JP4048293B2 - Diagnosis of rheumatism and diagnostic agent for rheumatism - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the diagnosis of rheumatism.
[0002]
[Prior art]
Rheumatoid arthritis (RA) is an unexplained destructive and progressive inflammatory disease whose main symptoms are multiple joint pain and joint swelling. Initially, it is synovitis, but the inflammation repeatedly relieves and worsens, destroying the articular cartilage and bone tissue, resulting in joint deformation and dysfunction. RA is generally very well known, and despite the large number of patients, it still meets four or more of the following American College of Rheumatology diagnostic criteria (1987, American College of Rheumatology) to make a definitive diagnosis: There is a need. 1. Morning stiffness (morning stiffness lasts at least 1 hour). 2.3 Arthritis over 3 joint areas (swelling of soft tissue or pooling of joint fluid in at least 3 joint areas). 3. Hand arthritis (swelling in one joint area of the hand). 4). Symmetric arthritis. 5. Rheumatoid nodule. 6). Serum rheumatoid factor positive. 7). Change in X-ray image. However, an X-ray change, which is one of diagnostic criteria, appears to take about half a year for adults after the appearance of arthritis with swelling in the joint, and is difficult as a material for actual early diagnosis. The diagnostic criteria of 1-4 also have conditions such as symptoms lasting for 6 weeks or more, and it usually takes time until diagnosis after arthritis occurs.
RA is refractory, and joint destruction and dysfunction are prominent, so it is well-known and many patients visit orthopedics because they are concerned about RA. Many people show arthritis but rarely have RA. Therefore, X-ray tests and rheumatoid factor tests for blood show negative results, and they are excluded. However, considering the current diagnostic criteria, this is not a condition that can be completely excluded. In addition, in the case of RA, the importance of early diagnosis and treatment is asserted that active rheumatic drugs should be aggressively treated from an early stage for active symptoms.
[0003]
[Problems to be solved by the invention]
The purpose of this study is to quickly diagnose rheumatism.
[0004]
[Means for Solving the Problems]
Midkine is a growth factor that promotes cell proliferation, migration, and survival (Muramatsu, T., Wiley Encyclopedia Mol. Med., Pp 2086-2088, 2002). An immunochemical assay for midkine has been established (Muramatsu, H., et al., J. Biochem., 119, 1171-1175, 1996). Moreover, it has been reported that the rheumatoid arthritis and osteoarthritis both increase the midkine level in joint fluid (Takeda, T. et. Al., J. Biochem., 122, 453-458, 1997). However, the number of cases was small and the relationship with the pathological condition was unknown, and it was necessary to review it. As a result, this study unexpectedly found that high midkine values characterize rheumatism.
[0005]
That is, the present invention uses a midkine value as a rheumatic marker.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 shows a comparison of midkine levels in the joint fluid of patients with rheumatoid arthritis and osteoarthritis, and FIG. 2 shows a midkine level in serum of RA patients.
[0007]
1. The determination of midkine value will be described. It can be quantified enzymatically using a specific antibody against midkine (Muramatsu, H. et al., J. Biochem., 119, 1171-1175, 1996). In addition, quantification (Takei, Y. et al., Cancer Res., 61, 8486-) after Western blot (Muramatsu, H. et al., Dev. Biol., 159, 392-402, 1993). 8491, 2001). Is also possible.
[0008]
2. The determination of rheumatoid factor will be described. It is judged by whether polystyrene latex particles adsorbed with human γ-globulin react with rheumatoid factor (RF) and show aggregation. It can be measured using a kit called RA77 Eiken sold by Eiken Chemical Co., Ltd.
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[0009]
(Example 1) The midkine value of the joint fluid of patients with rheumatoid arthritis or osteoarthritis was measured. For this purpose, 1 μg of affinity purified anti-human midkine antibody (Muramatsu, H. et al., J. Biochem., 119, 1171-1175, 1996) was dissolved in 50 μl of 50 mM Tris-HCl buffer pH 8.0, Falcon 3915 was placed in a microtiter plate hole and adsorbed on the plate surface. The solution was removed and the wells were washed with phosphate buffered saline without divalent ions [PBS (−)] and washed buffer [100 mM Tris-HCl buffer pH 7.5, 0.4 M NaCl, 0.1% Bovine serum albumin, 0.1% NaN ₃ , 1 mM MgCl ₂ ] was incubated for 1 hour. After removing the washing buffer, a 50 ml joint fluid specimen diluted 2-fold with PBS (−) was added to the well and kept at room temperature for 3 hours. After removing the sample solution, 0.7 ng biotinylated anti-midkine antibody (Muramatsu, H. et al., J. Biochem, 119, 1171-1175) in 50 μl washing buffer was added, overnight at 4 ° C., Reacted. 5 mU of streptavidin β-galactosidase complex (Boehringer Mannheim) was dissolved in 50 μl of washing buffer, added to the well, and allowed to react at room temperature for 1 hour. After removing the solution and washing the wells, 5 μg of 4-methylumbelliferyl β-D-galactoside was added in 50 μl of washing buffer and allowed to react at room temperature. The enzyme reaction was stopped with 200 μl of 0.1 M glycine buffer pH 10.3, and the released 4-methylumbelliferone was measured with a microplate fluorescence reader (Cytoflow II, Biosearch). Based on this measurement value, the midkine value in joint fluid was determined from a calibration curve using standard midkine. When rheumatoid patients were divided into rheumatoid factor positive and negative, midkine levels were high in all positive cases and 55% even in negative cases (FIG. 1). In patients with osteoarthritis, midkine levels were low (FIG. 1). A high midkine level in synovial fluid can be an important factor in the diagnosis of rheumatism.
[0010]
(Example 2) Serum midkine value was measured by the method of [0009] using 0.1 ml of serum. When the cut-off value of a normal person is 300 pg / 0.5 ml (Muramatsu, H. et al., J. Biochem., 119, 1171-1175, 1996), 8 out of 32 patients who are rheumatoid factor negative are positive, Among the rheumatoid factor positive patients, 85 out of 88 patients were positive. In combination with rheumatoid factor measurement in serum, a measurement with high accuracy (higher two values) or high coverage (higher value of either) can be performed. Although there are about 5% of rheumatoid factor positive in healthy people, midkine levels are low in healthy people.
[0011]
[Brief description of the drawings]
FIG. 1 is a comparison diagram of midkine levels in synovial fluid of patients with rheumatoid arthritis and patients with osteoarthritis. % Indicates the percentage of cases with midkine or higher in osteoarthritis. Rheumatoid factor (RF) negative (−), positive (+), and strong positive (++) groups are illustrated.
FIG. 2 is a graph showing the relationship between serum midkine level and serum rheumatoid factor (RF).

Claims (2)

A method for testing rheumatism, comprising measuring midkine in serum collected from a subject immunochemically and further measuring rheumatoid factors. The method according to claim 1 , wherein when the concentration of midkine in serum collected from a subject is 600 pg / mL or more, it is determined to be positive for rheumatism.

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