JP2014167446A - Evaluation method and diagnostic kit of liver disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel marker of liver disease.SOLUTION: By measuring a soluble LR11 concentration in a blood-derived sample originated from a mammal, severity and prognosis prediction of liver disease (except liver cancer) in the mammal are evaluated.

Description

  The present invention relates to a novel liver disease marker in blood.

  Among liver diseases, viral hepatitis is caused by HBV (hepatitis B virus) and HCV (hepatitis C virus) infection, and the number of people infected with hepatitis B and hepatitis C in Japan is 3 million. Reach around people. Infection with HBV causes transient acute hepatitis, but in about 80-90% of infected people, seroconversion causes the infected virus to change to a less proliferative virus, and in many cases does not develop hepatitis Transition to an asymptomatic carrier. On the other hand, the remaining 10 to 20% of persistently infected persons transition to chronic hepatitis and further progress to cirrhosis. It is said that many HCV-infected persons have subclinical infection, and 60-80% thereafter become chronic. Chronic hepatitis is transferred to cirrhosis in about 30 to 40% of those affected in about 20 years. As a cause of recent cirrhosis in Japan, about 72% (HCV infection: about 60%, HBV infection: about 12%) is caused by virus infection, and alcoholic hepatitis accounts for half of the remaining 28%. Other than that, non-alcoholic steatohepatitis, autoimmune hepatitis, and primary biliary cirrhosis, which have been increasing recently, account for.

  In any of the above-mentioned causes, it is difficult to treat when it progresses to cirrhosis, and a radical treatment by liver transplantation is necessary. Furthermore, since the risk of developing hepatocellular carcinoma also increases, it is important to prevent progression from chronic hepatitis to cirrhosis. Currently, markers for liver function tests (hereinafter sometimes referred to as liver function markers) include AST and ALT, which are enzymes that deviate from hepatocytes due to hepatocyte damage, albumin and cholinesterase produced only by hepatocytes, and hepatocytes. Type IV collagen and hyaluronic acid, which increase with fiber formation, are clinically applied. However, even in cases where HCV infection is positive and the liver function marker test value is lower than the reference value, there are many cases in which mild liver fibrosis is observed as a result of liver biopsy, which is currently applied clinically. The presence of liver disease and the state of liver disease (for example, severity) are not sufficiently evaluated only by the examination of the liver function marker, and the early stage is difficult to detect by the existing liver function marker test. Thus, a new liver disease marker that can accurately grasp the onset and progression of liver disease has been desired.

  On the other hand, as a severity classification when liver disease progresses to cirrhosis, the Child-Pugh score calculated by scoring five factors (albumin and bilirubin concentrations, prothrombin time, ascites and hepatic encephalopathy degree) is prognostic. Has been applied. In addition, the MELD score defined by the three factors of creatinine, bilirubin, and prothrombin time is applied to predict the prognosis of liver transplantation. In addition to these existing prognostic prediction methods, most recently, serum soluble uPAR (abbreviation of urokinase plasminogen activator receptor, also referred to as PLUAR, CD87, hereinafter sometimes referred to as suPAR) concentration is used to predict prognosis of chronic liver disease. It was reported to be a factor (Non-Patent Document 1). In this report, the suPAR concentration has a strong negative correlation with the cholinesterase activity, which is a liver function marker, and a strong positive correlation with the fibrosis marker, hyaluronic acid, and further predicts the prognosis of liver transplantation. Since it showed a strong positive correlation with a useful MELD score, it is described that the suPAR concentration can be used as an index for predicting the prognosis of liver disease.

  On the other hand, LR11 (LDL receptor relative with 11 binding-binding repeats) has been identified as a novel LDL receptor-like protein having a structure characteristic of the LDL receptor family (Patent Document 1, Non-Patent Document 2). LR11 has a ligand-binding region with 11 repeat structures with apoE as a common ligand and is hardly expressed in normal vascular wall cells, but is specifically expressed in thickened intimal smooth muscle cells. It is known that it is recognized (nonpatent literature 3). Furthermore, from the study using cultured smooth muscle cells, the expression level of LR11 increased with the proliferation of smooth muscle cells, and secretion into the culture medium was observed. In a cuff-injured mouse model, anti-LR11 antibody was Intraperitoneal administration is known to inhibit intimal thickening caused by smooth muscle cell migration and proliferation (Non-patent Document 4).

The present inventors have recently found that the concentration of soluble LR11 (soluble LR11, hereinafter sometimes referred to as sLR11) in the blood of patients with malignant tumors is significantly increased compared with that of healthy individuals. Obtain knowledge that the concentration has been normalized by treatment, and has risen again at the time of recurrence, etc., and determine the presence, severity, choice of treatment method or effect of malignancy, risk of recurrence, or the presence or absence of recurrence And found a patent application (Patent Document 2). In this application, as malignant tumors to be applied as tumor markers, epithelial malignant stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer Colon cancer, colon cancer, gallbladder cancer and the like, and it has been confirmed that the soluble LR11 concentration in the serum derived from a specimen of a liver cancer patient, which is one of liver diseases, is increased. However, the fluctuation of soluble LR11 concentration in the serum of patients with liver disease who does not develop cancer is not described, and the concentration of soluble LR11 in blood is increased in liver diseases other than cancer. It has not been known that it can be applied to predict the prognosis of disorder status and liver disease.
In addition, the function of LR11 in vascular smooth muscle has been analyzed for the pathway through which signals are transmitted into cells via uPAR, and LR11 has been reported to be a ligand for uPAR (Non-patent Document 5). However, there was no report that directly examined the relationship between these two markers.

Japanese Patent Laid-Open No. 9-163988 International Publication No. 2011/074594

Liver Int. 2012; 32,500-509 J. et al. Biol. ChEm. 1996; 271, 24761-24768. Arterioscler. Thromb. Vasc. Biol. 1999; 19, 2687-2695. CirCres. 2004; 94; 752-758. J. et al. Clin. Invest. 2008; 118; 2733-2746

  An object of the present invention is to provide a new marker for liver disease.

The present inventors received serum samples from patients with various liver diseases under treatment, measured soluble LR11 concentration and usPAR concentration in serum, and verified the relationship with various liver function markers. Both usPAR were found to be significantly correlated with various liver function markers. Furthermore, as a result of obtaining the concentration level of healthy subjects and comparing it with the concentration in various liver diseases, the soluble LR11 concentration was significantly increased in most liver diseases, whereas the suPAR concentration was unexpectedly outside the cirrhosis group. There was no significant difference in liver disease. Furthermore, a reference standard range was set from the concentration level of healthy subjects, samples exceeding the upper limit were set as positive, soluble LR11 and suPAR, and type IV collagen as a marker for liver fibrosis were added for each liver disease 3 Comparison of positive rates by item was performed. As a result, suPAR had a very low positive rate because no significant difference was observed, but soluble LR11 was almost 100% in the cirrhosis group and also had a high positive rate in other liver diseases. Surprisingly, the positive rate was similar to that of type IV collagen. Moreover, it discovered that the positive rate which combined soluble LR11 and type IV collagen improved further.
Based on the above-mentioned findings, the present inventor conducted an animal experiment in a rat model of acute liver injury caused by galactosamine. As a result, hepatic injury caused by galactosamine administration was caused, and at the same time, the soluble LR11 concentration in the blood increased. It has been confirmed that the increasing trend continues after the disorder has converged, and it has been found that soluble LR11 can be a marker that can accurately grasp the pathological condition even in the early stage of liver disease exhibiting liver fibrosis. It came to do.

That is, the present invention provides the following [1] to [4].
[1] A method for evaluating the severity and prognosis prediction of liver disease (excluding liver cancer) in the animal, wherein the soluble LR11 concentration in a blood-derived sample derived from a mammal is measured.
[2] Soluble LR11 concentration is determined by measuring the soluble LR11 concentration in a blood-derived sample by an immunological method, setting a cut-off value, and risk for cirrhosis or liver cancer when the cut-off value is exceeded. The evaluation method according to [1], wherein the liver disease is determined to be increasing.
[3] Liver disease is viral hepatitis, cirrhosis, alcoholic hepatitis, fatty liver, non-alcoholic steatohepatitis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, idiopathic portal hypertension The evaluation method according to [1] or [2], which is a symptom.
[4] A diagnostic kit for liver diseases (excluding liver cancer) containing a soluble LR11 measurement reagent.

  ADVANTAGE OF THE INVENTION According to this invention, the marker of a new liver disease is provided and it becomes possible to grasp a disease state exactly also in the liver disease of the initial stage which exhibits liver fibrosis.

It is the figure which compared sLR11 density | concentration in the plasma of various liver disease patients except a healthy person's serum and liver cancer. It is the figure which compared the suPAR density | concentration in the plasma of various liver disease patients except a healthy person's serum and liver cancer. It is the figure which showed the fluctuation | variation of the sLR11 density | concentration in the serum of the acute hepatitis model rat by galactosamine administration or non-administration. It is the figure which showed the fluctuation | variation of the AST activity in the serum of the acute hepatitis model rat by galactosamine administration or non-administration.

  The mammal-derived blood to be tested in the present invention can be collected from humans, monkeys, mice, rats, rabbits, dogs, sheep, goats, horses, etc., but humans are preferred. As a blood-derived sample when measuring soluble LR11, any of whole blood, serum, or plasma can be used according to the characteristics of the measurement system.

  Measurement of soluble LR11 in a blood-derived sample is performed using a substance having affinity for soluble LR11. The affinity substance is not particularly limited as long as it has a binding ability to soluble LR11. For example, apo E, β-VLDL, RAP, uPA, PAI-1, or a complex thereof (uPA-PAI-1) And anti-soluble LR11 antibody, and RAP and anti-soluble LR11 antibody are preferable, and anti-soluble LR11 antibody is more preferable.

  The anti-soluble LR11 antibody may be either a polyclonal antibody or a monoclonal antibody, and the antibody can be prepared by a known method. Moreover, these antibodies can also be obtained as commercial products and can also be used in the present invention. Examples thereof include Mouse anti-LR11 monoclonal antibody (BD biosciences), Rabbit anti-LR11 Polyantibodies (CHEMICON), and anti-LR11 antibody described in International Publication No. 2009/116268.

  The method for measuring soluble LR11 in the present invention is preferably an immunological measurement method using an anti-LR11 antibody. Examples of immunological methods include immunostaining (Western blot), enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry (TIA and LTIA), enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay, and the like. ELISA is preferred. As an ELISA kit, those commercially available from Sekisui Medical can be used.

  Liver diseases to be evaluated for severity and prognosis prediction in the present invention include viral hepatitis (types B and C), cirrhosis, alcoholic hepatitis, fatty liver, nonalcoholic steatohepatitis, autoimmunity Examples include hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and idiopathic portal hypertension.

  As an evaluation method, for example, when the soluble LR11 concentration in a blood-derived sample is measured by an immunological method, and a cut-off value is set and becomes equal to or higher than the cut-off value, the risk of cirrhosis or liver cancer is increasing. The method of determining with a certain liver disease can be mentioned.

  Moreover, if the evaluation by the soluble LR11 concentration in the blood-derived sample of the present invention and the evaluation by type IV collagen are evaluated together, it is useful as a method for evaluating the degree of liver disease seriousness and prognosis prediction. It is particularly useful as a method for evaluating liver fibrosis.

  In addition, the soluble LR11 measurement reagent is useful as a diagnostic kit for liver diseases (excluding liver cancer). When the diagnostic kit is an ELISA kit, an immunoassay plate on which an anti-soluble LR11 antibody is immobilized, an anti-soluble LR11 antibody having an antigen recognition site different from the immobilized anti-soluble LR11 antibody (such as an enzyme or biotin) A reagent containing a reagent that contains an antibody for detection) is used as a main component, and a buffer solution for diluting or pretreating a blood-derived sample and an anti-soluble LR11 antibody are immobilized. Cleaning solution for washing the immunoassay plate, components that bind to the detection antibody (enzyme-labeled antibody that recognizes the detection antibody, enzyme-labeled avidin, etc.), and components that detect the enzyme reaction To convert and calculate the contained detection reagent (sometimes called by the name of substrate agent, color former, etc.) and soluble LR11 concentration Calibration standard substances (such as containing the purified soluble LR11 and soluble LR11 known concentrations in serum) is formed by combining as appropriate.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to a following example.

[Example 1] Detection of blood soluble LR11 in various liver diseases Materials and methods:
(1) Patients with various liver diseases under treatment, viral hepatitis [CH] (42 B type, 204 C type), cirrhosis [LC] 43 people, alcoholic hepatitis [AL] 10 people, fatty liver [FL] 21 people, non-alcoholic steatohepatitis [NASH] 12 people, autoimmune hepatitis [AIH] 44 people, primary biliary cirrhosis [PBC] 88 people, primary sclerosing cholangitis [PSC] 3 people, and idiopathic The concentration of soluble LR11 (sLR11) in plasma collected from 4 patients with portal hypertension [IPH] was measured using a soluble LR11 measuring reagent (manufactured by Sekisui Medical). As a comparison, the soluble LR11 concentration in the serum of 57 healthy volunteers was also measured. Furthermore, the soluble uPAR (suPAR) concentration in these specimens was measured by the following ELISA method. The other liver function markers were quoted from clinical test results.

(2) SuPAR concentration measurement method To a 96-well ELISA plate, 50 μL of anti-human uPAR monoclonal antibody (R & D systems, MAB807) diluted to 10 μg / mL with PBS was added per well, and allowed to stand at room temperature for 2 hours. Solid phase. Thereafter, the plate was washed with PBS containing 0.05% Tween (registered trademark) 20 (PBST), and then PBST containing 1% BSA (BSA-PBST) was added at 200 μL per well and allowed to stand at room temperature for 1 hour. The plate was then blocked with BSA. After removing the blocking solution, 50 μL of a solution obtained by diluting the sample 5 times with BSA-PBST was added per well and reacted at room temperature for 1 hour. Separately, a dilution series was prepared with BSA-PBST using Recombinant Human uPAR (R & D systems, 807-UK), and the reaction was performed under the same conditions as the sample. After washing the plate with PBST, a solution obtained by diluting the anti-human uPAR polyclonal antibody (R & D systems, AF807) with a biotin-labeled antibody to 0.1 μg / mL with BSA-PBST was obtained at 50 μL per well. The mixture was added and allowed to react at room temperature for 1 hour. After washing the plate with PBST, 50 μL of HRP-labeled streptavidin (Thermo Scientific, 21126) diluted to 0.5 μg / mL with BSA-PBST was added, and 30 minutes at room temperature. Reacted. After washing the plate with PBST, a substrate solution in which tetramethylbenzidine was dissolved to 0.3 mg / mL using a citrate buffer containing hydrogen peroxide was added at 50 μL per well, and at room temperature. After reacting for 10 minutes, 50 μL of diluted sulfuric acid solution was added per well to stop the reaction. The absorbance at 450 nm was measured, and the concentration of suPAR in the sample was calculated using a calibration curve prepared from a dilution series of a calibrator.

2. Results and Discussion (1) Analysis 1: Correlation with liver function markers General liver function marker tests, 7 items (AST, ALT, alkaline phosphatase [ALP], cholinesterase [ChE], albumin [Alb], platelets [ In addition to PLT] and type IV collagen [IVc]), the renal function test 2 items (urea nitrogen [UN] and creatinine [Cr]) were confirmed to correlate with sLR11 and suPAR concentrations. Table 1 shows the calculated correlation coefficient and the regression analysis result.

The sLR11 concentration was significantly positively correlated with AST, ALT, and ALP, which reflected hepatocyte damage, while negatively correlated with ChE, Alb, and PLT, which reflected protein synthesis in the liver. . Interestingly, it has been found that there is the strongest positive correlation with IVc, a marker of liver fibrosis. The relationship between the suPAR concentration and each liver function marker was the same as the sLR11 concentration except for ALT.
In contrast, the suPAR concentration was significantly positively correlated with the renal function marker, but the sLR11 concentration was not correlated at all. Thus, it was newly found that the PAR concentration is hardly affected by the renal function.
Also, a relatively strong positive correlation was observed between the sLR11 concentration and the suPAR concentration, indicating that both the sLR11 concentration and the suPAR concentration are useful markers for liver disease.

(2) Analysis 2: Setting of reference standard values for sLR11 and suPAR concentrations in healthy subjects The reference standard values for sLR11 and suPAR are expressed as the mean value ± 2 × standard deviation (2SD) of the serum concentrations of 57 healthy volunteers Calculated. sLR11 was calculated to be 7.8 ± 3.2 ng / mL (4.6 to 11.0 ng / mL), and suPAR was calculated to be 2.4 ± 0.7 ng / mL (1.0 to 3.8 ng / mL).

(3) Analysis 3: Comparison of sLR11 and suPAR concentrations by liver disease The results of sLR11 and suPAR concentration measurement in various liver disease samples were compared with the respective concentrations of normal volunteers (normal) obtained in Analysis 2 by disease. . The results are shown in Table 2, FIG. 1, Table 3, and FIG.

  From the results in Table 2, it was confirmed that the sLR11 concentration was significantly increased in the liver disease groups other than AL and FL as compared with the normal group.

  The results in Table 3 unexpectedly confirmed that the suPAR concentration was not significantly increased in the liver disease group other than LC as compared with the normal group.

(4) Analysis 4: Comparison of positive rates of sLR11, suPAR, and IVc by liver disease Reference reference ranges of sLR11 and suPAR obtained in Analysis 1 (4.6 to 11.0 ng / mL, 1.0 to 3.8 ng) In addition, the IVc cut-off value for which a strong positive correlation was found in the analysis was determined to be 150 ng / mL, and specimens exceeding the upper limit were each positive, and the positive rate of each test item was determined for each liver disease. Calculated (Table 4). Furthermore, the positive rate when sLR11 and IVc were combined (when either was positive) was also determined.

  From the results of Table 4, it was confirmed that the positive rates of sLR11 and IVc were significantly higher than those of suPAR. On the other hand, the positive rates of sLR11 and IVc were the same in various disease groups. Interestingly, the positive rate is large when combining these two items and one of them is positive. Newly found to improve. Furthermore, the positive rate in the chronic viral hepatitis group was around 50% for both sLR11 and IVc, whereas in the cirrhosis group in which chronic hepatitis progressed with fibrosis, it became almost 100%, and sLR11 was used as a fibrosis marker. It has been found that it can be a new liver disease marker that not only has a detection performance equivalent to that of IVc that is clinically applied, but can also complement IVc, that is, can detect a patient that has been overlooked by IVc alone.

[Example 2] Examination of changes in sLR11 concentration in hepatitis model rats treated with galactosamine Materials and methods:
(1) After fasting four Fischer 344 rats (body weight 100-110 g) for 4 hours, D (+)-galactosamine hydrochloride (Wako Pure Chemical Industries, 075-) dissolved in PBS was added to two of them. 05013) was administered intraperitoneally to 800 mg / kg. The remaining two mice were given the same amount (approximately 1 mL) of PBS intraperitoneally to serve as a control group. After administration, the animals were further fasted for 4 hours and then switched to normal breeding. Blood was collected from the tail vein before galactosamine administration and at 3, 6, 9, 24, 48, 72 hours and 13 days after administration to obtain serum. The sLR11 in the serum was measured by the following ELISA method. The degree of liver cell damage was confirmed by measuring AST in rat serum using Autocera AST reagent (Sekisui Medical). The respective results are shown in FIG. 3 and FIG.

(2) Preparation of antibody specific for sLR11 measurement method in rat serum:
LR11 knockout mice (J. Clin. Invest., 2008, 118: 2733-2746) provided by Chiba University were provided with the LR11 partial protein (Actacryst. 2011, F67, 129-132) provided by the Institute for Protein Research, Osaka University. After the immunization, specific antibodies were prepared based on the usual method. Among them, an antibody that reacts with LR11 derived from animals including rats was selected, and a measurement system capable of quantifying sLR11 in rat serum was constructed by a combination in which a sandwich ELISA system was established. Details of the measurement conditions are shown below.
Sandwich ELISA:
50 μL of anti-LR11 monoclonal antibody (clone No. 93222) diluted to 10 μg / mL with PBS was added to a 96-well ELISA plate and allowed to stand at room temperature for 2 hours to solidify the antibody. Thereafter, the plate was washed with PBST, 200 μL of BSA-PBST was added per well, and the plate was allowed to stand at room temperature for 1 hour to block the plate with BSA. After removing the blocking solution, 50 μL of a solution obtained by diluting the sample 5 times with BSA-PBST was added per well and reacted at room temperature for 2 hours. Separately, as a calibrator, a dilution series of commercially available rabbit serum (Japan Bioserum) with BSA-PBST was prepared and reacted under the same conditions as the specimen. After washing the plate with PBST, a biotin-labeled anti-LR11 monoclonal antibody (clone No. 93213) was prepared, and 50 μL per well of a solution diluted to 1 μg / mL with BSA-PBST was added. And allowed to react at room temperature for 1 hour. After washing the plate with PBST, 50 μL of HRP-labeled streptavidin (Thermo Scientific, 21126) diluted to 0.5 μg / mL with BSA-PBST was added, and 30 minutes at room temperature. Reacted. After washing the plate with PBST, a substrate solution in which tetramethylbenzidine was dissolved to 0.3 mg / mL using a citrate buffer containing hydrogen peroxide was added at 50 μL per well, and at room temperature. After reacting for 10 minutes, 50 μL of diluted sulfuric acid solution was added per well to stop the reaction. Absorbance at 450 nm was measured, and the concentration of sLR11 in rat serum was calculated using a calibration curve prepared from a dilution series of a calibrator.

2. Results and Discussion From the results shown in FIG. 4, a marked increase in serum AST activity was observed in rats treated with galactosamine, and peaked after 24 hours. On the other hand, the sLR11 concentration in serum began to increase gradually from 24 hours after administration, and was still on an increasing trend even after 72 hours when liver cell damage with AST activity as an index almost converged. After that, at the stage when 13 days passed, the sLR11 concentration returned to the initial value (FIG. 3). From this result, an increase in sLR11 concentration in the blood was observed in the acute state, suggesting that it can be an excellent marker that can accurately grasp the pathological condition even in the early stage of hepatitis that presents with fibrosis. It was.
The galactosamine-induced liver injury rat is said to be a model exhibiting a pathological condition similar to human viral hepatitis. In viral hepatitis, the host lymphocytes destroy hepatocytes infected with the virus, and the state of eliminating the virus continues. Although the destroyed cells are immediately regenerated, according to the above data, the state in which hepatocytes are regenerated and the increase in the sLR11 concentration are almost the same. It could be an indicator that reflects the situation.

  The present invention provides a novel marker for liver disease.

Claims (4)

  A method for evaluating the degree of seriousness and prognosis prediction of liver disease (excluding liver cancer) in the animal, comprising measuring a soluble LR11 concentration in a blood-derived sample derived from a mammal.   Soluble LR11 concentration is measured by measuring the soluble LR11 concentration in a blood-derived sample by an immunological method, and setting a cut-off value. If the cut-off value is exceeded, the risk of cirrhosis and liver cancer is increasing. The evaluation method according to claim 1, wherein the evaluation method is to determine that a liver disease is present.   Liver disease is viral hepatitis, cirrhosis, alcoholic hepatitis, fatty liver, non-alcoholic steatohepatitis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or idiopathic portal hypertension The evaluation method according to claim 1 or 2.   A diagnostic kit for liver diseases (excluding liver cancer) containing a soluble LR11 measurement reagent.