Biomarkers applicable for diagnosing hepatic fibrosis. RU patent 2505821.

FIELD: medicine.

SUBSTANCE: invention refers to diagnostic techniques for hepatic fibrosis in an individual involving urokinase plasminogen, matrix metalloproteinase 9 and β-2-microglobulin expression tests to derive a score and to diagnose. Also, the present invention refers to a diagnostic kit for hepatic fibrosis comprising a first antibody specifically bound to an urokinase plasminogen activator (uPA), a second antibody specifically bound to matrix metalloproteinase 9 (MMP9) and a third antibody specifically bound to β-2-microglobulin (β-2-MG).

EFFECT: higher diagnostic accuracy.

17 cl, 33 tbl, 3 ex

A related application

This application claims the priority of the initial application, US no 61/119,077, filed December 2, 2008; its contents are fully included as a link.

Background of the invention

Liver fibrosis includes excessive accumulation of extracellular matrix proteins (for example, collagen) on the cells of the liver, which leads to the formation of scar tissue. Most chronic liver disease, type in metabolic diseases and liver diseases associated with infections of hepatitis b or C, as well as in the case of alcohol. Developed liver fibrosis leads to cirrhosis of the liver, liver cancer, liver failure and portal hypertension.

Currently liver biopsy is the best way to detect liver fibrosis and determination of its gravity. However, liver biopsy, as an invasive procedure, is not an ideal way to diagnose.

For the diagnosis of liver fibrosis were developed non-invasive serological tests based on associated with fibrosis of serum . The sensitivity and precision of such analyses strongly depends on the used biomarkers. It is therefore extremely important to identify a reliable biomarkers that with high sensitivity distinguish patients with fibrosis of people who are not affected by them.

Summary of the invention

The present invention is based on unexpected identify three new serum biomarkers, namely, plasminogen activator type (uPA), matrix metalloproteinase 9 (9; GenBank Accession Number) and beta-2-microglobulin (beta-2-MGB) for the diagnosis of liver fibrosis.

Thus, one aspect of the present invention characterizes method for the diagnosis of liver fibrosis based on the level of expression of one or more of the three above-mentioned biomarkers, on an optional combination with one or more additional whey biomarker, type glutamic GOT (glutamic pyruvic transaminase (GPT) and alpha-fetoprotein (AFP).

Described diagnostic method consists of at least 4 stages: (i) obtaining a blood sample from the subject, the suspect in the fibrosis of the liver (for example, the carrier of hepatitis b or C, or of a patient suffering from alcohol-related liver disease, or a metabolic liver disease); (ii) determination of the obtained sample blood level of expression of one or more of the listed previously biomarkers; (iii) calculation of the scoring of the disease based on the level of expression; (iv) on the basis of the scoring to identify whether the specified patient fibrosis. This diagnostic method includes (optional) after the specified stage (iv) additional step (v): assessment of the stage of fibrosis in the specified patient on the basis of the scoring compared with the previously established limit concentration values defining the various stages of fibrosis. When used herein, the term «diagnostics for detection of presence/absence of the subject (for example, a person) of liver fibrosis or evaluation stage of the disease. A sample of blood can be any sample obtained from the blood, type specimen serum or plasma samples.

Specified scoring can determine if the level of expression of a biomarker analyze discriminators, analysis method raised regression analysis method logical regression.

Another aspect of the present invention describes diagnostic kit containing at least two antibodies (for example, the whole immunoglobulin molecule), and one of them is specific in relation to the uPA, MMP9 or β 2MGB, and the rest are specific in relation to the uPA, MMP9, b-2MGB, GOT, GPT, and AFP. These two antigen have different antigen specificity. Preferably, for diagnosis of liver fibrosis specified set consisted mainly of the above antibodies, namely contained only antibodies specific to antigen (for example, fibrosis-related biomarkers). In one example of a specified set contains antibody against the uPA, antibody against MMP9 and antibody against β 2MGB.

The present invention also includes the application of any of the mentioned above antibodies in the diagnosis of liver fibrosis or in the manufacture of the kit for the diagnosis of liver fibrosis.

Details of one or more variants of the present invention are listed below in the description. Other signs or advantages of the present invention will be apparent from the following detailed description of some of the options, as well as of the accompanying claims.

A detailed description of the present invention

One aspect of the present invention relates to a method of diagnostics of liver fibrosis on the basis of the level in the blood of the subject of biomarkers uPA, MMP9, b-2MGB, combination, or a combination of (a) one or more of uPA, MMP9 and beta-2MGB, and (b) one or more additional biomarkers fibrosis (for example, GOT, GPT, or AFP). This method applies to the patient to identify his or her presence/absence of liver fibrosis or for the assessment of the stage of the disease. Specified, the patient may be a carrier of the hepatitis b virus or hepatitis C virus, or a patient suffering from alcohol-related diseases (e.g., fatty infiltration of liver, alcoholic hepatitis), metabolic liver disease or liver cancer.

uPA person has two isoforms, both of them can be used in method of diagnosis of the present invention. Inventory number 1 isoforms in GenBank NP_002649.1 (18-OCT-2009)as 2 isoforms in GenBank - NP_001138503.1 (18-OCT-2009). Inventory numbers in GenBank two other new markers, MMP9 and beta-2MGB-NP_004985.2 (18-OCT-2009), (22-NOV-2009) and NP_004039.1 (OCT 25, 2009) respectively.

For the application of the method of the present invention a sample of blood can be obtained from the subject, the suspect in the fibrosis of the liver, and the level of one or more of the above mentioned biomarkers you can define a standard method, such as enzyme-linked immunosorbent assay (ELISA) or Western-. To get the scores that characterize the blood profile, data indicating the level of biomarkers, subject to the analysis of discriminators, analysis method raised regressions or logical analysis of regressions. If necessary, consideration may be included clinical factors (such as age and gender characteristics of the patient). Then, to analyze whether a specific subject fibrosis of the liver, and if it does, what its stage, the obtained scores compared with the value of the maximum concentration of determining the presence or absence of liver fibrosis, or with set values of the utmost concentration, which recognizes the various stages of fibrosis. The specified values of the maximum concentration you can determine examining the same assay blood profile of the same biomarkers of the entities, which are not suffering from liver fibrosis in patients with different stages of fibrosis. For example, you might have a mid-point between grades for subjects not suffering fibrosis of the liver, and for patients with fibrosis.

Below is the approximate method of determining the values listed above marginal concentration on the basis of factors that are determined to be associated with different stages of fibrosis:

(1) the assignment of patients with fibrosis of the liver in different groups according to the status of the disease (for example, the degree of fibrosis and risk factors);

(2) the identification of these patients of potential factors that may be correlated with the stages of fibrosis;

(3) the selection using invariant analysis of the installed potential factors the factors that differ significantly for different groups of patients;

(4) installed factors is analyzed by the method of discriminators, method raised regressions logical regression, or the method of generalized linear models to assess the independent significance of each of these factors in the diagnosis of fibrosis;

(5) creation of model, raised regressions or models of logistic regressions for, on the basis of the identified factors (including the associated with fibrosis biomarkers, and clinical factors providing their applicability) calculate the scoring of the disease; and

(6) determination of the maximum concentration for each stage of the disease on the basis of the obtained score (for example, its average value in each group of patients, as well as other relevant factors such as sensitivity, specificity, and predictive value of a positive result (PPV) and negative predictive value of the (NPV).

Diagnostic significance of the developed model, raised regressions or models of logistic regressions can be assessed by analysis of the characteristic curve detection, to create the corresponding curve (ROC-response). In this analysis the optimal multivariant model provides a large area of the specified under the curve (AUC). These models are described below in examples 1-3.

The specified set according to the present invention includes antibodies, which can be obtained from suppliers. Or, antibodies can be obtained by standard methods. To produce antibodies against a specified above biomarker, this marker linked (optional) with a protein carrier, can be mixed with adjuvant and make him an injection in the host organism of an animal. these animals antibodies purified by affinity chromatography. Standard used by the owners, the animals are rabbits, rats, Guinea pigs and mice. To enhance the immune response can be used by various adjuvants, it depends on the type of animal. These adjuvants include: adjuvant Freud (complete or incomplete, mineral gels (type aluminium hydroxide), surface-active substances (type ), polyols, , peptides, emulsions, lymph snails, dinitrophenol. Serum with animals polyclonal antibodies are present, namely heterogeneous population of molecules antibodies.

Monoclonal antibodies, namely homogeneous populations of molecules, antibodies can be obtained using the standard methods of a hybrid (see, for example Kohler et al. (1975) Nature, 256, 495; Kohler et al. (1976). Eur.j.Immunol. 6, 511, Kohler et al. (1976). Eur.j.Immunol. 6, 292; Hammerling et al (1981) Monoclonal Antibodies and T cell Hybridomas, Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained by any technology, which ensures production of antibody molecules continuous cell lines in culture, the culture type described in Kohler et al. (1975) Nature, 256, 495 and in U.S. patent 4,376,110. These antibodies can be any immunoglobulin of any class, including IgG, IgM, IgE IgA, IgD, or any subclass. , monoclonal antibodies according to the present invention, can be cultivated in vivo and in vitro. The ability of monoclonal antibodies to produce high titres in vivo makes them particularly useful way of production.

In addition, antibody fragments can be obtained by known methods. For example, such fragments which include fragments of the F(ab') 2 , but not limited to, you can get splitting molecules, antibodies, and Fab fragments can be obtained restoration bridges fragments F(ab') 2 .

Without further clarification, it is assumed that based on the above description is qualified in this area the person is capable to apply the present invention in its entirety. The following variations are made just illustrative. They are not in any way limit the disclosure of the invention. All cited publications incorporated by reference.

Example 1

Diagnosis of liver fibrosis virusinfizierovannah hepatitis C patients on the basis of levels of uPA, MMP9, b-2MGB and other related fibrosis markers in the blood serum.

Materials and methods

(i) Patients.

The study involved 140 virusinfizierovannah hepatitis C patients and 93 healthy volunteers. And infected patients and healthy volunteers were assigned randomly in the study group (n=148) and in the testing group (n=85). They were all subjected to the usual laboratory, including the study of liver panel (GOT/AST,GPT/ALT, total bilirubin, alkaline phosphatase and albumin, prothrombin index (INR), AFP, tests to exclude other types of liver disease, liver ultrasound, endoscopy and modified .

Conducted a biopsy of the liver of all patients, histological characteristics biopsy were assessed on a scale METAVR. These samples (more than 10 mm in length) recorded, covered with paraffin, held staining with hematoxylin and . Stage of fibrosis for each sample biopsy were assessed on the following criteria:

F0: no fibrosis

F1: portal fibrosis without

F2: small Sept

F3: numerous Sept without cirrhosis, and

F4: cirrhosis

The resulting scale (namely, the level of inflammation caused by infection of viral hepatitis C) for each specimen biopsy have also a standard way.

(iii) Serological analysis

Each patient was taken to 10 ml of venous blood, she was placed in a test tube without additives, and tubes kept fixed within 30 minutes Then the samples were centrifuged, the process is conducted at 4 OC, 1600 g for 15 min; was assembled.

The level of serum 9 determined immunological analysis using Quantikine MMP9 Immunoassay (R&D Systems, Minneapolis, MN). This analysis is designed to measure total number of MMP9, including 92 form predecessor, and 82 original form. Diluted serum (1:100) were placed on a plate in the microwells, pre-coated with a monoclonal antibody directed against a person in respect of MMP9. Two hours later, the plate is washed, and it entered handled antibody and Biotin rights of MMP9. After incubation at room temperature for 1 h plastic washed and added a can of drained to streptavidin horseradish peroxidase (HRP) and then /3,3,5,5-tetramethylbenzidine (TMB). Enzymatic reaction completed, adding a 1 mol/litre sulfuric acid. Absorption was measured at a wavelength of 450 nm on the device Spectramax M5 (Molecular Devices). Then on the basis of the obtained values of the absorption from the standard curve determined the level of serum MMP9 as described above. All measurements, in accordance with the manufacturer's instructions, were three times. about

The level of serum β-2MG determined by enzyme immunoassay in the following way. 20 ml of the sample of diluted sera (1:100) were placed on , pre-coated with mouse monoclonal antibody against β-2MG and mixed with 200 cells diluted sample. The mixture was incubated at 37 C for 30 min, then record 4 times washed with distilled water and added fused with horseradish peroxidase (HRP) antibody sheep against β-2MG. After incubation at 37 C for 30 hours plate again washed and added /3,3,5,5-tetramethylbenzidine (TMB). After 20 min reaction was stopped with IN HCl. Absorption was measured at a wavelength of 450 nm on the device Spectramax M5 (Molecular Devices). The level of serum B-2MG determined as described above. All measurements, in accordance with the manufacturer's instructions, were three times.

The serum level of the other associated with fibrosis markers, namely, GOT, GPT, or AFP defined in a standard way.

(iv) definition score disease

Correlation between the levels of expression of uPA, MMP9, b-2MG or their combinations and stages of fibrosis was determined by analysis of the discriminators, analysis by the method of raised regressions or logical analysis of regressions, if possible, taken into account clinical factors. Diagnostic values for each of the three biomarkers or their combination was evaluated on the basis of sensitivity, specificity, and predictive value of a positive result (PPV) and negative predictive value of the (NPV). Values of sensitivity, specificity, and predictive value of a positive result and predictive value of a negative result was determined by testing, where the points on the curve detection, corresponding to different values of the maximum concentration indicate a positive test.

(i) Characteristics of patients

Characteristics of patients, including clinical factors obtained in the above-mentioned tests, and serum levels for biomarkers associated with fibrosis, are given below in table 1.

Characteristics of patients

Trained group (n=148)

Test group (n=85)

The value of P (univariate analysis)

Mean age (standard deviation)

45,87 (14.53) 84

50,26 (13,61)

Women, n (%)

Stage of fibrosis, n (%)

Fibrosis is missing(healthy F0)

Fibrosis (F1+F2+F3)

Cirrhosis (F4)

Serum biochemical markers (standard deviation)

GOT/AST, IU/L

45,28 (KZT 51.78)

57,63 (55,94)

GPT/ALT,IU/L

57,73 (81,9)

72,95 (87,08)

Total bilirubin (/l)

15,94 (20.55)

23,76 (19,46)

Albumin (g/l)

44,6 (5,6)

40,04 (6,2)

AFP, ng/ml

to 8.85 (17,09)

19,96 (53,74)

New serum markers (standard deviation)

uPA, ng/ml

0,82 (0,59)

0,96 (0,78)

9, mg/ml

0,22 (0,2)

0,22 (0,2)

β-2MG mg/ml

2,17 (3,14)

1,82 (0,95)

(ii) the relation of serum uPA, MMP9 or β 2MG, and other clinical factors with fibrosis of the liver,

As shown below in table 2 serum levels of uPA, MMP9 or β 2MG correlate with the presence/absence of fibrosis in patients and his degree as in the study group and in the tested group,

Patients with fibrosis or absent his degree was low, had a low level of uPA and beta-2MG while this level is significantly increased in patients with moderate or severe stage of fibrosis. In particular, it was found that in a training group average serum values of uPA in groups F0, F1, F2, F3 and F4 were 0,46 ng/ml, 0.61 ng/ml 0.75 ng/ml, 0,86 ng/ml, and $ 1.66 ng/ml, respectively, and the mean serum values β-2MG in groups F0, F1, F2, F3 and F4 were 1,26 mcg/ml, 1,86 mcg/ml, 2,22 mcg/ml, 2,3 8 mcg/ml and 4 mcg/ml, respectively. On the other hand, have a healthy patients or patients with minor fibrosis serum level 9 was significantly higher than in patients with fibrosis of moderate or severe degrees. It was found that the mean serum levels of this marker amounted to 0.33 mcg/ml, 0,16 mg/ml, 0,19 mg/ml, 0,14 mcg/ml and 0.1 mcg/ml, respectively. Very similar results were obtained in the tested group. These results show that the markers of uPA, 9 and beta-2MG separately are reliable markers for the diagnosis of liver fibrosis.

(ii) model for the diagnosis of liver fibrosis Results of this study show that the Union of any two levels of markers of a number of uPA, 9, b-2MG, GOT, GPT and AFP with clinical factors can be used as a reliable markers for the diagnosis of liver fibrosis. Below are two sample model, namely uPA+MMP 9 and uPA+GPT, as well as the equation for calculating scoring disease on the basis of the United levels for any pair of two markers. These equations were developed using the analysis of discriminators, analysis by the method of raised regressions or logical analysis of regressions. In the following tables (3-8) specified concentration limits, sensitivity, specificity, predictive positive result (PPV) and negative predictive result (NPV), and area under the characteristic (ROC) curve detection (AUROC) for these models, uPA and MMP9

Analysis of discriminators:

Score disease = 1,4829 x uPa(ng/ml)-3,2605 x 9(mcg/ml)+5

The value of the coefficient of uPA was of 0.741 up to 1,763, preferably from 1.26 to 1,705, and the value of the coefficient 9 from -7,553 to -2,839, preferably from -3,75 to -2,771.

The analysis of the logical regressions;

Score disease = exp (Logitvalue)/(1+exp(Logitvalue)), where

Logit_vaiue=-2,2416+3,2059 x uPA (ng/ml)-5,6316 x 9 (mcg/ml)

Coefficient crossing ranged from -3 to -1,48, preferably from -2,578 to -1,905, the coefficient for the uPA was from 2.49 to 3,91, preferably from 2,725 to 3,687, and the coefficient for 9 ranged from -8,01 to -3,24, preferably from -6,477 to -4,787.

Analysis by raised regressions;

Score disease = 1,6641+1,7227 x uPA (ng/ml) - 1,9821 x 9 (mcg/ml)

Coefficient crossing ranged from 1,430 up to 2,531, preferably from 1,414 to 1,914, the coefficient for the uPA was 1,191 to 1,938, preferably from 1,464 to 1,895, and the coefficient for 9 ranged from -4,428 to -1,501, preferably from -2,279 to-1,685.

uPA and GPT

Discriminant functional analysis:

Score disease = 1,5351 x uPA (ng/ml)+0,0083 x GPT (IU/L)+5

The value of the coefficient of uPA was 0,949 to 1,750, preferably from 1,305 to 1,719, and the coefficient for the GPT was 0,006 to 0.017, preferably from 0,007 to 0.01.

The analysis of the logical regressions:

Score disease = exp(Logit_value)/(1+exp(Logitvalue)), where Logit_value=-3,7206+3,8376 x uPA (ng/ml)+(-0,0001)x GPT (IU/L)

Coefficient crossing was -4,30 to -3,14, preferably from -4,279 to -3,274, the coefficient for the uPA was 3,11 to 4.57, preferably from 3,262 to 4,413, and the coefficient for the GPT ranged from 0.01 to 0,002, preferably from -0,00012 to -0,00008,

Analysis by raised regressions:

Score disease = 0,9199+1,8321 x uPA (ng/ml)+0,0034 x GPT(IU/L)

Coefficient crossing was 0,705 to 1,281, preferably from 0,782 to 1.03, the coefficient for the uPA was 1,303 to 2,052, preferably from 1,557 to 2,107, and the coefficient for GPT from 0.002 to 0,009, preferably from 0,0029 up to 0.0039.

(iv) model for the diagnosis of liver fibrosis.

Below for an example describes models based on the integrated level three serum markers selected from the uPA, 9, b-2MG, GOT, GPT and AFP for the analysis of liver fibrosis. If necessary, consideration was given to clinical factors. These models have been developed using the analysis of discriminators, analysis by the method of raised regressions or logical analysis of regressions. These three models were calculated scores diseases that were analyzed in terms of the degree of severity. Was the linear correlation between the METAVIR stages of fibrosis and point estimates.

Four concentration limits, indicating (i) some fibrosis (Healthy from F1-F4); (And) moderate fibrosis (Healthy ~of F1 F2-F4); (iii) serious degree of fibrosis (Healthy ~of F2 F3-F4); and (iv) cirrhosis (Healthy ~F3 of F4) were identified in the study group and confirmed in the tested group.

uPA, 9, and beta-2MG

Discriminant functional analysis:

Score disease = 1,4159 x uPA (ng/ml)-3,0399 x 9 (mcg/ml)+0,0897 x β-2MG (mcg/ml)+5

The value of the coefficient of uPA was 0,389 to 1,604, preferably from 1,204 to 1,586, the coefficient for 9 ranged from -7,321 to -2,302, preferably from -3,496 to -2,584, and the coefficient for beta-2MG - from 0,048 to 1,114, preferably from 0,076 to 0,103.

Analysis by logical regressions:

Score disease = exp(Logit_value)/(1+exp(Logit_yalue)), where Logit_value=-3,8614+2,8761 x uPA (ng/ml)-4,0100 x 9 (mcg/ml)+0,7853 x β-2MG (mcg/ml)

Coefficient crossing was -4,9 to -2,28, while the coefficients for the uPA, MMP9, and beta-2MG were from 2.15 to 3.6, from -6,4 to -1,61 and from 0.47 to 1.1 respectively, Mainly coefficient crossing and the coefficients for uPA, MMP9 and beta-2MG ranged from -4,441 to -3,282, from 2,454 to 3,308, from -4,611 to -3,409 and from 0,668 to 0,903 respectively.

Analysis by raised regressions:

Score disease = 1,4645+1,6683 x uPA (ng/ml)-1,7868 x MMP9 (mcg/ml)+0,0926 x β-2MG (mcg/ml)

Coefficient crossing was 0,558 to 2,418 (for example, from 1,245 to 1,684); the coefficients for the uPA, MMP9 and beta-2MG ranged from 0,818 to 1,907 (for example, from 1,418 to 1.835 squa), from -4,677 to -0,997 (for example, from -2,055 to -1,519) and from 0,076 to 0,825 (for example, from 0,079 to 0,106) respectively.

Below in table 12 shows the results obtained for the tested group for the previously described model:

Table 13.

Estimated concentration for different stages of fibrosis

Stage of fibrosis

Numerical score ( model)

Numerical score (Model for logical regression),

Numerical score (Model raised regressions)

Healthy ~F1

to 5.21-5,26

0,09-0,11

2,00-2,05

5,26-5,55

0,11-0,28

2.05-2,40

5,55-5,63

0,28-0,35

2,40-2,50

5,63-6,01

0,35-0,50

2,50-2,90

6,01-6,10

0,50-0,54

2,90-3,00

6,10-6,62

0,54-0,75

3,00-3,80

6,62-6,78

0,75-0,85

3,80-3,90

of 0.85 to 1.00

uPA, 9, and OPT

Discriminant functional analysis:

Score disease = 1,2295 x uPA (ng/ml)+(-2,6571)x 9 (mcg/ml)+0,0072 x GPT(IU/L)+5

The coefficients for the uPA, and MMP9 GPT ranged from 0,539 to 1,456 (for example, from 1,045 to 1,414), from -6,988 to -2,053 (for example, from -3,056 to -2,391) and from 0.004 to 0.014 (for example, from 0,006 0,008) respectively.

Analysis by logical regressions:

Score disease = exp(Logit-value)/(1+exp(Logit-value)), where Logit-value=-2,1715+3,3171 x uPA (ng/ml)+(-6,2008)x 9 (mcg/ml)+(-0,0018)x GPT (IU/L)

Coefficient crossing was -2,95 to -1,38 (for example, from -2,497 to -1,846), while the coefficients for the uPA, and MMP9 GPT were from 2.56 to 4,07 (for example, from 2,82 to 3,649), from -8,73 to -3,66 (for example, from -7,131 to -5,271) and from -0,02 to 0.001 (for example, from -0,0021 to -0,0015) respectively.

Analysis by raised regressions:

Score disease = 1,5020+1,6479 x uPA (ng/ml)-1,7885 x MMP9 (mcg/ml)+0,0028 x GPT (IU/L)

Coefficient crossing ranged from 1,154 to 2,300 (for example, from 1,277 to 1,727), while the coefficients for the uPA, and MMP9 GPT ranged from 1,075 to 1,941 (for example, from 1,401 to 1,895), from -4,192 to -1,218 (for example, from -2,057 to -1,52) and from 0.001 up to 0.007 (for example, from 0,0024 to 0,0032) respectively.

(v) 4-marker model for the diagnosis of liver fibrosis. Results obtained in this study indicate that the combination of any of the four factors of group uPA, MMP9, b-2MG, GOT, GPT, AFP (and possible consideration of clinical factors) is a reliable markers for the diagnosis of liver fibrosis. Below is described the approximate variant of the token, composed of the uPA, MMP9, b-2MG and GPT. The results are shown in tables 17-19.

Discriminant functional analysis:

Score disease = 1,1645 x uPA (ng/ml)-2,4312 x 9 (mcg/ml)+0,0957 x β-2MG (mcg/ml)+0,0073 x GPT (IU/L)+5

The coefficients for the uPA, MMP9, b-2MG and GPT ranged from 0,196 to 1,376 (for example, from 0.99 to 1,339), from -6,684 to -1,623 (for example, from -2,796 to -2,067), from 0.055 to 0,974 (for example, from 0,081 to 0.11) and from 0.004 to 0,012 (for example, from 0,0062 to 0,0084) respectively.

Analysis by logical regressions:

Score disease = exp(Logit_value)/(1+exp(Logit_value)), where Logit_value=-3,6742+3,0107 x uPA (ng/ml)-4,4549 x 9 (mcg/ml)+0,7074 x β-2MG (MK/ml)+(-0,0017)x GPT(IU/L)

Coefficient crossing was -4,74 to -2,61 (for example, from -4,225 to -3,123), while the coefficients for the uPA, MMP9, b-2MG and GPT ranged from 2,24 to 3.77 (for example, from 2.559 inches to 3,462), from -6,99 OED -1,92 (for example, from -5,123 to -3,787), from 0.39 to 1.02 (for example, from 0,6013 to 0,8135) and from -0,004 to 0.001 (for example, from -0,002 to -0,001) respectively.

Analysis by raised regressions;

Score disease = 1,2866+1,5874 x uPA (ng/ml)--1,5725 x 9 (mcg/ml)+0,0955 x β-2MG (mcg/ml)+0,0029 x GPT(IU/L)

Coefficient crossing was 0.297mm to 2,109 (for example, from 1,094 to 1.48), while the coefficients for the uPA, MMP9, b-2MG and GPT ranged from 0,748 to 1,800 (for example, from 1,349 to 1,778), from -3,919 to -0,776 (for example, from -1,808 to -1,337), from 0,077 to 0,830 (for example, from 0,0812 to 0,1098) and from 0.001 up to 0.007 (for example, from RUR 0.0025 to 0,0033) respectively.

(vi) 5-marker model for the diagnosis of liver fibrosis. Results obtained in this study indicate that the combination of any of the four factors of group uPA, MMP9, b-2MG, GOT,

GPT, AFP (and possible consideration of clinical factors) is a reliable markers for the diagnosis of liver fibrosis. Below is described the approximate variant of the 5-marker model composed of uPA, 9, b-2MG, GPT and GOT. The results are shown in tables 20-22.

Discriminant functional analysis:

Score disease = 1,1009 x uPA (ng/ml)-2,2941 x 9 (ng/ml)+0,0974 x β-2MG (mcg/ml)+0,0065 x GPT(IU/L)+0,0024 X GOT(IU/L)+5

The coefficients for the uPA, 9, b-2MG, GPT and GOT were from -0,070 to 1,512 (for example, from 0,936 to 1,266), from -6,453 to -1,468 (for example, from -2,638 to -1,95), from 0,058 winter to 1,209 (for example, from 0,083 to 0,112), from -0,002 to 0,019 (for example, from 0,0055 to 0.0075) and from -0,011 to 0.025 (for example, from 0.002 to 0,0028) respectively.

Analysis by logical regressions:

Score disease = exp(Logit_value)/(1+exp (Logitvalue)),

Logit_value=-3,4751+2,7416 x uPA (ng/ml)-4,5237 x 9 (ng/ml)+0,6952 x β 2(mcg/ml)-0,0021 x GPT(IU/L)+0,0007 X GOT(IU/L)

Coefficient crossing was -4,54 to-2.4 (for example, from -3,996 to -2,954), while the coefficients for the uPA, MMP9, b-2MG, GPT and GOT were from 1.87 to 3.61 (for example, from 2.33 to 3,153), from -7,13 to -1,9 (for example, from -5,202 to -3,845), from 0.38 to 1.01 (for example, from 0,5909 to 0,7995), from-0.01 0.01 (for example, from -0,0024 to -0,0018) and from-0.01 0.01 (for example, from 0.0006 to 0,0008) respectively.

Analysis by raised regressions:

Score disease = 1,2750+1,3505 x uPA (ng/ml)-1,4346 x MMP9 (mcg/ml)+0,0978 x β-2MG (mcg/ml)+0,0004 x GPT(IU/L)+0,0056 X GOT(IU/L)

Coefficient crossing was 0,145 to 1,909 (for example, from 1,084 to 1.466), while the coefficients for the uPA, MMP9, b-2MG, GPT and GOT were from 0,576 to 1,826 (for example, from 1,148 to 1,553), from -3,676 to -0,603 (for example, from -1,65 to -1,219), from 0,077 to 0,862 (for example, from 0,0831 to 0,1125), from -0,005 to 0,009 (for example, from 0,0003 to 0.0004) and from -0,008 to 0,021 (for example, from 0,0047 to 0,0065) respectively.

(vii) 6-marker model for the diagnosis of liver fibrosis Results obtained in this study indicate that the combination of uPA, MMP9, b-2MG, GOT, GPT, and AFP (and if necessary are taken into account clinical factors) is a reliable markers for the diagnosis of liver fibrosis. Below is the equation for the calculation of the score (developed methods of discriminant analysis, analysis of logical regression and analysis raised regression), on the basis of these 6-marker combinations, as well as the values of the maximum concentration for different stages of liver fibrosis (see tables 23-25).

Discriminant functional analysis:

Score disease=of 1.4401 x uPA (ng/ml)-1,2831 x 9 (mcg/ml)+0,0921 x β-2MG (mcg/ml)-0,0099 x AFP(ng/ml)+0,0129 x GPT(IU/L)-0,0004 X GOT(IU/L)+5

The coefficients for the uPA, MMP9, b-2MG, AFP, GPT and GOT were from 0,141 to 1,923 (for example, from 1,224 to 1,656), from -5,052 to -0,393 (for example, from -1,476 to -1,091), 0,069 to 1,303 (for example, from 0,078 to 0,106), from -0,032 to 0,054 (for example, from -0,0114 to -0,0084), from 0.002-0.032 (for example, from 0,011 to 0,0148) and from -0,023 to 0,021 (for example, from -0,00046 to -0,00034) respectively.

Analysis by logical regressions:

Score disease = exp (Logit_value)/(1+exp(Logit value)), where Logit_value=-4,1023+2,4436 x uPA (ng/ml)-6,8921 x MMP9 (mcg/ml)+1,2869 x β-2MG (mcg/ml)-0,0112 x x AFP (ng/ml)-0,0015 x GPT(IU/L)+0,0018 X GOT(IU/L)

Coefficient crossing was -5,6 to -2,61 (for example, from -4,718 to -3,487), while the coefficients for the uPA, MMP9, b-2MG, GPT, GOT and AFP ranged from 1,38 to 3.5 (for example, from 2,077 to 2,81), from -10,86 to -2,92 (for example, from -7,926 to -5,858), from 0.82 to 1.75 (for example, from 1,0939 to 1,4799), from-0.01 0.01 (for example, from -0,0017 to -0,0012), from-0.01 0.01 (for example, 0,0015 to 0.002) and from-0.01 to 0.02 (for example, from 0.01 to -0,0095) respectively.

Analysis by raised regressions:

Score disease=0,9632+1,4215 x uPA (ng/ml)-1,0722 x 9 (mcg/ml)+0,0986 x β-2MG (mcg/ml)-0,0053 x RDA(ng/ml)+0,0019 x GPT(IU/L)+0,0058 X GOT(IU/L)

Coefficient crossing was -0,336 to 1,587 (for example, from 0,819 to 1,108), while the coefficients for the uPA, MMP9, b-2MG, AFP, GPT and GOT were from 0,396 to 2,024 (for example, from to 1.208 to 1,635), from -2,763 to -0,256 (for example, from -1,239 to -0,916), from USD 0.087 to 1,034 (for example, from 0,088 up to 0.113), from -0,021 to 0.037 (for example, from -0,0061 to -0,0045), from -0,006 to 0.015 (for example, from 0,0016 to 0,0021) and from -0,014 to 0,024 (for example, from 0,0049 to 0,0066) respectively.

All mentioned above models were confirmed in the tested group and observed similar results, including concentration limits, sensitivity, specificity, NPV, PPV and AUROC.

Example 2: Diagnosis of liver fibrosis in patients with positive reactions to hepatitis b virus is made on the basis of serum levels of uPA, MMP9 and beta-2MG.

In this study, 30 patients carriers of HBV and 30 healthy patients were also confirmed by the marker and 3-marker model. The following table 26 lists the data obtained.

Table 26.

Characteristics of patients

Data (n=60)

Mean age (standard deviation)

44,23 (9,27)

Women, n (%)

Serum biochemical markers, mean (standard deviation)

GOT/AST, IU/l

54,53 (59,56)

GPT/ALT, IU/l

87,02 (129,47)

Total bilirubin, mkmol/l

21,52 (21,78)

Albumin (g/l)

42,3 (5,88)

AFP, ng/ml

8,73 (26,62)

New serum markers, mean (standard deviation)

uPA, ng/ml

0,73 (0,6)

9, mcg/ml

0,27 (0,22)

β-2MG, mcg/ml

1,44 (1,16)

As shown below in table 27, serum levels of each of the uPA, MMP9 and beta-2MG correlate with the degree of fibrosis:

Table 27.

Serum levels of uPA, MMP9 and beta-2MG patients test positive for The hepatitis b virus

uPA (ng/ml)

MMP9 (mcg/ml)

β-2MG (mcg/ml)

Healthy (n=30)

0,46 (0,18)

0,4 (0,23)

1,1 (0,18)

to 0.87 (0,53)

0,18 (0,04)

1,17 (0,4)

0,42 (0,1)

0,08 (0,01)

1,13 (0,26)

0,77 (0,35)

0,13 (0,06)

1.42 (0,42)

F4 (n=11)

1,45 (0,93)

0,11 (0,05)

2,68 (2,34)

* The number of healthy: 30 subjects (test group)

In the tables below 28-30 presents data on the limit concentration, representing different stages of fibrosis, on the basis of the score, which were calculated using the equations described in 3-marker model example 1:

Example 3: the Diagnosis of liver fibrosis in patients with alcohol-dependent liver fibrosis, on the basis of serum levels of uPA, 9 b-MG.

In this study took 53 patients with alcohol-dependent liver fibrosis and 30 healthy subjects. Patient characteristics are shown below in table 31. These patients were subjected to regular laboratory tests described in example 1, and the special survey to determine their level of alcohol consumption

Table 31.

Characteristics of patients

Data (n=83)

Mean age (standard deviation)

43,83 (9,23)

Women, n (%)

Serum biochemical markers, mean (standard deviation)

GOT/AST, IU/l

67,88 (71,87)

GPT/ALT, IU/l

46,28 (57,22)

Total bilirubin, /l

53,31 (50,8)

Albumin (g/l)

35,02 (7,15)

AFP, ng/ml

103,81 (892,5)

New serum markers, mean (standard deviation)

uPA, ng/ml

1,11 (1,07)

9, mcg/ml

0,3 (0,24)

β-2MG, mcg/ml

1,77 (1,11)

Serum levels of uPA, 9 and beta-2MG of these patients were studied enzyme-linked immunosorbent assay (ELISA), as described in example 1. The results are shown below in table 32.

Table 33.

Concentration limits, representing different stages of fibrosis in 3-marker model for patients with alcohol-dependent disease

model

Model logical regression

Model raised regressions

Stage of fibrosis

Number of patients (%)

Healthy, fatty liver, alcoholic hepatitis of F4

Concentration

Sensitivity (%)

Specificity (%)

* The number of healthy: 30 subjects (test group);

the number of subjects with a fatty liver, alcoholic hepatitis: 22

Other options

All of the features disclosed in this description can be combined in any combination. Every sign, disclosed in this description may be replaced by an alternative sign, with the same equivalent or similar purpose. Thus, if not otherwise specified, each character represents the only example of a number of equivalent or similar signs.

From the above description of each qualified in this area of the person is able to easily evaluate the significant features of the present invention. It may also, without straying from the boundaries and the essence of the present invention to make various changes and modifications to adapt it to different applications and conditions. Therefore, other options are also included in the claims.

1. Method for the diagnosis of liver fibrosis patient, including: (a) obtaining a blood sample from the subject, the suspect in the fibrosis of the liver; (b) identify in the specified sample of blood level (levels) of the expression of one or more from a series: plasminogen activator type (Ira), matrix metalloproteinase 9 (9) and beta-2-microglobulin (beta-2-MG); (c) calculation of the scoring of the disease, exposing the received levels of analysis method discriminators, method of logical regression method or raised regressions; and (d) identify whether the specified patient fibrosis, if a points-based assessment above a pre-defined limit-point evaluation obtained by using the same methods, and the methods used at the stage of (C) to determine the levels of expression of the same biomarkers, and that biomarkers, levels of expression of which are determined at the stage of (b) in groups of subjects without fibrosis.

2. The method according to claim 1, characterized in that said a sample of blood serum is.

3. The method according to claim 1, characterized in that the subject is chosen from the group consisting of: a carrier of The hepatitis C virus, a bearer of the hepatitis b virus, a patient suffering from alcohol-related liver disease, and the patient suffering from metabolic liver disease.

4. The method according to claim 1, characterized in that the stage of determination by analyzing the levels of expression of two of the uPA, MMP9 and beta-2MG in the obtained sample of blood.

5. The method according to claim 1, characterized in that the definition stage is the analysis of the levels of expression of uPA, MMP9 and beta-2MG in the obtained sample of blood.

6. The method according to claim 5, wherein the specified sample of blood serum is.

7. The method according to claim 5, including in case of availability of the specified subject of liver fibrosis assessment of the stage of fibrosis by comparing the obtained scoring with a previously defined the utmost concentration, indicating a stage of fibrosis, and a previously defined concentration is established using the same techniques used and the methods used at the stage of (C) to determine the levels of expression of the same biomarkers that and biomarkers, levels of expression of which are determined at the stage of (b)in groups of patients with different stages of fibrosis, and where entities is diagnosed the presence of a certain stage of fibrosis in case, if a points-based assessment is within the range previously defined utmost concentration.

8. Method for the diagnosis of liver fibrosis the subject, including: (a) obtaining a blood sample from the subject, the suspect in the fibrosis of the liver; (b) identify in the specified sample of blood levels of expression of (a) one or more tokens, selected from the group consisting of plasminogen activator type (uPA), a matrix metalloproteinase 9 (9) and beta-2-microglobulin (beta-2-MG), and (b) one or more tokens, selected from the group consisting of glutamic (GOT), glutamic pyruvic transaminase (GPT) and alpha-fetoprotein (AFP); (c) calculation of the scoring of the disease, exposing received the levels of analysis method discriminators, method of logical regression method or raised regressions; and (d) identify whether the specified patient fibrosis, if a points-based assessment above a pre-defined limit-point evaluation obtained by using the same methods, and the methods used at the stage of (C) to determine the levels of expression of the same biomarkers that and biomarkers, levels of expression of which are determined at the stage of (b) in groups of subjects without fibrosis.

9. The method of claim 8, wherein the above sample of blood serum is.

10. Kit for the diagnosis of liver fibrosis, containing the first antibody specifically associated with activator plazminoguena type (Ira), the second antibody that specifically associated with the matrix 9 (9), and the third antibody specifically associated with β-2-microglobulin (beta-2-MG).

11. Set to 10, containing also antibody specifically associated with glutamic (GOT)antibody that specifically associated with glutamic pyruvic (GPT)and antibody specifically associated with alpha- (AFP), or a combination thereof.

12. Set to 10, characterized in that the antibodies are a whole immunoglobulin molecule.

13. Kit for the diagnosis of liver fibrosis, consisting of the first antibodies specifically associated with activator plazminoguena type (Ira), second antibody specifically associated with the matrix 9 (9), and the third antibodies specifically associated with β-2-microglobulin (beta-2-MG).

14. Set on a 13, wherein the specified collection also contains antibody specifically associated with glutamic (GOT)antibody that specifically associated with glutamic pyruvic (GPT)and antibody specifically associated with alpha- (AFP), or a combination thereof.

15. Kit for the diagnosis of liver fibrosis, containing (a) antibody specifically associated with activator plazminoguena type (Ira)antibody that specifically associated with the matrix 9 (9), and antibody specifically associated with β-2-microglobulin (beta-2-MG), or a combination thereof; and (b) antibody specifically associated with glutamic (GOT)antibody that specifically associated with glutamic pyruvic (GPT)and antibody specifically associated with alpha- (AFP), or their combination.

16. Set on item 15, characterized in that the antibodies are a whole immunoglobulin molecule.

17. Kit for the diagnosis of liver fibrosis, consisting of (a) antibodies, specifically associated with activator plazminoguena type (Ira), antibodies specifically associated with the matrix 9 (9), antibodies specifically associated with β-2-microglobulin (beta-2-MG), or a combination thereof; and (b) antibodies specifically associated with glutamic (GOT), antibodies specifically associated with glutamic pyruvic (GPT), and antibodies specifically associated with alpha- (AFP), or their combination.