hotberg JC et al, “Characterization of anty-liver-kidney microsome antibody (anti-LKM-1) from hepatitis C virus-positive and negative sera // Gastroenterol., 104: 1762-1767 , 1993.

2. Choudhuri K., Gregorio G.V., Mieli-Vergani G. et al Immunjlogical cross-reactivity to multiple autoantigens in patients with liver kidney microsomal type 1 autoimmune hepatitis // Hepatology, 28: 1177-1181, 1998.

3. Barland P., Lipstein E., Selection and use of laboratory tests in the rheumatic diseases // Am. J. Med., V. 100 (suppl. 2A), 16s-23s.

4. Feltkamp T.E.W. Antinuclear antibody determination in a routine laboratory // Ann.Rheum. Dis., V.55, 723-727, 1996.

5. Alvarez F., Berg P.A., Bianchi F.B. et al., International autoimmune hepatitis group reports review of criteria for diagnosis // Hepatology, 1999, V.31, p.929-938.

6. Application RU 2003115886/15, G 01 N 33/564 dated 05/27/2003 - "A method for the diagnosis of autoimmune liver damage in patients with chronic hepatitis."

Claims (1)

A method for determining the presence of an autoimmune process in a human liver by examining blood serum by an enzyme-linked immunosorbent assay, characterized in that the content of autoantibodies IgM and IgG to ganglioside of liver tissue is determined, the ratio of the content of autoantibodies in the test serum is calculated in relation to their content in the negative control and its value more than one determines the presence of an autoimmune process in the human liver.