JPH10339730A - Discriminating method for chronic inflammation and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a kit for easily, speedily and highly sensitively discriminating the presence/absence or degree of chronic inflammation in safe at low cost, while avoiding the danger of X-ray exposure or infection without applying any pain to a patient. SOLUTION: The presence/absence or degree of chronic inflammation is discriminated by measuring the concentration of hyaluronic acid in the inspection sample of urine. As a method for measuring the concentration of hyaluronic acid in the inspection sample of urine, a measuring method to use competitive reaction to hyaluronic acid bonding protein is desired. The kit to be used for this discriminating method contains the hyaluronic acid binding protein and the hyaluronic acid symbolized by a symbolic material at least.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for judging the presence or absence or degree of chronic inflammation, in particular, a method for judging the degree of progression of the disease state of rheumatism, a method for judging the degree of cartilage destruction in osteoarthritis, and a method for judging viral liver disease. It relates to a method of determining the presence or absence. The present invention also relates to kits used for these determination methods.

[0002]

2. Description of the Related Art As a method for determining the presence or degree of chronic inflammation, there are known methods such as X-ray photography and a method for measuring substances in serum.

For example, for rheumatoid arthritis, which is one of the chronic inflammations, X-ray imaging, measurement of rheumatoid factor in serum, measurement of hyaluronic acid in blood, and the like are known as methods for determining the degree of progression of the disease state. Have been.

Also, "Steinbrocke (Steinbrocke)
As in r) et al.'s class classification, there is known a method of judging the degree of progression of the disease state of rheumatoid arthritis using an index of a possible degree of movement or exercise in a patient's daily life. According to this method, it is possible to roughly determine the degree of progress of the disease state, but it is not suitable for determining the delicate progress of the disease state because it is not a quantitative determination method.

[0005] As for the method of determining viral liver disease, GPT, GO, which are serum enzymes in blood, are used.
Methods for measuring T, ALP, γGTP, LDH, and the like, and measurement of blood hepatitis virus antigens and antibodies against the antigens are known.

[0006] Methods for determining chronic hepatitis and cirrhosis include measurement of N-terminal peptide of hyaluronic acid, type IV collagen and type III procollagen in blood. Although the presence or degree of chronic inflammation can be determined by these methods, a further improved determination method is required as follows. In other words, (1) it is not affected by radiation (X-rays), (2) it does not cause pain in blood collection to patients, and it is possible to collect samples for determination at any time, and it is difficult to collect blood, especially for newborns, children and the elderly. Samples to be used for the determination can be collected from patients that are not necessary, (3) Pretreatment such as centrifugation such as blood is not required, and (4) The detection method is improved so that the subtle progress of the disease state can be detected. is there.

[0007]

Accordingly, the present invention provides a method for determining the presence or absence or degree of chronic inflammation without causing pain to a patient, avoiding the danger of radiation exposure or infection, etc., at a safe, low cost,
It is an object of the present invention to provide a method that can be simply, rapidly and highly sensitively determined, and a kit used for such a determination.

[0008]

Means for Solving the Problems The present inventors have found that the concentration of hyaluronic acid in a urine sample increases with the progress of the disease state of rheumatism, which is one of the chronic inflammations. Rise with osteoarthritis cartilage destruction,
Furthermore, by measuring the concentration of hyaluronic acid in urine samples, it was found that the degree of progression of rheumatic disease, the degree of cartilage destruction in osteoarthritis, and the presence or absence of viral liver disease can be determined with high sensitivity. The present invention has been completed.

That is, the present invention provides a method for judging the presence or absence of chronic inflammation, which is characterized by measuring the concentration of hyaluronic acid in a urine sample (hereinafter, also referred to as the "judging method of the present invention"), and in particular, the pathological condition of rheumatism. A method for determining the degree of progress (hereinafter, also referred to as “the method for determining rheumatism of the present invention”), a method for determining the degree of cartilage destruction of osteoarthritis (hereinafter, also referred to as “the method for determining arthropathy of the present invention”), and viral liver A method for determining the presence or absence of a disease (hereinafter, also referred to as “the method for determining liver disease of the present invention”), and at least a hyaluronic acid-binding protein and hyaluronic acid labeled with a labeling substance (also referred to as “labeled hyaluronic acid”) And a kit (hereinafter, also referred to as "the kit of the present invention") used in these determination methods. In this specification, "chronic inflammation"
Is also intended to include "viral liver disease."

[0010] According to the present invention, the presence or degree of chronic inflammation can be determined by measuring a urine sample, so that it does not cause any pain to the patient, and avoids the dangers of radiation exposure and infection, and safely.
The determination can be easily performed at low cost.

[0011] Conventionally, it has been known that the concentration of hyaluronic acid in serum is increased in patients with liver diseases and RA patients.
(The Bone, Vol. 8 No. 4, pp. 115-123, 1994). Hyaluronic acid contained in serum is a high molecular weight and hyaluronic acid in urine is a low molecular weight.Therefore, these two hyaluronic acids are very different in molecular weight and are completely different. I can say.

From this, the concentration of hyaluronic acid (low molecular weight) in urine is not necessarily the same as the concentration of hyaluronic acid (high molecular weight) in serum.
It does not always reflect the concentration. However, the present inventors have found that not only serum hyaluronic acid (high molecular weight) but also urinary hyaluronic acid (low molecular weight) concentration increases in chronic inflammation, and urinary hyaluronic acid (low molecular weight) concentration Is R
The present invention has been found to be a very good indicator of the progress of the disease state of A, the degree of cartilage destruction of OA, and the presence or absence of viral liver disease, thereby completing the present invention.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) Determination method of the present invention The determination method of the present invention is a method for determining the presence or degree of chronic inflammation, which comprises measuring the concentration of hyaluronic acid in a urine sample.

The urine sample used in the determination method of the present invention is not particularly limited as long as it is a mammalian urine sample, but is preferably a human urine sample. The urine sample used in the present invention does not necessarily need to be subjected to pretreatment such as purification. That is, although it depends on the method of measuring the concentration of hyaluronic acid used in the determination method of the present invention, generally, even when other impurities are present in the urine sample, the low-molecular-weight hyaluronic acid in the urine sample is selectively measured. The measurement result is not affected.

In the determination method of the present invention, the molecular weight of the hyaluronic acid to be measured is not particularly limited as long as it is in the range of the molecular weight of the hyaluronic acid contained in urine, but is preferably 40,000 or less, more preferably the average molecular weight. 200
0 to 40,000, particularly preferably an average molecular weight of 2000 to 30,000,
It is extremely preferable to measure hyaluronic acid having an average molecular weight of 2,000 to 10,000.

The method for measuring the concentration of hyaluronic acid in the determination method of the present invention quantitatively measures the concentration of such low-molecular-weight hyaluronic acid since urine has a lower molecular weight than that contained in plasma. The method is not limited as long as it can be performed.For example, (1) hyaluronic acid and labeled hyaluronic acid in a urine sample are subjected to a competitive reaction with hyaluronic acid-binding protein, and labeled hyaluronic acid bound to the protein or bound A method of measuring any of the unlabeled hyaluronic acid and measuring the concentration of hyaluronic acid in a urine sample (for example, JP-A-63-150669), (2) a method using liquid chromatography (for example, Analyt.Bioch
em. 157, 93-99 (1986)) and (3) electrophoresis (capillary electrophoresis, electrophoresis using a cellulose acetate membrane, etc.).

A more specific method of the method (2) using liquid chromatography and (3) the electrophoresis method will be described below, but the method is not limited thereto. In order to remove impurities in the urine sample, for example, dialysis, ion exchange chromatography, gel filtration and the like are performed. Next, hyaluronic acid in the urine sample is decomposed by a hyaluronic acid-degrading enzyme to obtain a hyaluronic acid oligosaccharide. The resulting hyaluronic acid oligosaccharide is separated and analyzed by high performance liquid chromatography (HPLC) equipped with an anion exchange column and the like. Labeling is performed with a fluorescent labeling substance (2-cyanoacetamide, dansyl hydrazine, or the like) as needed to increase the sensitivity in HPLC. These labeled hyaluronic acid oligosaccharides can be detected by a fluorescence detection system or the like.

Here, instead of HPLC, separation analysis may be performed by electrophoresis (capillary electrophoresis, electrophoresis using a cellulose acetate membrane, or the like). Also,
Hyaluronic acid and labeled hyaluronic acid in the urine sample of (1) are subjected to a competitive reaction with a hyaluronic acid-binding protein,
A method for measuring either labeled hyaluronic acid bound to the protein or unlabeled hyaluronic acid and quantifying the low molecular weight hyaluronic acid concentration in the urine sample will be described in more detail below. However, the present invention is not limited to this. According to the method (1), the concentration of hyaluronic acid in a urine sample can be measured at a low cost, with good quantification, with high sensitivity, quickly and easily. It is particularly preferable as a method for measuring the concentration.

The labeled hyaluronic acid used here is:
Binding of labeling substance to hyaluronic acid by known means
Can be obtained by Of hyaluronic acid labeled
Molecular weight is measured in urine samples for hyaluronic acid binding protein
Is particularly limited as long as it competes with hyaluronic acid
Although not, average molecular weight 2000 to 2,000,000, preferably
Average molecular weight 10,000 to 2,000,000, more preferably average molecular weight
About 40,000 are exemplified. Used for labeling hyaluronic acid
Labeling substances used include radioisotopes ( ¹²⁵I,
¹³⁵I,^ThreeH), fluorescent substances (fluorescein
Anate (FITC), umbelliferone, 7-amino-4-
Methyl coumarin-3 acetic acid, etc.) and chemiluminescent substances (lumino
), Enzymes (peroxidase, alkaline phosph
Tatase, β-galactosidase, luciferase,
Cetylcholinesterase) or other substances (bi
Otin, avidin (eg streptavidin)
Is exemplified. The labeling method for hyaluronic acid depends on the labeling substance.
Any suitable known method may be used, for example, using biotin.
When using, use a hydrazide derivative of biotin.
Method (Avidin-Biotin Chemistry: A Handbook, p57-63, PIER
CE CHEMICAL COMPANY, published in 1994)
When using CEJN isothiocyanate,
The method can be appropriately selected from the methods described in JP-A-7843.

The hyaluronic acid-binding protein used herein is not particularly limited as long as it has a property of binding to hyaluronic acid and labeled hyaluronic acid in urine samples, and is not limited to proteoglycan (eg, cartilage). Proteoglycan, tryptic digest of cartilage proteoglycan, chondroitinase ABC-treated cartilage proteoglycan, core protein of proteoglycan (eg, cartilage proteoglycan core protein, etc.), link protein, hyaluronectin, CD4
4, partial proteins containing a hyaluronic acid binding site of these proteins, or fusion proteins of the partial proteins with other proteins, and the like. Particularly, tryptic digest of cartilage proteoglycan, and further trypsin of bovine nasal cartilage proteoglycan Digestions are preferred.

Here, a method for allowing a hyaluronic acid in a urine sample to react with a labeled hyaluronic acid in a competitive reaction with a hyaluronic acid-binding protein includes, for example, a hyaluronic acid-binding protein immobilized on a solid phase in advance, a labeling method, and the like. Contacting the labeled hyaluronic acid and the urine sample, then separating the solid phase and the liquid phase, and labeling hyaluronic acid bound to the hyaluronic acid-binding protein fixed to the solid phase or labeled hyaluronic acid not bound (Included in the liquid phase).

Examples of the solid phase on which the hyaluronic acid-binding protein is immobilized include plates (eg, wells of a microplate), tubes, beads, membranes, gels, and the like. As the material, polystyrene, polypropylene, nylon, polyacrylamide, and the like can be used.

As a method for immobilizing a hyaluronic acid-binding protein on these solid phases, a general method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bonding method, and an entrapment method (immobilized enzyme, 1975) Kodansha, pages 9 to 75) can be applied. Especially physical adsorption method,
This is preferable because the operation is simple.

Specific examples of the physical adsorption method include the following methods: dissolving the hyaluronic acid-binding protein in a buffer solution (eg, phosphate buffer solution, carbonate buffer solution, etc.) having a pH of about 7 to 9; And add it to the solid phase.
Store for 2 hours or overnight at 4 ° C. to fix.

Further, the portion where the hyaluronic acid-binding protein is not fixed may be blocked by serum albumin, gelatin, casein, skim milk, etc.
And preferred.

Specific examples of the blocking method include the following methods. Add a blocking substance (eg, serum albumin, gelatin, casein, skim milk, etc.) and store at about 37 ° C. for 30 minutes to 2 hours or at room temperature for 1 to 2 hours.

The method of contacting the hyaluronic acid-binding protein (a), the labeled hyaluronic acid (b) and the urine sample (c) fixed on the solid phase involves contacting (a), (b) and (c) simultaneously. Method, (a) and
(c) and then contacting (b) with this,
Either a method in which (b) and (c) are brought into contact and then (a) is brought into contact therewith, or a method in which (a) and (b) are brought into contact and then (c) is brought into contact therewith may be used. Contact time, temperature and pH
Are conditions that can be appropriately determined by those skilled in the art.

Specific examples of the method of contacting the hyaluronic acid-binding protein, the labeled hyaluronic acid, and the urine sample fixed on the solid phase include the following methods. The urine sample is added to the solid phase to which the hyaluronic acid-binding protein is fixed, and the mixture is allowed to stand or stirred at 25 to 40 ° C. for about 20 to 90 minutes to bind the hyaluronic acid in the urine sample to this hyaluronic acid-binding protein. . Further, labeled hyaluronic acid is added, and the mixture is allowed to stand or stirred at 25 to 40 ° C. for about 20 to 90 minutes to bind the labeled hyaluronic acid to the hyaluronic acid-binding protein. Thereafter, the solid phase is preferably washed with a buffer (for example, a phosphate buffer to which a surfactant is added) or the like to remove non-specifically adsorbed substances. Examples of the washing liquid include polyoxyethylene alkyl phenyl ethers (Triton surfactant; Triton X-100, etc.)
(For example, phosphate buffer, phosphate buffered saline, and Tris-HCl buffer) to which a nonionic surfactant such as polyoxyethylene sorbitan alkyl esters (Tween-based surfactant) is added. Is done. The pH of this buffer is preferably near neutral.

Next, the concentration of hyaluronic acid in the urine sample is measured by detecting the labeling substance of the labeled hyaluronic acid bound to the hyaluronic acid-binding protein. The concentration of hyaluronic acid in a urine sample was determined using a known hyaluronic acid standard solution, and a calibration curve was prepared in advance for the relationship between the hyaluronic acid concentration in the standard solution and the detection intensity of the labeled hyaluronic acid labeling substance. The measurement can be performed by a method using the detection intensity of the labeling substance when a urine sample is used and the above-mentioned calibration curve.

For example, when hyaluronic acid labeled with biotin is used, a labeled substance can be detected by a conventional method for measuring enzyme activity using avidin (streptavidin or the like) to which an enzyme is bound.

According to this method, the concentration of hyaluronic acid in a urine sample, particularly, the concentration of hyaluronic acid having an average molecular weight of 2000 to 40,000 can be determined with high sensitivity (in the range of about 0.1 to 1000 ng / ml). It is.

The measured value of the hyaluronic acid concentration obtained by the above measuring method may be corrected, if necessary, by the concentration of other components such as creatinine contained in the urine sample.

The method for measuring the hyaluronic acid concentration is as follows: (1) fixing the hyaluronic acid-binding protein to a solid phase;
Then, a sample hyaluronic acid is added to bind to the protein, a labeled hyaluronic acid-binding protein is further added, and hyaluronic acid is sandwiched between the protein immobilized on a solid phase and the labeled protein, A method for measuring hyaluronic acid by forming a sandwich-like conjugate and measuring the labeling substance of the binding protein (Japanese Patent Publication No. 6-41952, Clin.chim.acta., 181, 317-327 (1989)); (2) A sample containing sample hyaluronic acid is added to the chondroitinase ABC treated product of cartilage proteoglycan fixed to the solid phase, and the treated product is combined with the sample hyaluronic acid. The binding protein is added, the sample hyaluronic acid is sandwiched between the treated product and the labeled hyaluronic acid binding protein, and the sample hyaluronan is measured by measuring the labeled substance of the sandwich-like conjugate. A so-called sandwich method such as a method for quantifying an acid (JP-A-4-62797) is also known. However, such a sandwich method is suitable for measuring high-molecular-weight hyaluronic acid,
It is not suitable for measuring low molecular weight hyaluronic acid as found in urine samples.

Utilizing that the concentration of hyaluronic acid in a urine sample increases with the presence or degree of chronic inflammation,
The presence or absence or degree of chronic inflammation can be determined. For example, when the concentration of hyaluronic acid in the urine sample is high, it is determined that the inflammation is chronic. Further, for example, when an anti-chronic inflammatory drug is administered to a chronic inflammation patient, the hyaluronic acid concentration in the urine sample of the patient is continuously measured, and if the hyaluronic acid concentration is reduced, the pathology of chronic inflammation is reduced. Progress can be stopped and it can be determined that it is in the direction of improvement. Similarly, if the hyaluronic acid concentration in the urine sample does not change, the progress of the disease state is stopped and the disease state is maintained, and if the hyaluronic acid concentration in the urine sample is increased, the disease state progresses and worsens Is determined to be in the direction of.

As described above, the method of the present invention can be used not only for determining the degree of chronic inflammation in patients with chronic inflammation, but also for determining the effect of the anti-chronic inflammatory drug, and changing or selecting the anti-chronic inflammatory drug to be administered. It provides useful information for the policy.

In addition, the concentration of hyaluronic acid in the urine sample of a patient whose degree of chronic inflammation is unknown, such as a patient with chronic inflammation at the first visit, is measured, and the degree of chronic inflammation of the patient is determined from the hyaluronic acid concentration. Can be determined. This also provides useful information for treatment of chronic inflammation, such as selection of an anti-chronic inflammatory drug to be administered.

The concentration of hyaluronic acid in a urine sample, which serves as a criterion for determining the presence or degree of chronic inflammation, is appropriately determined depending on the subject to be determined, and is determined based on the measured concentration of hyaluronic acid in the urine sample.

The subject to be judged by the judgment method of the present invention is the presence or absence or degree of chronic inflammation. Among them, particularly preferable judgment objects are the degree of progression of the disease state of rheumatism, the degree of cartilage destruction of osteoarthritis, and the degree of viral liver disease. The presence or absence of the disease. That is, the determination method of the present invention includes a method of determining the degree of progression of the disease state of rheumatism (the method of determining rheumatism of the present invention) and the degree of cartilage destruction of osteoarthritis, which comprises measuring the concentration of hyaluronic acid in a urine sample. (The method for determining arthropathy of the present invention) and the method for determining the presence or absence of viral liver disease (the method for determining liver disease of the present invention). The rheumatism to be determined by the rheumatism determination method of the present invention is preferably rheumatoid arthritis. The viral liver disease to be determined by the method for determining liver disease of the present invention is preferably hepatitis C.

The term "hepatitis C" also includes "chronic active hepatitis C" and "chronic inactive hepatitis C".

(2) Kit of the Present Invention The kit of the present invention is a kit used in the determination method of the present invention, which comprises at least a hyaluronic acid-binding protein and a labeled hyaluronic acid. More preferably,
A kit for use in the method for judging rheumatism of the present invention, the method for judging arthrosis of the present invention, or the method for judging a hepatic disease of the present invention, comprising at least a hyaluronic acid-binding protein and labeled hyaluronic acid.

The subject of use of the kit of the present invention is the method of determining the present invention, the method of determining rheumatism of the present invention, the method of determining arthrosis of the present invention, and the method of determining liver disease of the present invention, and these are described in detail in (1) above. It is.

The kit of the present invention contains at least a hyaluronic acid-binding protein and a labeled hyaluronic acid as constituent reagents. The hyaluronic acid-binding protein and the labeled hyaluronic acid that can be used here are as described in the above (1).

The hyaluronic acid-binding protein is preferably fixed on a solid phase. The solid phase, the fixing method, and the like that can be used here are as described in the above (1).

As the constituent reagents of the kit of the present invention, a standard solution of hyaluronic acid of various concentrations for preparing a calibration curve (preferably having an average molecular weight of 2000 to 40,000,
More preferably, the average molecular weight is 200,000 to 30,000.
And a reagent for detecting a labeling substance. Further, in addition to these constituent reagents, a blocking substance, a washing solution, a sample diluent, an enzyme reaction stop solution, and the like may be contained.

These constituent reagents can be stored in separate containers, respectively, and stored as a kit that can be used according to the determination method of the present invention at the time of use. If necessary, a kit for measuring other components such as creatinine contained in the urine sample may be included for use in correcting the hyaluronic acid concentration in the urine sample.

According to the kit of the present invention, it is possible to measure the concentration of hyaluronic acid in a urine sample with extremely high sensitivity (it can measure a very small amount of hyaluronic acid of 0.1 ng / ml), easily and with good reproducibility. it can. This makes it possible to determine the presence or absence or degree of chronic inflammation with high sensitivity, simply and with good reproducibility, and to accurately grasp chronic inflammation or its degree. That is, the kit of the present invention can also be provided as a diagnostic agent or a diagnostic kit for examining chronic inflammation or its degree.

[0047]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In addition, in Examples 4-6, all were implemented with respect to the person or the patient who has no renal disorder / disease.

[0048]

[Preparation Example 1] (Preparation of hyaluronic acid-binding protein) A hyaluronic acid-binding protein was prepared according to the production example (hyaluronic acid-binding protein) described in JP-A-4-262797. That is, the bovine nasal septum cartilage was subjected to an extraction operation using a 4M guanidine hydrochloride solution, a supernatant of the extract was collected, dialyzed with deionized water, and lyophilized to obtain a crude extract. . This crude extract was digested with trypsin and then purified by affinity chromatography using a hyaluronic acid-binding resin.

[0049]

Preparation Example 2 (Preparation of Biotin-Labeled Hyaluronic Acid) Hyaluronic acid (average molecular weight: 40,000) was dissolved in 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.5). To 1 ml of the 10 mg / ml solution, add Biotin
-Solution 2 in which LC-Hydrazide (trade name, manufactured by Pierce) was dissolved in dimethyl sulfoxide to 50 mM.
5 μl was added, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was further added to 0.1 M
Dissolved in MES buffer (pH 5.5) at 100 mg /
12.5 μl of the resulting solution was added thereto, and reacted at 20 ° C. for 16 hours with stirring.

Thereafter, dialysis was carried out at 20 ° C. for 2 hours using a dialysis membrane having a molecular weight of 3500 cuts and distilled water as an external solution for dialysis. Thereafter, the dialysis solution was exchanged, and dialysis was performed twice more.

Thereafter, the dialysis solution containing biotin-labeled hyaluronic acid was adjusted to 5 mg / ml with distilled water and stored at -20 ° C.

[0052]

[Comparative example]

(Measurement of Hyaluronic Acid Concentration by Sandwich Method) Using a commercially available kit for measuring hyaluronic acid (trade name: Hyaluronic acid "Chugai", manufactured by Chugai Pharmaceutical Co., Ltd .; kit for measuring high molecular weight hyaluronic acid by the sandwich method) The concentration was measured.

As samples, the average molecular weight was 3,000, 30,000, 30
10,000, 800,000 and 2,000,000 hyaluronic acid are dissolved in the reaction buffer attached to the kit to prepare a 50-1000 ng / ml solution of each molecular weight of hyaluronic acid, and measured according to the protocol described in the package insert of the kit. did. In addition,
The package insert also states that the coefficient of variation (CV value) of the measured value when the control serum is measured 10 times simultaneously as a sample is 15% or less, and the measurement range is 10 to 10.
It is described as 800 ng / ml.

FIG. 1 shows the obtained results. From these results, a commercially available hyaluronic acid measurement kit (sandwich method)
According to the measurement by the method described above, the quantitativeness of hyaluronic acid having a molecular weight of 30,000 or less was extremely low.

[0055]

Example 1 (Measurement of low-molecular-weight hyaluronic acid concentration) The solution of the hyaluronic acid-binding protein purified in Preparation Example 1 was diluted to 800 ng / ml with a sodium carbonate solution, and 50 μl of the diluted solution was added to a Nunc immunoplate. Add 1 well at 4 ° C to each well
Coating was uniform by storage for 6 hours.

Next, the plate was washed twice with 200 μl of phosphate buffered saline (PBS), and PBS (−) containing 3% bovine serum albumin (BSA) (available from Seikagaku Corporation) as a blocking substance was used. 200 μl of the solution was added to each well and allowed to stand at room temperature for 2 hours.

Next, the plate was washed three times with 200 μl of a washing solution (PBS containing 0.05% Tween 20),
A hyaluronic acid standard solution (0.1 to 1000 ng / pH) prepared using a sample diluent (PBS (-) containing 1% BSA)
ml of each concentration; average molecular weight of 10,000) and a sample diluent as a control, 50 μl of each solution was added to the plate, and reacted at 37 ° C. for 60 minutes.

Thereafter, 10 μl of a solution prepared by dissolving the biotin-labeled hyaluronic acid of Preparation Example 2 in a sample diluent to a concentration of 200 ng / ml was added to each well and reacted at 37 ° C. for 60 minutes.

Then, after washing three times with 200 μl of a washing solution,
A solution obtained by diluting peroxidase-conjugated streptavidin (manufactured by Seikagaku Corporation) 5000 times with a sample diluent was added in 50 μl portions, and reacted at 37 ° C. for 30 minutes.

Thereafter, the plate was washed three times with a washing solution, and 50 μl of a tetramethylbenzidine (TMB) solution (manufactured by Moss) was added thereto, followed by reaction at 37 ° C. for 15 minutes to develop color. After color development, the reaction was stopped by adding 50 μl of 1 M hydrochloric acid, and TMB was added.
The absorbance at a wavelength of 450 nm of the colored liquid due to the decomposition of was measured using a well reader SK601 (available from Seikagaku Corporation). FIG. 2 shows the obtained results.

Thus, according to the method for measuring hyaluronic acid using a competitive reaction for hyaluronic acid-binding protein, low-molecular-weight hyaluronic acid (average molecular weight 10,000) in the range of 0.1 to 1000 ng / ml can be quantified with high sensitivity. Has been shown to be possible.

[0062]

Example 2 (Measurement of Concentration of Hyaluronic Acid of Various Molecular Weights) Example 1
The relationship between the molecular weight of hyaluronic acid and the quantitativeness was examined in the method for measuring hyaluronic acid described in (1). Tetrasaccharide,
0.1 to 1000 ng / ml for hexasaccharide, octasaccharide, decasaccharide (molecular weight 2,000) and hyaluronic acid of dodecasaccharide, and hyaluronic acid having an average molecular weight of 10,000, 40,000, 80,000, 380,000 and 1,000,000.
Was examined for each concentration. The results are shown in FIGS.

From these results, it can be seen that in the present measurement method, 0.1 to 100
In the range of 0 ng / ml, low molecular weight hyaluronic acid, particularly hyaluronic acid having an average molecular weight of 40,000 to 10 sugars (average molecular weight of 2000) can be quantified with high sensitivity, and the measurement of the concentration of hyaluronic acid contained in a urine sample can be performed. Was shown to be a very preferred method.

[0064]

Example 3 (Evaluation of reproducibility) In the method of measuring hyaluronic acid described in Example 1, when a urine sample having a hyaluronic acid concentration of 254 ng / ml was measured simultaneously six times, the coefficient of variation (CV value) was 4 %, Indicating that this method is extremely excellent in reproducibility.

[0065]

Example 4 (Measurement of Urinary Hyaluronic Acid Concentration in Healthy Subjects and Patients with Chronic Inflammation) Healthy subjects (19 subjects), rheumatoid arthritis (hereinafter abbreviated as RA) patients (107 subjects), viral liver disease (type C) Hepatitis) patients (in Table 1, simply referred to as “liver disease patients”; 31)
The concentration of hyaluronic acid present in each urine sample in Example) was measured by the method for measuring hyaluronic acid described in Example 1. The creatinine concentration in each urine sample was measured using a commercially available kit (Wako Pure Chemical Industries, Ltd.), and the results are shown in Table 1 as hyaluronic acid concentration corrected by the creatinine concentration. Compared with healthy individuals, the average value of hyaluronic acid concentration in urine samples of RA patients is about 3.6 times, and about 2.6 in viral liver disease patients.
It was twice as high.

In addition, a patient whose hyaluronic acid concentration (corrected by creatinine concentration) in a urine specimen was “positive” than “normal (average value + 2 SD)” was determined as “positive”, and the positive rate was examined for each disease. . As a result, RA patients showed a high positive rate of 87.9%, and viral liver disease patients showed a high positive rate of 75.0%. From these results, it was shown that the above-mentioned disease can be determined by measuring the concentration of hyaluronic acid in a urine sample.

[0067]

[Table 1] 数 Subjects Number of subjects Age Hyaluronic acid concentration (ng / mg creatinine) Positive rate (%) (mean ± SD) (mean + 2SD <) ────────────────────────────── ────── Healthy person 19 36.5 ± 5.0 383 ± 80 − RA patient 107 58.9 ± 10.8 1379 ± 993 87.9 Liver disease patient 31 59.9 ± 11.5 978 ± 1206 75.0 ────── ───────────────────────

[0068]

Example 5 (Relationship between Urinary Hyaluronic Acid Concentration and RA Disease Stage) In RA patients without viral liver disease, RA disease stage and hyaluronic acid concentration in urine specimen (corrected by creatinine concentration) Was examined by the method for measuring hyaluronic acid described in Example 1. FIG. 5 shows the results. In FIG. 5, "*" indicates that there is a significant difference at the risk rate of 0.01 or less (p <0.01), and "**" indicates that the risk rate is 0.001 or less (p <0.001).
Indicates a significant difference. In addition, the identification of the stage of RA pathological condition is described in “Steinbrocker et al.
Class classification "(see" Treatment for rheumatoid arthritis ", p54-55, Nankodo Co., Ltd. (1988), etc.), and a doctor with experience in diagnosing RA was performed. "Stein brokers clas
In the “s classification”, in stages 1 to 4, the larger the number is, the more the disease state of rheumatism is progressing.

As a result, it was shown that the higher the pathological stage of RA, the higher the concentration of hyaluronic acid in the urine sample. In addition, the RA patient in stage 1 had a significantly increased hyaluronic acid concentration in urine specimens at a risk factor of 0.01 or less compared to healthy individuals. In addition, the stage 3 patients and the stage 4 patients have a risk factor of 0.
Hyaluronic acid concentrations in urine samples were significantly increased below 001 and below 0.01. From these results, it was shown that by measuring the concentration of hyaluronic acid in the urine sample, it was possible to determine the degree of progression of the disease state of a rheumatic patient. In particular, it was shown that each of the stages 1 to 4 in the “Stein brokers' class classification” can be determined with high sensitivity. It was also shown that the stages 1 and 2 and the stages 3 and 4 can be clearly distinguished.

[0070]

Example 6 (Relation between urinary hyaluronic acid concentration and degree of cartilage destruction in osteoarthritis) In patients with osteoarthritis (hereinafter abbreviated as OA) without viral liver disease, the degree of OA cartilage destruction And the hyaluronic acid concentration in the urine sample (corrected with the creatinine concentration) was examined by the hyaluronic acid measurement method described in Example 1. In addition, identification of the degree of cartilage destruction
Based on the X-ray image, a physician experienced in the diagnosis of OA was performed. Those with a low degree of cartilage destruction were identified as Stage 1, those with a moderate degree were identified as Stage 2, and those with a high degree of cartilage destruction were identified as Stage 3.

[Mean value ± SD] of hyaluronic acid concentration (corrected by creatinine concentration) in urine samples of healthy subjects (19)
Is 383 ± 80 (ng / mg creatinine), and the concentration of hyaluronic acid in the urine sample (corrected by the creatinine concentration) is
Patients larger than (mean value + 2 SD) were determined to be "positive," and the positive rate was checked for each stage.

As a result, the positive rate in stage 1 patients (3) was 33.3%, the positive rate in stage 2 patients (5) was 80.0%, and the positive rate in stage 3 patients (14) was 85.7%. Met.

The results show that the higher the stage of OA cartilage destruction, the higher the concentration of hyaluronic acid in the urine sample and the higher the positive rate. In addition, the hyaluronic acid concentration in the urine sample was significantly increased in stage 3 OA patients at a risk factor of 0.001 or less compared to healthy individuals. from this result,
It was shown that the degree of cartilage destruction in osteoarthritis patients can be determined by measuring the concentration of hyaluronic acid in urine samples.

[0074]

Example 7 (Relationship between Serum Hyaluronic Acid Concentration and Urinary Hyaluronic Acid Concentration) In patients with viral liver disease, serum hyaluronic acid concentration measured by a conventional method (method of comparative example)
Hyaluronic acid concentrations in urine samples measured by the hyaluronic acid measurement method described in Example 1 were compared. As a result, it was found that there was a relatively high correlation between the two (correlation coefficient: 0.87).

[0075]

Example 8 (Kit of the Present Invention) The kit of the present invention comprises the following. 1. 1. One microplate with immobilized hyaluronic acid binding protein 2. Biotin-labeled hyaluronic acid Standard hyaluronic acid solution (average molecular weight 10,000) 0.1ng / ml 0.5ml Lyophilized product 1 bottle 0.5ng / ml 0.5ml Lyophilized product 1 1.0ng / ml 0.5ml Lyophilized product 1 5.0ng / ml 0.5ml Freeze Dried product 1 bottle 10.0ng / ml 0.5ml Lyophilized product 1 bottle 50.0ng / ml 0.5ml Lyophilized product 1 100.0ng / ml 0.5ml Lyophilized product 1 500.0ng / ml 0.5ml Lyophilized product 1 1000.0ng 3./ml 0.5 ml freeze-dried product 1 (All were prepared using PBS (-) containing 1% BSA and then freeze-dried.) Peroxidase-conjugated streptavidin solution 10
ml 1 bottle 5. TMB (tetramethylbenzidine) solution 10ml 1
Book 6. Stock solution of sample diluent (5-fold concentration of PB containing 5% BSA)
S (-)) 40ml 1 bottle (diluted 5 times with pure water for use) Wash solution (PBS containing 0.05% Tween 20)
40ml 1 bottle 8. Color reaction stop solution (1M HCl) 10ml 1 bottle

[0076]

By using the method of the present invention and the kit of the present invention, the presence or absence or degree of chronic inflammation can be safely and inexpensively determined without causing pain to the patient and avoiding the danger of radiation exposure and infection. Thus, the determination can be made simply, quickly and with high sensitivity.

[Brief description of the drawings]

FIG. 1 shows the relationship between the molecular weight of hyaluronic acid and the reactivity in the measurement method of Comparative Example.

FIG. 2 shows a standard curve for the determination of hyaluronic acid in the measurement method of Example 1.

FIG. 3 shows the relationship between the molecular weight of hyaluronic acid and the reactivity in the measurement method of Example 1.

FIG. 4 shows the relationship between the molecular weight of hyaluronic acid and reactivity in the measurement method of Example 1.

FIG. 5 shows the relationship between the stage classification of RA pathological conditions and the concentration of hyaluronic acid in urine samples.

Continued on the front page (72) Inventor Tsuyoshi Ishimaru 3-6-6 Nakahara, Higashiyamato-shi, Tokyo 203 Amenity Nakahara

Claims (7)

[Claims] 1. A method for determining the presence or degree of chronic inflammation, which comprises measuring the concentration of hyaluronic acid in a urine sample. 2. A method for determining the degree of progression of a disease state of rheumatism, comprising measuring the concentration of hyaluronic acid in a urine sample. 3. A method for determining the degree of cartilage destruction in osteoarthritis, comprising measuring the concentration of hyaluronic acid in a urine sample. 4. A method for determining the presence or absence of a viral liver disease, comprising measuring the concentration of hyaluronic acid in a urine sample. 5. A hyaluronic acid in a urine sample and a hyaluronic acid labeled with a labeling substance are subjected to a competitive reaction with a hyaluronic acid-binding protein, and labeled hyaluronic acid bound to the protein or unlabeled hyaluronic acid. The method according to any one of claims 1 to 4, wherein the concentration of hyaluronic acid in the urine sample is measured by measuring any of hyaluronic acid. 6. The kit for use in the determination method according to claim 1, comprising at least a hyaluronic acid-binding protein and hyaluronic acid labeled with a labeling substance. 7. The kit according to claim 6, wherein the hyaluronic acid binding protein is fixed on a solid phase.