JP2009257842A - Examination method of osteoarthritis and diagnostic kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel examination method of osteoarthritis (OA). <P>SOLUTION: The examination method of osteoarthritis is characterized by performing discrimination analysis respectively using the measured values of C2C, CTX-II, CPII, NTx and hyaluronic acid obtained from a subject and a C2C/CPII ratio as variables. This diagnostic kit of osteoarthritis is characterized by performing discrimination analysis respectively using the measured values of C2C, CTX-II CPII, NTx and hyaluronic acid obtained from a subject and the C2C/CPII ratio as variables. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for testing osteoarthritis and a diagnostic kit.

Osteoarthritis (hereinafter also referred to as “OA”) is a joint disease with chronic arthritis, and is a disease that causes destruction of cartilage and proliferative changes in bone and cartilage due to degenerative degeneration of joint components. . The number of patients increases with age, and at the age of 60 years or older, it is said that OA symptoms are observed in knees, elbows, hip joints, and vertebrae, with more than 80% of the degree being different.
Currently, treatment of OA is centered on coping therapy that suppresses pain, and therefore development of a treatment method that suppresses degenerative changes in joints is required. On the other hand, various diagnosis methods are required from the viewpoint of early diagnosis. It is being considered.

As a method of grasping the state of the joint, morphological diagnosis such as visual inspection and X-ray examination is common in addition to interrogation, but since these cannot be determined that the disease does not progress to some extent, other diagnostic methods are proposed Has been. For example, focusing on the cartilage matrix components that have migrated into the joint fluid due to the decomposition of the cartilage matrix, normal aggrecan that has the ability to bind hyaluronic acid is quantified, and normal joints and pathological joints are related to other proteoglycans. (Patent document 1), focusing on a gene whose expression is significantly increased in osteoarthritic cartilage, and using the protein or DNA as a diagnostic marker for arthritis (patent document 2), Runx3 gene and Diagnosis of joint diseases including the step of analyzing bone-joint related diseases such as OA or diagnostic agents (Patent Document 3), analyzing N-linked glycoprotein sugar chains of articular cartilage by analyzing protein expression and A method (Patent Document 4) for providing useful information in treatment has been proposed.
PCT International Publication No. WO95 / 22765 JP 2004-187680 A JP 2005-204557 A JP 2007-205777 A

  The pathological evaluation of OA is usually performed using scores such as JOA, VAS, and WOMAC. These scores are excellent for assessing pain and symptoms, but may depend on patient subjectivity. On the other hand, hyaluronic acid, aggrecan, and the like are used as an index for objectively evaluating the pathological condition of OA, but these substances are not necessarily reflected on cartilage metabolism because they are secreted from tissues other than cartilage.

  The present invention provides a novel OA testing method and diagnostic kit characterized by performing discriminant analysis using a plurality of specific biomarkers as variables, which have been obtained in the course of OA research. The task is to do.

As a result of intensive studies, the present inventors have made a discriminant analysis using six biomarkers obtained from a subject: C2C, CTX-II, CPII, NTx, hyaluronic acid, and C2C / CPII ratio as variables. As a result, it was found that the OA pathological condition can be easily and objectively evaluated with good accuracy, and the present invention has been completed.
That is, the present invention
(1) A deformable joint characterized by performing discriminant analysis using measured values of C2C, CTX-II, CPII, NTx, and hyaluronic acid obtained from a subject, and C2C / CPII ratio as variables, respectively. (2) C2C, CTX-II, CPII, NTx, and hyaluronic acid measured values obtained from a subject, and C2C / CPII ratio are used as variables, respectively, and discriminant analysis is performed. And an osteoarthritis diagnostic kit.

  According to the present invention, compared to conventional pathological evaluation using pain and X-ray findings, pathological evaluation of OA can be performed easily, objectively, and with good accuracy. That is, in addition to the determination of whether or not OA is affected, it can be used for pathological evaluation such as staging and malignancy classification of OA.

Biomarkers used in the present invention include cartilage destruction markers C2C and CTX-II, cartilage synthesis marker CPII, bone metabolism marker type I collagen cross-linked N-terminal peptide (NTx), and measured values of hyaluron, And 6 items of the C2C / CPII ratio.
In the present invention, for example, a biological sample (serum, urine, etc.) is collected from a subject by a conventional technique, and biomarkers existing in the biological sample: C2C, CTX-II, CPII, NTx, and hyaluronic acid are conventionally used. These biomarkers may be obtained by measurement by an immunological method such as the enzyme antibody method (Enzyme Linked Immunosorbent Assay). Then, the C2C / CPII ratio is obtained by calculation from the measured values of C2C and CPII thus obtained.

  In the present invention, discriminant analysis is performed using the six biomarker items obtained from the subject as described above as variables. Discriminant analysis is a method of multivariate analysis that analyzes which group an individual belongs to by disassembling multiple pieces of information of a given individual into several elements and weighting the elements. Widely used. In the present invention, this discriminant analysis is used to examine the OA of the subject.

The inspection of the present invention may be performed as follows, for example.
First, for the subjects whose OA pathology has been evaluated, for example, grouped according to staging or malignancy classification, the above-described 6 biomarkers are acquired, and a rule for identifying the groups is constructed based on this. . Next, the rule is applied to a new subject, and the group to which the subject belongs is determined. As a result, it has been possible to easily evaluate OA pathological conditions such as staging or malignancy classification of a subject, which has been conventionally performed by a complicated method, by discriminant analysis using six forward lean biomarkers. Can be done.

  In the present invention, the subject refers to a human or a mammal that is evaluated for the pathological condition of OA. Examples of mammals include pets such as dogs and cats and livestock such as cows and horses. The subject should be the same species as the animal species used in constructing the rules. For example, if the subject is a human, the rules should be constructed in humans.

  Osteoarthritis examined or diagnosed in the present invention is not particularly limited in its onset site, and can occur in knee joints, hip joints, elbow joints, finger joints, ankle joints, cervical vertebrae, spine, lumbar vertebrae, and the like. The onset site is not limited to one, and can develop at multiple sites. Examples of osteoarthritis include knee osteoarthritis, lumbar spondylosis, hip osteoarthritis, and elbow arthropathy. From the viewpoint that the pathological condition of OA can be evaluated with good accuracy by the method of the present invention, osteoarthritis of the knee, lumbar spondylosis, and hip osteoarthritis are preferable, and knee osteoarthritis is particularly preferable.

  The diagnostic kit according to the present invention includes a reagent for measuring a biomarker by the measurement method described above. The diagnostic kit of the present invention may include a biological sample collection tool, a preparation tool, a biomarker measurement tool, such as a column cartridge. Further, the diagnostic kit of the present invention records discriminant analysis means, for example, a document or manual describing the procedure of discriminant analysis, a program for causing a computer to execute the discriminant analysis procedure, the program list, and the program. A computer-readable recording medium (for example, a flexible disk, an optical disk, a CD-ROM, a CD-R, and a CD-RW), a device or a system (such as a computer) that performs discriminant analysis may be included.

  EXAMPLES Hereinafter, although an Example etc. are given and this invention is demonstrated in more detail concretely, this invention is not limited to these.

1. Purpose of the test The subject was diagnosed by a doctor, OA patient (pain in the knee and obvious deformation by X-ray), Pre-OA (1) (pain in the knee and deformation by X-ray) 4 groups: Pre-OA (2) (no pain in the knee, obvious deformation by X-ray), and healthy people (no pain in the knee, no deformation by X-ray) (Table 1), each group was subjected to discriminant analysis using a biomarker. The presence or absence of knee pain was determined by the subject's self-report.

2. Table 2 shows the types of biomarkers used in the test and items to be marked. First, serum and urine were obtained from a subject by blood collection and urine collection, respectively. In the obtained serum, C2C is a measurement kit sold by Seikagaku Biobusiness, CPII is a measurement kit sold by Seikagaku Biobusiness, and NTx is a measurement kit sold by Mochida Pharmaceutical Co., Ltd. (Osteomark NTx Serum , Manufactured by WAMPOLE), and hyaluronic acid was measured with a measurement kit (Elpiace A HA, manufactured by Mitsubishi Chemical Yatron) sold by Fujirebio Inc., respectively. In the obtained urine, CTX-II was measured with a measuring kit sold by Sumisho Pharma Co., Ltd., and creatinine was measured with a measuring kit manufactured by Daiichi Chemicals Co., Ltd. (Pure Auto S CRE-L), and creatinine was measured. Corrections were made to obtain CTX-II measurements. The C2C / CPII ratio was calculated from the measured values of C2C and CPII thus obtained (Table 2).

3. Discriminant analysis (1)
(A) Subject A person who is likely to appear OA was selected as a subject. Healthy individuals are 10 males and 15 females aged 58 to 80 years (average 68.6 ± 5.8 years), those who have pain in the knee but do not deform (Pre-OA (1)) are 40 to 40 years old Four men and seven women aged 76 years (average 62.2 ± 10.4 years), those with no knee pain but deformed (Pre-OA (2)) age 59-81 years (average 68.68). (2 ± 7.4 years old) 1 male and 5 females, OA patients were 48-87 years old (average 74.3 ± 9.8 years old) 7 males and 14 females (Table 3) .

(A) Results Discriminant analysis was performed on four groups of subjects using six biomarker items “C2C, CPII, C2C / CPII ratio, CTX-II, NTx, hyaluronic acid” as variables. Discriminant analysis was performed using the SPSS discriminant analysis program. Specifically, according to Analysis Example 1 (pages 248 to 257) described in “Statistical Data Analysis by SPSS” (Hario Yanai, Hiromitsu Ogata, second edition issued July 3, 2007, Hyundai Mathematics, Kyoto) I went.
The classification results are shown in Table 4. As a result of discriminant analysis from the viewpoint of the declaration of pain and deformation of the subject and the marker, 71.4% were correctly classified, but 28.6% could not be correctly classified. The reason is that, as shown in the canonical discriminant function in FIG. 1, those who did not have pain in the knee but had deformation (Pre-OA (2)) and the healthy people almost matched. Therefore, discriminant analysis was performed again with the two groups as the same group.

4). Discriminant analysis (2)
(A) Grouping As described above, the subjects of 4 groups were changed to 3 groups. Pre-OA (2) (no pain in the knee, apparent deformation by X-ray) and normal subjects (no pain in the knee, no deformation by X-ray) were grouped together. That is, patients with OA (pain in the knee and obvious deformation by X-ray), Pre-OA (pain in the knee and no deformation by X-ray), and those without pain (in the knee) There was no pain and it was divided into 3 groups (table 5).

(A) Subject Eleven persons who have no pain in the knee are 58 to 81 years old (average 68.5 ± 6.0 years old) and 20 females, who have pain in the knee but do not deform (Pre -OA) are 4 males and 7 females aged 40-76 years (average 62.2 ± 10.4 years), and OA patients are males 48-87 years old (average 74.3 ± 9.8 years) There were 7 and 14 women (Table 6).

(C) Results of discriminant analysis The above-mentioned discriminant analysis (1) was performed on three groups of subjects using six biomarkers, “C2C, CPII, C2C / CPII ratio, CTX-II, NTx, hyaluronic acid” as variables. Discriminant analysis was performed as shown in the figure. As a result, it was found that 92.1% was correctly classified as a result of discriminant analysis from the viewpoint of the declaration of pain of the subject, the presence or absence of deformation and the marker (Table 7, FIG. 2).

The method and diagnostic kit of the present invention can be used for OA discrimination, pathological evaluation, and diagnosis.
The method of the present invention can also be used for screening for a therapeutic or prophylactic agent for osteoarthritis.

It is a figure which shows a canonical discriminant function. It is a figure which shows a canonical discriminant function.

Claims (4)

  A test for osteoarthritis, characterized by performing discriminant analysis using measured values of C2C, CTX-II, CPII, NTx, and hyaluronic acid obtained from a subject, and C2C / CPII ratio as variables, respectively. Method.   The method according to claim 1, wherein the osteoarthritis is knee osteoarthritis, hip osteoarthritis, or lumbar spondylosis.   For diagnosis of osteoarthritis, characterized in that discriminant analysis is performed using measured values of C2C, CTX-II, CPII, NTx, and hyaluronic acid obtained from a subject, and C2C / CPII ratio as variables, respectively. kit.   The diagnostic kit according to claim 3, wherein the osteoarthritis is osteoarthritis of the knee, hip osteoarthritis, or lumbar spondylosis.

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