WO2009090882A1 - Diagnosis method for non-alcoholic fatty liver disease - Google Patents

Abstract

Disclosed are: a method for determining the occurrence of progressive non-alcoholic steatohepatitis; and a method for determining the occurrence of hepatic fibrosis in non-alcoholic fatty liver disease. Specifically disclosed are: a method for determining the occurrence of progressive non-alcoholic steatohepatitis, which is characterized by measuring the PTX3 level in a sample collected from a patient suffering from non-alcoholic fatty liver disease; and a method for determining the occurrence of hepatic fibrosis, which is characterized by measuring the PTX3 level in a sample.

Description

Diagnostic method for nonalcoholic fatty liver disease

The present invention relates to a method and a diagnostic agent for determining the occurrence of liver fibrosis or progressive nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the presence of predominantly large droplets of liver fat similar to alcoholic liver injury as liver histological findings despite no apparent alcohol consumption It is a general term for liver damage. NAFLD is a disease concept including simple fatty liver with good prognosis and progressive non-alcoholic fatty liver (NASH) (Non-patent Document 1), and it is considered that a severe disease type of NAFLD is NASH. . However, the prognosis of simple fatty liver and NASH is very different, and the pathological condition rarely progresses in simple fatty liver, whereas in NASH, 5-20% of cases progress to cirrhosis in 5-10 years. (Non-patent document 2), and further, it is supposed to develop liver cancer (non-patent document 3).
NASH often has few subjective symptoms, and about 10 to 20% of NASH cases have already progressed to cirrhosis at the time of diagnosis. In other words, it is very important to diagnose NASH at an early stage and start treatment. Although NAFLD patients have been increasing in recent years, NASH, which has a poor prognosis, accounts for approximately 10% of NAFLD, and the number of patients is estimated to be approximately 8.5 million in the United States.

Clinical findings, imaging tests, biochemical tests, etc. are used for diagnosis of NAFLD and NASH, but liver biopsy is necessary for definitive diagnosis. Search for blood markers useful for diagnosis of NAFLD and NASH instead of liver biopsy with invasion is desired. In particular, blood markers for differential diagnosis of NASH among NAFLD are very useful.

Liver fibrosis refers to a disease state that shows an increase in connective tissue resulting from repeated necrosis and repair due to chronic inflammation. In the process of progression from chronic hepatitis to cirrhosis and liver cancer, hepatic fibrosis is considered to be an important index, and type IV collagen, hyaluronic acid, etc. are used as liver fibrosis markers. Therefore, there is a need for a marker that is low in invasiveness and that can be detected sensitively and accurately at an early stage of liver fibrosis.

By the way, PTX3 is also called Pentraxin, Pentaxin, TSG-14, and MPTX3, and is found in the Pentraxin family that was discovered to be expressed in human umbilical cord endothelial cells stimulated with interleukin 1 (IL-1). It belongs to a secreted protein (Non-patent Document 4).

The Pentraxin family is broadly divided into Long Pentraxin and Short Pentraxin. C reactive protein (CRP) and serum amyloid P component (SAP), known as inflammatory proteins, belong to Short Pentraxin and are produced in the liver in response to IL-6 caused by inflammation. However, PTX3 is known not to be induced by IL-6, unlike CRP and SAP. (Non-Patent Documents 4 and 5).

However, sufficient knowledge about the relationship between PSH3 concentration and morbidity of NASH, which is a severe form of NAFLD, and liver fibrosis, has not been obtained.

An object of the present invention is to provide a method for determining liver fibrosis and a method for determining NASH.

The present inventors have measured the concentration of PTX3 in blood using an anti-PTX3 monoclonal antibody and investigated the relationship between the concentration and various diseases. As a result, NASH, which is a severe form of NAFLD, increases PTX3 in the blood. In particular, it was found that NASH can be diagnosed separately from simple fatty liver. Moreover, the result that PTX3 can become an index of liver fibrosis was also obtained. As a result, it was found that an appropriate treatment guideline can be determined for each NAFLD patient by measuring the PTX3 concentration, and the present invention has been completed.

That is, the present invention provides a method for determining progressive nonalcoholic fatty liver, characterized by measuring PTX3 concentration in a test sample derived from a patient with nonalcoholic fatty liver disease.
In addition, the present invention measures the PTX3 concentration in a test sample derived from a patient with nonalcoholic fatty liver disease, and a reference value obtained from the distribution of PTX3 concentration in a healthy person or a patient diagnosed with simple fatty liver In contrast, the present invention provides a method for determining progressive non-alcoholic fatty liver, characterized by distinguishing from progressive non-alcoholic fatty liver at a higher concentration.

The present invention also provides a diagnostic agent for progressive nonalcoholic fatty liver containing a PTX3 measurement reagent.
The present invention also provides the use of an anti-PTX3 antibody for the manufacture of a diagnostic agent for progressive nonalcoholic fatty liver.
The present invention also provides the use of an anti-PTX3 antibody for the diagnosis of progressive nonalcoholic fatty liver.

The present invention provides a method for determining liver fibrosis, which comprises measuring the PTX3 concentration in a test sample.
The present invention also provides a method for determining liver fibrosis, characterized by measuring PTX3 concentration in a test sample and determining that the higher the PTX3 concentration is, the higher the severity of liver fibrosis is. To do.

The present invention also provides a diagnostic agent for liver fibrosis containing a PTX3 measurement reagent.
The present invention also provides a diagnostic agent for liver fibrosis in nonalcoholic fatty liver disease containing a PTX3 measurement reagent.
The present invention also provides use of an anti-PTX3 antibody for producing a diagnostic agent for liver fibrosis or a diagnostic agent for liver fibrosis in nonalcoholic fatty liver disease.
The present invention also provides use of an anti-PTX3 antibody for diagnosis of liver fibrosis in liver fibrosis or nonalcoholic fatty liver disease.

According to the present invention, simple fatty liver, which is a non-alcoholic fatty liver disease, and NASH can be differentiated and diagnosed. According to the present invention, the severity of liver fibrosis can be diagnosed. Furthermore, it is useful for formulating an appropriate treatment policy for each NAFLD patient by making diagnosis in a short time with low invasiveness.

The plasma PTX3 concentrations of 17 patients with simple fatty liver and 22 patients with NASH are shown. The plasma PTX3 density | concentration of a liver fibrosis mild group and a liver fibrosis progression group is shown. The plasma PTX3 concentrations of the patients of liver fibrosis determination stage 0 (16 persons), stage 1 (12 persons), stage 3 (6 persons), and stage 4 (5 persons) are shown. The plasma PTX3 concentrations of 28 patients with simple fatty liver and 42 patients with NASH are shown. The plasma PTX3 density | concentration of a liver fibrosis mild group and a liver fibrosis progression group is shown. The plasma PTX3 concentrations of stage 0 (28 patients), stage 1 (21 persons), stage 2 (4 persons), stage 3 (11 persons), and stage 4 (6 persons) patients with liver fibrosis are shown. The plasma PTX3 concentrations of hepatic steatosis grades 1 to 3 are shown. The PTX3 values of grade 0 to 3 groups of necrotic inflammation findings are shown. The receiver operating characteristic analysis (ROC analysis) by the PTX3 measurement value of a simple fatty liver and a NASH patient is shown. The receiver operation | movement characteristic analysis (ROC analysis) by the PTX3 measurement value of the patient of a light case of NAFLD fibrosis and a progressive case is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, measurement includes quantitative or non-quantitative measurement. For example, as non-quantitative measurement, whether or not PTX3 protein is present, whether PTX3 protein is present in a certain amount or more is used. Measurement of whether or not, measurement of comparing the amount of PTX3 protein with other samples (for example, control samples, etc.) can be mentioned. Examples of quantitative measurement include measurement of the concentration of PTX3 protein, measurement of the amount of PTX3 protein, and the like. Information on the base and amino acid sequence of the PTX3 gene can be obtained from a public database such as Genbank, and is disclosed in Genbank accession number NM_002852, for example. Furthermore, it is disclosed in International Publication Gazette WO2007 / 055340.

The test sample is not particularly limited as long as it may contain PTX3 protein, but is preferably a sample collected from the body of an organism such as a mammal, more preferably a sample collected from a human. . Specific examples of the test sample include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, liver tissue, and the like. Preferred are blood, serum, and plasma. A sample obtained from a test sample such as a collected liver tissue culture solution is also included in the test sample of the present invention.

Collect a test sample from a NAFLD patient, measure the PTX3 concentration of the test sample, and be higher than the reference value obtained from the distribution of PTX3 concentration in healthy subjects or patients diagnosed with simple fatty liver. NASH is suspected. In addition, a cutoff value can be set in advance, and NASH can be diagnosed based on this cutoff value. The reference value may be set to an appropriate value in consideration of sensitivity and specificity for each measurement system.
Specific examples are given below, but the cutoff value is not limited to the following numerical values.
As an example, NASH can be used if the cutoff value is set to 1.85 to 1.93 ng / mL and the test sample has a PTX3 concentration of 1.85 to 1.93 ng / mL or more, as described in Examples below. Judgment is high.

Moreover, it is known that the reference value of a healthy person is 1.98 to 2.28 ng / mL or less (Inoue K et al., Aterioscler. Thromb. Vasc. Biol. 27; 161-167, 2007), As described in Examples below, the distribution of PTX3 concentration in the group of simple fatty liver patients was almost the same as the distribution of PTX3 concentration in healthy individuals.
For this reason, you may compare with the reference value calculated | required from the distribution of PTX3 density | concentration of a healthy person, and presence of NASH is doubted if it is a high value compared with the reference value of a healthy person in this case. The reference value may be set to an appropriate value in consideration of sensitivity and specificity for each measurement system.
Specific examples are given below, but the cutoff value is not limited to the following numerical values.
As an example, if the cutoff value is set to 1.98 to 2.28 ng / mL, and the PTX3 concentration of the test sample is 1.98 to 2.28 ng / mL or more, it is determined that the possibility of NASH is high. May be.

Here, NAFLD patients may be identified according to a conventional method, and a case of fatty liver disorder similar to alcoholic liver disorder is diagnosed as NAFLD even though there is no clear alcohol drinking calendar (Japanese Society of Hepatology) Hen, NASH / NAFLD Medical Guide). As an example, it can be performed as follows.
(1) Non-drinker (20 g / day or less in terms of ethanol).
(2) Clear chronic liver diseases are excluded (metabolic diseases such as viral hepatitis, autoimmune liver disease, hemochromatosis, Wilson's disease are excluded).
In addition to satisfying (1) and (2), a definite diagnosis is made by liver biopsy in cases where fat deposits are detected by image examination. In addition, even when fat deposition is recognized in the image examination before performing a liver biopsy, it is good also as a test subject as a patient suspected of NAFLD.

In addition, at the time of liver biopsy in (3) above, hepatic steatosis grade (grade 0 to 3) is determined by the proportion of hepatocytes in which large lipid accumulation is observed, and those who are grade 1 to 3 are identified as NAFLD. It may be certified as a patient.
Grade 0: No fatty liver.
Grade 1: Less than 33% hepatocytes with large droplet fat accumulation.
Grade 2: 33-66% hepatocytes with large droplet fat accumulation.
Grade 3: More than 66% hepatocytes with large droplet fat accumulation.

When a NAFLD patient is determined with a hepatic steatosis grade, the following cutoff value may be set, but is not limited thereto.
As an example, if the cutoff value is set to 1.6 ng / mL, and the PTX3 concentration of the test sample is 1.6 ng / mL or more, it may be determined that the possibility of NASH is high.

In the present invention, a test sample is collected from a patient, the PTX3 concentration of the test sample is measured, and the higher the PTX3 concentration, the more severe the liver fibrosis can be diagnosed. Hepatic fibrosis occurs due to chronic inflammation of the liver, so that the accuracy of the diagnosis is improved, so that it originates from a patient who has developed or suspected to have chronic inflammation of the liver (also simply referred to as a “liver disease patient”). It is preferable to use a test sample.
Here, diseases that cause chronic inflammation of the liver include viral chronic hepatitis, autoimmune hepatitis, hereditary / metabolic diseases (Wilson disease, hemochromatosis, α1 antitrypsin deficiency, amyloidosis, etc.), alcoholic liver Disorder, drug-induced hepatic disorder, NASH, NAFLD, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) (by Michio Imanaka, Hiromitsu Kumada, Hirohito Tsubouchi, Norio Hayashi Liver Pathology Asakura Shoten (2006) )). In these diseases, fibrosis is observed as the disease progresses, and the mechanism of liver fibrosis is common (Gresser OA et al .: J. Cell. Mol. Med. 11 (5), 1031-1051). (2007)). Actually, in any disease, liver fibrosis markers such as type IV collagen and hyaluronic acid are commonly used.

In the present invention, it is particularly preferable to collect a test sample from a NAFLD patient and measure the PTX3 concentration of the test sample. Liver fibrosis is found in both NASH and non-NASH patients, but is the most important indicator of NASH pathology and prognosis, and the more severe the fibrosis, the worse the prognosis and the more severe NASH (Farrell GC et al .: Hepatology. 43 (2 Suppl 1), S99-112 (2006), Hashimoto E et al .: Hepatol Res. 200533 (2), 72-76 (2005)) The higher the value, the worse the prognosis and the more severe NASH can be diagnosed.
Here, NAFLD patients may be identified according to a conventional method, and a case in which fatty liver disorder similar to alcoholic liver disorder is observed despite the absence of an apparent drinking calendar is diagnosed as NAFLD (edited by the Japan Society of Liver Studies). NASH / NAFLD medical care guide) can be performed in the same manner as described above.

When determining the severity of liver fibrosis of the patient by the method of the present invention, it can be determined that the higher the PTX3 concentration, the higher the severity of fibrosis. It can also be determined based on the off value. This cut-off value can be set for each fibrosis stage, or can be set based on another fibrosis index. The cutoff value may be set to an appropriate value in consideration of sensitivity / specificity for each measurement system.

Specific examples are given below, but the cutoff value is not limited to the following numerical values.
As an example, when determining the severity of liver fibrosis as a light case (stage 0 to 2) or an advanced case (stages 3 and 4), the cut-off value is set to 2.5 ng / mL. If the PTX3 concentration is less than 2.5 ng / mL, it is determined that there is a high possibility that it is a light case, and if it is 2.5 ng / mL or more, it is likely to be an advanced case.

As an example, if the cutoff value is set to 2.13 to 2.37 ng / mL and the PTX3 concentration in the test sample is 2.13 to 2.37 ng / mL or more from the description in Examples below, the stage 3 or It can also be determined as stage 4. If the cut-off value is set to 4.0 ng / mL, and the PTX3 concentration in the test sample is 4.0 ng / mL or more, it can be determined as stage 4.

Here, when the severity of liver fibrosis is evaluated as a stage (stage 0 to 4) as described above, the following is based on the classification of Brunt (Brunt EM. Semin Liver Dis 2004; 24: 3-20) Can be determined.
Stage 0: No fibrosis Stage 1: Perivenular / perisinusoidal / pericellular fibrosis is present in part or extensively around Zone 3 Stage 2: Fibrosis is observed in the portal vein region in addition to Stage 1 Stage 3: Bridging fibrosis Partial or widespread Stage 4: Liver cirrhosis

In the method of the present invention, the measurement of PTX3 concentration is preferably an immunological measurement method using an anti-PTX3 antibody. Hereinafter, the measurement method using the anti-PTX3 antibody will be described in detail.

The anti-PTX3 antibody used in the present invention may specifically bind to the PTX3 protein. Preferably, the antibody exhibits a high binding affinity for the three-dimensional structure of PTX3, more preferably a high binding affinity for the three-dimensional structure of PTX3, and does not cross-react with CRP or SAP. More preferred are PPMX0102 (FERM P-10326), PPMX0104 (FERM BP-10719) and PPMX0105 (FERM BP-10720), and most preferred are PPMX0104 (FERM BP-10719) and PPMX0105 (FERM BP-10720). is there.
The PPMX0102 (FERM P-10326), PPMX0104 (FERM BP-10719), and PPMX0105 (FERM BP-10720) described in this specification are the National Institute of Advanced Industrial Science and Technology as of September 22, 2005. (Address: Tsukuba City, Ibaraki Pref. 1-1-1 Central 6th)

The origin, type (monoclonal, polyclonal) and shape of the antibody are not limited. Specifically, known antibodies such as mouse antibodies, rat antibodies, human antibodies, chimeric antibodies, humanized antibodies can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.

In addition, the anti-PTX3 antibody immobilized on the support in the immunoassay and the anti-PTX3 antibody labeled with the labeling substance may recognize the same epitope of the PTX3 molecule or different epitopes.

The anti-PTX3 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-PTX3 antibody used in the present invention, a mammal-derived monoclonal antibody is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.

Monoclonal antibody-producing hybridomas can be basically produced using known techniques as follows. That is, using PTX3 as a sensitizing antigen, this is immunized according to a normal immunization method, and the resulting immune cells are fused with a known parent cell by a normal cell fusion method, and monoclonal antibodies are obtained by a normal screening method. It can be produced by screening antibody-producing cells.

Specifically, the monoclonal antibody can be produced as follows.
First, PTX3 used as a sensitizing antigen for obtaining an antibody is obtained by purifying it from an available cell culture supernatant. Alternatively, it can be obtained according to the method disclosed in JP-T-2002-503642.

Next, this purified PTX3 protein is used as a sensitizing antigen. Alternatively, a partial peptide of PTX3 can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human PTX3, or a part of the PTX3 gene can be incorporated into an expression vector, and further, natural PTX3 is degraded by a proteolytic enzyme. Can also be obtained. The part and size of PTX3 used as the partial peptide are not particularly limited.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, rabbits, monkeys, etc. are used.

Immunization of animals with a sensitizing antigen can be performed according to a known method. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if desired, and emulsified. The mammal is dosed several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize by binding to a carrier protein such as albumin or keyhole limpet hemocyanin.

Thus, after immunizing a mammal and confirming that the desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Cell.

Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeloma cell is known from various known cell lines such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81,1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6,511-519), MPC-11 (Margulies DHet al., Cell (1976) 8,405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SFet al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) and the like. Preferably used.

Cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler. G. and stein Milstein, C., Methods Enzymol. (1981) 73, 3- 46) etc.

More specifically, the cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency if desired.

The usage ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used. Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.

In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a polyethylene glycol (PEG) (for example, an average molecular weight of about 1000 to 6000) solution preheated to about 37 ° C. is usually 30. The target fused cell (hybridoma) is formed by adding and mixing at a concentration of ˜60% (w / v). Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

Screening and single cloning of the target antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, an antigen is bound to a carrier such as beads made of polystyrene or a commercially available 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, washed with the carrier, and then reacted with an enzyme-labeled secondary antibody or the like. Thus, it can be determined whether or not the target antibody reacting with the sensitizing antigen is contained in the culture supernatant. A hybridoma producing the target antibody can be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used.

In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes are sensitized to PTX3 in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having permanent mitotic potential, A desired human antibody having binding activity to PTX3 can also be obtained (see Japanese Patent Publication No. 1-59878). Further, PTX3 as an antigen may be administered to a transgenic animal having all repertoires of human antibody genes to obtain anti-PTX3 antibody-producing cells, and human antibodies against PTX3 may be obtained from the immortalized cells. (See WO94 / 25585 pamphlet, WO93 / 12227 pamphlet, WO92 / 03918 pamphlet, WO94 / 02602 pamphlet).

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution and can be stored for a long time in liquid nitrogen.

In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites is obtained. The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

A method can be used in which genes encoding these antibody fragments are constructed, introduced into an expression vector, and then expressed in an appropriate host cell.

In addition, these antibodies may be low molecular weight antibodies such as antibody fragments (fragments) or modified antibodies as long as they do not lose the property of recognizing the full length or part of the protein encoded by the PTX3 gene. . Specific examples of the antibody fragment include Fab, Fab ', F (ab') 2, Fv, Diabody, and the like. Such antibody fragments can be obtained by digesting the Fc part of IgG with pepsin or papain, constructing genes encoding these antibody fragments, introducing them into expression vectors, and expressing them in appropriate host cells. (E.g., Co, MSet al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methodszy Enzymol. (1989) 178,476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird REandREWalker, BW, Trends Biotechnol. (1991) 9, 132-137).

The antibody produced as described above can be isolated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F.F. (manufactured by GE Healthcare) and the like can be mentioned. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the affinity column, filters, ultrafiltration, salting out, dialysis and the like (Antibodies A Laboratory Manual.Ed Harlow, David Lane). , Cold Spring Spring Harbor Laboratory, 1988).

An anti-PTX3 antibody bound to various molecules such as a labeling substance can also be used as a modified antibody. The “antibody” in the present invention includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field.

The PTX3 measured in the present invention is not particularly limited, and may be the full length PTX3 or a fragment thereof.

Although the detection method of PTX3 protein contained in the test sample is not particularly limited, it is preferably detected by an immunological method using an anti-PTX3 antibody. Examples of the immunological method include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, etc. An immunoassay is particularly preferred, which is an enzyme-linked immunosorbent assay (ELISA) (for example, a sandwich ELISA). The above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.

As a general detection method using an anti-PTX3 antibody, for example, the anti-PTX3 antibody is immobilized on a support, a test sample is added thereto, and incubation is performed to bind the anti-PTX3 antibody and the PTX3 protein, followed by washing. A method of detecting PTX3 protein in a test sample by detecting PTX3 protein bound to a support via an anti-PTX3 antibody can be mentioned.

Examples of the support used in the present invention include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resins, polystyrene resins, polyacrylamide resins, nylon resins, and polycarbonate resins, and insoluble supports such as glass. Can be mentioned. These supports can be used in the form of beads or plates. In the case of beads, a column packed with these can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used. The anti-PTX3 antibody and the support can be bound by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be commercially available.

The binding between the anti-PTX3 antibody and the PTX3 protein is usually performed in a buffer solution. As the buffer solution, for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like are used. Incubation is performed under conditions often used, for example, incubation at 4 ° C. to room temperature for 1 to 24 hours. The washing after the incubation may be anything as long as it does not interfere with the binding between the PTX3 protein and the anti-PTX3 antibody. For example, a buffer containing a surfactant such as Tween 20 is used.

In the method for measuring PTX3 protein of the present invention, a control sample may be installed in addition to the test sample for detecting PTX3 protein. Examples of the control sample include a negative control sample not containing PTX3 protein and a positive control sample containing PTX3 protein. In this case, it is possible to detect the PTX3 protein in the test sample by comparing the result obtained with the negative control sample not containing PTX3 protein and the result obtained with the positive control sample containing PTX3 protein. . In addition, a series of control samples with varying concentrations are prepared, the detection results for each control sample are obtained as numerical values, a standard curve is created, and a test curve is created based on the standard curve from the test sample values. It is also possible to quantitatively detect the PTX3 protein contained in the sample.

As a preferred embodiment of the measurement of the PTX3 protein bound to the support via the anti-PTX3 antibody, there can be mentioned a method using an anti-PTX3 antibody labeled with a labeling substance.

For example, the test sample is brought into contact with the anti-PTX3 antibody immobilized on the support, and after washing, detection is performed using a labeled antibody that specifically recognizes the PTX3 protein.

The labeling of the anti-PTX3 antibody can be performed by a generally known method. As the labeling substance, labeling substances known to those skilled in the art such as fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, radioactive substances and the like can be used. As specific examples, radioisotopes ( ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide Examples include oxidase, microperoxidase, and biotin. When biotin is used as the labeling substance, it is preferable to further add avidin to which an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody. For binding of the labeling substance and the anti-PTX3 antibody, a known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid method can be used.

The enzyme enzyme labeling method includes, but is not limited to, a hinge method and a non-hinge method. In the hinge method, Fab ′ and an enzyme molecule are separated from each other by using a thiol group generated by reducing a disulfide bond in a portion called a hinge portion between an F (ab ′) 2 portion having an antigen-binding ability of antibody IgG. It is a way to combine. On the other hand, the non-hinge method does not specify which reactive group of the antibody is used, but in many cases, it is a method of binding the antibody molecule and the enzyme molecule using the amino group of the antibody.

Specifically, a solution containing an anti-PTX3 antibody is added to a support such as a plate, and the anti-PTX3 antibody is fixed to the support. After washing the plate, it is blocked with, for example, BSA, gelatin, albumin or the like in order to prevent nonspecific binding of proteins. Wash again and add the test sample to the plate. After incubation, wash and add labeled anti-PTX3 antibody. After moderate incubation, the plate is washed and the labeled anti-PTX3 antibody remaining on the plate is detected. Detection can be performed by methods known to those skilled in the art. For example, in the case of labeling with a radioactive substance, it can be detected by liquid scintillation or RIA. In the case of labeling with an enzyme, a substrate is added, and an enzymatic change of the substrate, such as color development, can be detected with an absorptiometer. Specific examples of the substrate include 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine), 3,3 ′ , 5,5′-tetramethylbenzidine (TMB) and the like. In the case of a fluorescent substance, it can be detected by a fluorometer.

As a particularly preferred embodiment of the method for measuring PTX3 protein of the present invention, there is a method using an antibody labeled with a method described in a general enzyme labeling method after removing an Fc portion unrelated to the antigen-binding ability of antibody IgG. Can do.

Specifically, a solution containing an anti-PTX3 antibody is added to a support such as a plate to fix the anti-PTX3 antibody. After washing the plate, it is blocked with, for example, BSA in order to prevent non-specific protein binding. Wash again and add the test sample to the plate. After incubation, wash and add peroxidase directly labeled anti-PTX3 antibody. After appropriate incubation, the plate is washed, a substrate corresponding to the enzyme is added, and the PTX3 protein is detected using an enzymatic change of the substrate as an indicator.

As another embodiment of the method for measuring PTX3 protein of the present invention, there may be mentioned a method using one or more primary antibodies that specifically recognize PTX3 protein and one or more secondary antibodies that specifically recognize the primary antibody. Can do.

For example, the test sample is brought into contact with one or more types of anti-PTX3 antibody immobilized on a support, incubated, washed, and the PTX3 protein bound after washing is identified with the primary anti-PTX3 antibody and the primary antibody. It is detected by one or more secondary antibodies that are recognized automatically. In this case, the secondary antibody is preferably labeled with a labeling substance.

As another embodiment of the method for measuring PTX3 protein of the present invention, a detection method using an agglutination reaction can be mentioned. In this method, PTX3 can be detected using a carrier sensitized with an anti-PTX3 antibody. As the carrier for sensitizing the antibody, any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles and the like can be used, and polystyrene latex particles are preferably used. The sensitized particles are mixed with the sample and stirred for a certain time. The higher the concentration of the anti-PTX3 antibody in the sample, the greater the degree of aggregation of the particles. Therefore, PTX3 can be detected by viewing the aggregation with the naked eye. It is also possible to detect turbidity due to aggregation by measuring with a spectrophotometer or the like.

As another aspect of the method for measuring PTX3 protein of the present invention, for example, a method using a biosensor utilizing the surface plasmon resonance phenomenon can be mentioned. A biosensor using the surface plasmon resonance phenomenon can observe a protein-protein interaction as a surface plasmon resonance signal in real time using a trace amount of protein and without labeling. For example, it is possible to detect the binding between the PTX3 protein and the anti-PTX3 antibody by using a biosensor such as BIAcore (Pharmacia). Specifically, a test sample is brought into contact with a sensor chip on which an anti-PTX3 antibody is immobilized, and PTX3 protein that binds to the anti-PTX3 antibody can be detected as a change in resonance signal.

The measurement method of the present invention can be automated using various automatic inspection devices, and a large number of samples can be inspected at a time.

The present invention can also be used for immunohistochemical staining. Tissue obtained by biopsy or surgery can be stained with anti-PTX3 antibody by methods known to those skilled in the art.

The present invention is also aimed at providing a diagnostic agent for distinguishing liver fibrosis and simple fatty liver from NASH in NAFLD, and the diagnostic agent preferably contains an anti-PTX3 antibody. Here, the diagnostic agent includes a kit. When the diagnostic agent is based on the ELISA method, it may contain a carrier for immobilizing the antibody, and the antibody may be bound to the carrier in advance. When the diagnostic agent is based on an agglutination method using a carrier such as latex, it may contain a carrier to which an antibody is adsorbed. In addition, the diagnostic agent may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.

<Target of Example 1>
The study was conducted on 39 cases of NAFLD that were evaluated by performing liver biopsy.
A total of 39 NAFLD patients were studied, including 17 patients diagnosed with simple fatty liver and 22 patients diagnosed with NASH.
The diagnosis of NAFLD and NASH was performed as follows.
(1) Non-drinker (20 g / day or less in terms of ethanol).
(2) Clear chronic liver diseases are excluded (metabolic diseases such as viral hepatitis, autoimmune liver disease, hemochromatosis, Wilson's disease are excluded).
(3) In addition to satisfying (1) and (2), cases in which fat deposition was observed by imaging examination were confirmed by liver biopsy. After confirming that lipid droplets are present in hepatocytes and the nucleus has migrated to the edge of the cells by pathological diagnosis, hepatic steatosis is as follows depending on the percentage of hepatocytes in which large droplets of fat accumulate. A grade (0-3) was determined, and grades 1 to 3 were defined as NAFLD patients.
・ Determination of hepatic steatosis grade Grade 0: No fatty liver Grade 1: Less than 33% of hepatocytes with large droplets of fat accumulation Grade 2: 33-66% of hepatocytes with large droplets of fat accumulation
Grade 3: More than 66% hepatocytes with large droplet fat accumulation Furthermore, a pathological diagnosis was made regarding the degree of fibrosis and whether it was NASH.

Determination of liver fibrosis Fibrosis was evaluated using the Brunt classification (Brunt EM. Semin Liver Dis 2004; 24: 3-20).
Stage 0: No fibrosis Stage 1: Perivenular / perisinusoidal / pericellular fibrosis is present in part or extensively centering on Zone 3 Stage 2: Fibrosis enlargement in the portal vein region in addition to Stage 1 Stage 3: bridging fibrosis Partial or widespread Stage 4: Liver cirrhosis

-NASH determination Fatty (large droplet> small droplet, often in the center of the leaflet), inflammation in the leaflet (mild, neutrophil or mononuclear cell infiltration), balloon-like degeneration of hepatocytes (peripheral periphery, leaflet) NASH was determined when three findings of fibrosis were observed (mostly in the center) (American Liver Society Single Topic Conference 2002, Neuschwander-Tetri BA, et al: Nonalcoholic steatohpatitis: summary of an AASLD Single Topic Conference.).

Table 1 shows the PTX3 concentrations and determination results of the targeted patient groups.
The PTX3 concentration was determined by the ELISA method of Example 2 below.

<Example 2 Measurement of PTX3 concentration by ELISA method>
In order to detect PTX3 protein in blood, a PTX3 sandwich ELISA system was constructed as follows. That is, for the antibody coated on the 96-well plate, F (ab ′) 2 -modified PPMX0104 (FERM BP-10719) prepared according to WO2007 / 055340 was incubated overnight at 5 μg / mL, 100 μL / well, 4 ° C. Made.
PPMX0104 (FERM BP-10719) was deposited on September 22, 2005 at the National Institute of Advanced Industrial Science and Technology patent biological deposit center (address: 1-1-1 Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture). .

The next day, after washing 3 times with 300 μL / well wash buffer (0.05% (v / v) Tween 20, PBS), 150 μL of ABI immunoassay stabilizer (ABI # 10-601-001) was added to perform blocking. Stored overnight at 4 ° C. The ELISA method is disclosed in application number PCT / JP2006 / 322505. Plasma was diluted appropriately with a dilution buffer containing animal serum (50 mM Tris-Cl pH 8.0, 0.15 M NaCl), and incubated for 2 hours at room temperature. Subsequently, HRPO (horseradish peroxidase) -labeled Fab′-modified PPMX0105 (FERM BP-10720) antibody diluted to 20 μg / mL with PBS (−) containing animal serum and the like was added and incubated at room temperature for 2 hours. After discarding the reaction solution, the plate was washed 5 times with a 300 μL / well wash buffer, and then developed using Scytek's TMB (Cat # TM4999) according to the attached protocol, and the absorbance was measured with a microplate reader. The PTX3 protein concentration in the sample was converted using a spreadsheet software GraphPad PRISM (GlaphPad software Inc. ver. 3.0).
PPMX0105 (FERM BP-10720) was deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: 1-1-1 Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture) on September 22, 2005. .

<Example 3 Differential diagnosis between simple fatty liver and NASH>
Plasma PTX3 concentrations of 17 patients with simple fatty liver and 22 NASH patients described in Example 1 were measured by the ELISA method of Example 2, and the measured values were compared by t-test. The mean value ± standard deviation of the simple fatty liver patient group and the NASH group were 1.429 ± 0.512 ng / mL and 2.984 ± 2.173 ng / mL, and a significant difference was observed with p = 0.0006. (FIG. 1).

<Severity of liver fibrosis of Example 4 NAFLD>
In the NAFLD patient described in Example 1, the cases in which the determination of liver fibrosis was stage 0, stage 1, and stage 2 were the mild fibrosis group, and those in stage 3 and stage 4 were the fibrosis progression case group. The plasma PTX3 value was measured by the ELISA method of Example 2, and the measured value was compared by t-test. Mean value ± standard deviation of the mild fibrosis group is 1.628 ± 0.735 ng / mL, mean value ± standard deviation of the fibrosis progression group is 4.076 ± 2.548 ng / mL, and p <0.0001. A significant difference was observed (FIG. 2).

When fibrosis was determined for stage 0 (16 persons), stage 1 (12 persons), stage 3 (6 persons), and stage 4 (5 persons), the average value ± standard deviation was stage 0. 1.454 ± 0.518 ng / mL in the group, 1.819 ± 0.889 ng / mL in the stage 1 group, 3.968 ± 2.809 ng / mL in the stage 3 group, 4.206 ± 2. It was 516 ng / mL. (Stage 0 group and Stage 3 group (p = 0.0019), Stage 0 group and Stage 4 group (p = 0.0004), Stage 1 group and Stage 3 group (p = 0.0247), Stage 1 group and A significant difference was observed between the stage 4 groups (p = 0.0004) (FIG. 3).

<Target of Example 5>
Further cases were added and examined in the same manner as in Example 1 except that the patient group was changed.
Targeting 70 cases of NAFLD (28 patients with simple fatty liver and 70 NAFLD patients with NASH) evaluated using the same experiment and measurement method as in Examples 2 to 4 above. Various studies were conducted as in Examples 6-10.

<Example 6: Differential diagnosis between simple fatty liver and NASH>
The blood PTX3 concentrations of 28 simple fatty liver patients and 42 NASH patients described in Example 5 were measured, and the measured values were compared by t-test. As a result, in the simple fatty liver patient group and the NASH group, p = 0.0021 and a significant difference was recognized (FIG. 4). Moreover, the mean value ± standard deviation of the simple fatty liver patient group and the NASH group were 1.335 ± 0.515 ng / mL and 2.417 ± 1.739 ng / mL.

Example 7 Severity of NAFLD fibrosis and PTX3
The NAFLD patients described in Example 5 are classified as those in which fibrosis is determined as stage 0, stage 1, and stage 2 as mild fibrosis groups, and those that are as stage 3 and stage 4 as fibrosis progression cases. Then, plasma PTX3 value was measured by ELISA method and compared by t-test. The mean value ± standard deviation of the mild fibrosis group is 1.515 ± 0.617 ng / mL, and the mean value ± standard deviation of the fibrosis progression group is 3.447 ± 2.277 ng / mL. There was a significant difference between p <0.0001 and the progressing case group (FIG. 5).
On the other hand, the NAFLD patients of Example 5 were compared by t-test using existing fibrosis markers. When type IV collagen 7s is used as a marker, the mean ± standard deviation of type IV collagen 7s in the mild fibrosis group and the advanced group are 4.65 ± 0.93 ng / dL and 5.94 ± 1.53 ng / dL, respectively. The p value was 0.0018. When hyaluronic acid is used as a marker, the mean value ± standard deviation of the hyaluronic acid concentration in the mild fibrosis group and the advanced group are 32.7 ± 27.8 ng / dL and 72.4 ± 68.1 ng / dL, respectively. 0.009, when the platelet count is used as a marker, the mean ± standard deviation of the platelet count concentration in the mild fibrosis group and the advanced group is 25.8 ± 6.6 ng / dL and 20.0 ± 8.7 ng / mL, respectively. The p value was 0.0134.
In relation to the severity of fibrosis, PTX3 showed a highly significant difference compared to any existing liver fibrosis marker.

Further, when the PTX3 measurement value at the stage of fibrosis was subjected to the Kruskal-Wallis test, it was shown that there was a relationship between p <0.0001 and the PTX3 concentration and the stage of fibrosis (FIG. 6).
In addition, when fibrosis was determined for stage 0 (28 persons), stage 1 (21 persons), stage 2 (4 persons), stage 3 (11 persons), and stage 4 (6 persons), the average value was obtained. ± standard deviation is 1.328 ± 0.523 ng / mL in stage 0 group, 1.687 ± 0.714 ng / mL in stage 1 group, 1.768 ± 0.356 ng / mL in stage 2 group, and in stage 3 group It was 3.082 ± 2.320 ng / mL, and it was 4.117 ± 2.234 ng / mL in the stage 4 group. (Stage 0 and Stage 3 (p = 0,0009), Stage 0 and Stage 4 (p <0.0001), Stage 1 and Stage 3 (p = 0.136), Stage 1 and Significant differences were observed between stage 4 group (p = 0.0001), stage 2 group and stage 4 group (p = 0.0465) (FIG. 6).

<Example 8: Liver steatosis grade and necrotic inflammation findings and PTX3>
NAFLD patients described in Example 5 were grouped according to hepatic steatosis grade, and plasma PTX3 values were measured by ELISA as in Example 2 and compared by t-test. As a result, no association was found between hepatic steatosis grade and PTX3 measurement (FIG. 7).
Moreover, as a result of classifying the same patient group according to the grade of the necrosis inflammation findings and comparing the PTX3 values, there was no significant difference between the necrosis inflammation findings and PTX3 (FIG. 8).

<Example 9: Cutoff value of simple fatty liver and NASH>
Receiver operating characteristic analysis (ROC analysis) was performed from PTX3 measurement values of patients with simple fatty liver and NASH (FIG. 9). The cut-off value with the most excellent sensitivity and specificity in distinguishing between simple fatty liver and NASH patients was 1.61 ng / mL. Sensitivity was 66.7%, specificity was 78.6%, positive median was 82.4%, and negative median was 61.1%.

<Example 10: Cutoff value of mild case and advanced case of severity of NAFLD fibrosis>
Receiver operating characteristic analysis (ROC analysis) was performed from PTX3 measured values of patients with mild and advanced cases of NAFLD fibrosis (FIG. 10). The cut-off value with the highest sensitivity and specificity was 2.45 ng / mL in distinguishing between mild and severe cases of fibrosis of NAFLD. Sensitivity was 70.6%, specificity was 94.3%, positive median was 80.8%, and negative median was 90.9%.

Claims (17)

1. A method for determining progressive nonalcoholic fatty liver, characterized by measuring PTX3 concentration in a test sample derived from a patient with nonalcoholic fatty liver disease.
2. Measure PTX3 concentration in test samples from patients with non-alcoholic fatty liver disease and compare it with the reference value determined by the distribution of PTX3 concentration in healthy subjects or patients diagnosed with simple fatty liver. A method for determining progressive nonalcoholic fatty liver, characterized by distinguishing from progressive nonalcoholic fatty liver at a high concentration.
3. 3. The determination method according to claim 1, wherein the test sample is blood, serum or plasma.
4. Progressive non-alcoholic fatty liver diagnostic agent containing PTX3 measurement reagent.
5. A method for determining liver fibrosis, comprising measuring the PTX3 concentration in a test sample.
6. A method for determining liver fibrosis, characterized by measuring PTX3 concentration in a test sample and determining that the higher the PTX3 concentration, the higher the severity of liver fibrosis.
7. The determination method according to claim 5 or 6, wherein the test sample is derived from a liver disease patient.
8. The determination method according to claim 5 or 6, wherein the test sample is derived from a non-alcoholic fatty liver disease patient.
9. The determination method according to any one of claims 5 to 8, wherein the test sample is blood, serum or plasma.
10. Diagnostic agent for liver fibrosis containing PTX3 measurement reagent.
11. Diagnostic agent for liver fibrosis in non-alcoholic fatty liver disease containing PTX3 measurement reagent.
12. Use of anti-PTX3 antibody for the manufacture of a diagnostic agent for progressive nonalcoholic fatty liver.
13. Use of anti-PTX3 antibody for the production of a diagnostic agent for liver fibrosis.
14. Use of anti-PTX3 antibody for the production of a diagnostic agent for liver fibrosis in nonalcoholic fatty liver disease.
15. Use of anti-PTX3 antibody for diagnosis of progressive non-alcoholic fatty liver.
16. Use of anti-PTX3 antibody for diagnosis of liver fibrosis.
17. Use of anti-PTX3 antibody for diagnosis of liver fibrosis in nonalcoholic fatty liver disease.

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