JPH0526800B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a rheumatoid-specific protein (hereinafter referred to as RP) isolated from rheumatoid plasma such as rheumatoid arthritis. Diagnosis of rheumatism currently relies largely on the doctor's findings and the patient's chief complaint, and the reality is that an objective, quantitative diagnostic method is desired. This diagnostic method generally employs the American Rheumatism Association's diagnostic criteria consisting of 11 items, and rheumatism is diagnosed based on how many of these items are met. One of the standard items is the detection of rheumatoid factor, and because the measurement method is simple, detection of rheumatoid factor is widely performed as a routine test. By the way, rheumatoid factor is found at a high rate in the plasma of rheumatoid patients (depending on the report, rheumatoid factor has been detected in 40-70% of rheumatoid patients), but rheumatoid factor is detected in patients with non-rheumatoid conditions such as liver disease patients and cancer patients. It is frequently found in the plasma of patients with cirrhosis (more than 50% of patients with liver cirrhosis and nearly 20% of cancer patients are reported to be rheumatoid factor positive), and it is also found in 3-7% of healthy people. There is. Thus, the specificity of the presence of rheumatoid factors in rheumatism is low;
A diagnosis of rheumatism cannot be made immediately based on the presence of rheumatoid factor. Even in the diagnostic criteria of the American Rheumatology Association, the presence of rheumatoid factors is not recognized as a necessary or sufficient condition for the diagnosis of rheumatism. In other words, measuring rheumatoid factors has little practical utility in definitively diagnosing rheumatism, and therefore there is a need for parameters for rheumatoid diagnosis that can replace rheumatoid factors. As a result of research on the plasma of many rheumatism patients, patients with other diseases, and healthy people, the present inventors found that
Two-dimensional electrophoresis (hereinafter referred to as RP), which does not use a protein denaturing agent, was used to confirm that a novel protein (RP), which is not observed in the plasma of patients with other diseases or healthy individuals, is expressed at a high rate in the plasma of rheumatoid arthritis patients. 2-dimensional electrophoresis (all without using protein denaturants),
We succeeded in isolating this. According to our research results, RP affects 70-80% of rheumatism patients.
Moreover, it was not observed at all in healthy subjects, patients with renal failure, patients with liver disease, or patients with cancer. The previously known rheumatoid factor is positive in 40-70% of rheumatoid patients, and is also found at a high rate in healthy people, liver disease patients, and cancer patients. The positive rates for both cases are clearly high. Therefore, RP
Create an antibody (RP-Ab) specific to
Detection of RP as a routine clinical test using a diagnostic agent using RP-Ab can be an effective means for diagnosing rheumatism. The obtained RP is necessary for the production of this RP-Ab, and therefore both RP and RP-Ab are extremely useful in rheumatism diagnosis. When RP performs two-dimensional electrophoresis of rheumatoid plasma,
It is a protein with a molecular weight of approximately 170,000 as determined by SDS polyacrylamide gel electrophoresis, with an isoelectric point (pI) of 7.3 to 7.8 and a mobility (assuming the mobility of the leading edge of albumin to 1.0) as spots in the range of 0.40 to 0.55. , its amino acid composition is as follows (unit: mole%). Lys 6.6, His 1.9, Arg 4.0, Asp 7.0,
Thr 8.5, Ser10.0, Glu 10.4, Pro 7.9,
Gly10.4, Ala 4.7, Cys/2 1.0, Val 7.5,
Met 1.6, Ile 2.9, Leu 8.3, Tyr 4.5,
Phe 2.9 In two-dimensional electrophoresis of healthy human plasma, no proteins are observed in the range of isoelectric point (pI) of 7.3 to 7.8 and mobility (assuming the mobility of the leading edge of albumin to 1.0) of 0.40 to 0.55. First, it turns out that RP is a completely new, previously unknown protein found in rheumatoid plasma. Next, we will discuss how to obtain RP. RP is
Rheumatoid plasma obtained by a general method can be separated on a two-dimensional electrophoresis gel by a two-dimensional electrophoresis method in which rheumatoid plasma is first subjected to isoelectric focusing using a polyacrylamide gel, followed by concentration gradient gel electrophoresis. . RP has an isoelectric point (pI) of 7.3 to 7.8 on a two-dimensional electrophoresis gel and a mobility of 0.40 to 0.55 (assuming the mobility of the leading edge of albumin to be 1.0). It can be isolated by dividing and extracting with a buffer or the like. RP isolated at this time
The molecular weight of is approximately 170,000 as determined by SDS polyacrylamide gel electrophoresis. In addition, in order to isolate a large amount of RP, the precipitate salted out with 25-35% saturated ammonium sulfate from rheumatoid patient plasma should be prepared using DEAE
Perform ion exchange chromatography using an ion exchange gel such as 50 (manufactured by Pharmacia, Sweden), and collect the fraction eluted with 0.5 M sodium chloride. Furthermore, this fraction can be isolated by performing gel chromatography using a gel such as Sephacryl S-300 (manufactured by Pharmacia, Sweden) and then performing ion exchange chromatography using the above-mentioned ion exchange gel. When subjected to two-dimensional electrophoresis, the isolated RP has an isoelectric point (pI) of 7.3 to 7.8 and a mobility (assuming the mobility of the leading edge of albumin to 1.0) of 0.40 to 0.55.
It has a molecular weight of about 170,000 when measured by SDS polyacrylamide gel electrophoresis. RP-specific antibody RP-Ab can be purified, e.g.
It can be obtained by a general method for producing specific antibodies, which involves immunizing an immunizable animal with RP together with Freund's complete adjuvant, etc., and collecting and purifying antiserum from the immunized animal. For purification of antibodies from antiserum, for example, the antigen RP is bound to a gel such as Sepharose 4B (manufactured by Pharmacia, Sweden) using a ligand such as cyanogen bromide, and
Antiserum is passed through a column filled with the gel, and after thorough washing, the antibody is separated from the column using a glycine buffer, etc., using an immunoadsorption method or the like. The antibody RP-Ab obtained in this way can be confirmed to specifically react with purified RP to produce a precipitation line using general specific antibody confirmation methods such as immunoelectrophoresis and immunoprecipitation reaction. Can be confirmed. Measurement of RP in plasma using RP-Ab can be performed using currently commonly used antigen measurement methods using specific antibodies, such as the double immunodiffusion method using a gel such as agar, or the method of measuring RP in latex or red blood cells. This can be done by various agglutination reactions performed by immobilizing -Ab, or by immunoassay using enzymes, radioisotopes, etc. by adsorbing RP-Ab onto a solid phase such as beads or microplates. RP can be easily measured using RP-Ab by such a measurement method, and therefore RP-Ab is important as a routine test method for rheumatism diagnosis. Example <Method for isolating RP> A precipitate precipitated with 25% saturated ammonium sulfate from excreted plasma generated by plasma exchange in a rheumatoid arthritis patient with RP was collected at 8000 x g.
The supernatant was removed by centrifugation for 20 minutes, ammonium sulfate was further added to the supernatant, and the precipitate precipitated with 35% saturated ammonium sulfate was collected by centrifugation at 8000 xg for 20 minutes. This precipitate was dissolved in 0.02M phosphate buffer (PH7.2, hereinafter referred to as PBNa) and dialyzed against deionized water through a cellophane membrane to remove ammonium sulfate. Ion exchange chromatography was performed using an A-50 (manufactured by Pharmacia, Sweden) column. It was confirmed by two-dimensional electrophoresis that RP was contained in the fraction eluted with 0.5M sodium chloride solution,
This fraction was collected. The collected fractions were dialyzed twice against deionized water through a cellophane membrane to remove sodium chloride, concentrated by freeze-drying, and then subjected to gel chromatography using a Sephacryl S-300 (Pharmacia, Sweden) column. By collecting fractions corresponding to the fractional positions where RP was confirmed by two-dimensional electrophoresis, we separated substances whose molecular sieve behavior differs from the contaminated RP by ion-exchange chromatography. . After this, DEAE A-50 again
Separation based on ionic strength was further performed by ion exchange chromatography using a column (manufactured by Pharmacia, Sweden), and eluted with a concentration gradient of 0.0M to 0.6M sodium chloride.
Fractions corresponding to the fractional positions where RP was confirmed by dimensional electrophoresis were collected to obtain purified RP. Using this purified RP as a sample, perform the following two steps.
This was confirmed by dimensional electrophoresis. refined
Add 60μl of RP (protein concentration 0.40mg/ml) to Ampholine (manufactured by Pharmacia, 6.25w/v).
% (PH3.5-10:PH3.5-5=4:1)] on a polyacrylamide gel containing 0.01M phosphoric acid solution.
Using 0.04N sodium hydroxide solution, electrophoresis was performed at a constant current (2 mA) at first, and after the voltage reached 220 V, electrophoresis was performed at a constant voltage of 220 V for 20 hours. next,
This running gel was placed on a polyacrylamide slab gel with a concentration gradient of 4% to 17% and run at constant current (24 mA) for 20 hours. A 0.05M Tris-0.384M glycine buffer was used as the electrophoresis solution at this time. After two-dimensional electrophoresis as described above, 0.025% Coomersie Brilliant Blue (Coomersie Brilliant Blue R-250), 50v/v
% methanol, 7% acetic acid solution for 8 hours, 7%
FIG. 1 shows an electrophoretic image obtained by decolorizing with acetic acid and 10% methanol solution for 3 days. The electrophoresis image has an isoelectric point of 7.3 to 7.8,
It was confirmed that it was RP because the mobility was within the range of 0.40 to 0.55 (assuming the mobility of the leading edge of albumin to be 1.0). Also, the RP obtained at this time is SDS
The molecular weight was determined to be approximately 170,000 by polyacrylamide gel electrophoresis. For reference, two-dimensional electrophoresis images of plasma from patients with rheumatoid arthritis and plasma from healthy individuals are shown in FIGS. 2 and 3, respectively. In a two-dimensional electrophoresis image of plasma from a rheumatoid arthritis patient, spots of RP are seen within the range of isoelectric point 7.3 to 7.8 and mobility (assuming the mobility of the leading edge of albumin is 1.0) 0.40 to 0.55 (dashed line). ). However, in a two-dimensional electrophoretic image of plasma from a healthy person, there are not only RP but also protein spots within the isoelectric point of 7.3 to 7.8 and mobility (assuming the mobility of the leading edge of albumin to 1.0) of 0.40 to 0.55. is not accepted at all. Purified RP has maximum absorption at 280 nm in the UV spectrum, is ninhydrin positive, and
RA test, RAHA, and Immune Complex test were performed, and it was also confirmed that RP was not the previously known rheumatoid factor or Immune Complex. Furthermore, after hydrolyzing the purified RP with 6N hydrochloric acid at 110℃ for 24 hours,
Amino acid analysis was carried out by . The results obtained at this time were as follows (unit: mole%). Lys 6.6, His 1.9, Arg 4.0, Asp 7.0, Thr
8.5，Ser 10.0，Glu 10.4，Pro 7.9，Gly 10.4，
Ala 4.7, Cys/2 1.0, Val 7.5, Met 1.6, Ile
2.9, Leu 8.3, Tyr 4.5, Phe 2.9 <Creation of RP-Ab> 1 ml of purified RP using a domestic rabbit as the immunized animal
(protein content: 0.32 mg) was mixed with 1 ml of Freund's complete adjuvant to prepare a stable water-in-oil emulsion, which was injected subcutaneously into adult rabbits.
One month later, a stable water-in-oil emulsion was prepared again by mixing 1 ml of purified RP (protein content 0.40 mg) with 1 ml of Freund's complete adjuvant and injected subcutaneously into the same rabbit. After another week, blood was collected to obtain antiserum. Antibody was purified from antiserum as follows. First, Cepharose 4B (manufactured by Pharmacia,
Pre-wash 20 ml (Sweden) with distilled water on a Buchner funnel, add 20 ml of distilled water and aqueous cyanogen bromide solution (2 g/40 ml), and immediately adjust the pH to 11.0 with 4N sodium hydroxide.
And so. This was stirred for 8 minutes while maintaining the pH between 11.0 and 11.3, and after 8 minutes, the gel was suction-filtered using a Bützner funnel, and the gel on the funnel was ice-cooled.
Wash three times with sodium carbonate buffer (PH9.0),
Filter the water with suction and place in an ice water bath. Separately, 20 ml of purified RP was dialyzed against sodium carbonate buffer using a cellophane membrane, cooled on ice, and added to the prepared gel in an ice water bath for 10 minutes.
The antigen-binding gel was prepared by stirring for a minute. After packing this antigen-binding gel into a column and washing it with PBNa,
Pour 10 ml of antiserum obtained from a domestic rabbit to remove RP in the antiserum.
-Ab was adsorbed to antigen-binding gel. After thorough washing with PBNa, RP-Ab was collected by running a PH2.3 glycine buffer (0.17M) through the column, and immediately added 1/10 volume of a PH11.5 glycine buffer (1M). After neutralization, RP-Ab was purified and isolated. refined
RP-Ab was confirmed as follows. First, purified RP was developed by two-dimensional electrophoresis, and then a mixture of 0.5 ml of purified RP-Ab in 1% agar was poured onto the gel that had been developed by two-dimensional electrophoresis. After the agar solidified, it was left at room temperature for one day to perform an immunoprecipitation reaction. The precipitated spots formed on the agar after the immunoprecipitation reaction are
The isoelectric point was within the range of 7.3 to 7.8, and the mobility was within the range of 0.40 to 0.55 (assuming the mobility of the leading edge of albumin to be 1.0). The sedimentation spots obtained at this time are shown in Figure 4.
It was shown that the RP-specific antibody RP-Ab could be purified. <Examples of rheumatism diagnosis using RP-Ab> 60 patients with rheumatoid arthritis confirmed by comprehensive diagnosis by a doctor, 30 healthy people, 20 patients with renal failure, 12 patients with liver disease (cirrhosis, etc.), cancer patients ( For 40 cases (gastric cancer, colorectal cancer, etc.), double immunodiffusion method using RP-Ab and rheumatoid factor measurement using a commercially available RA test kit (manufactured by Nissui Pharmaceutical Co., Ltd.) (hereinafter referred to as RP-Ab method and RA Diagnosis of rheumatism was made using a test (referred to as a test). RP−
Positive/negative results in the Ab method were determined by the presence or absence of a sedimentation line. At this time, use purified RP as a positive control, RP
Plasma, which had been confirmed to be free of , was used as a negative control for each test. The RA test was determined according to the manufacturer's instructions. The results are shown in Table 1.

【table】

[Table] The positive rate of the RP-Ab method in patients with rheumatoid arthritis was 76.7% (23.3% false negative), while the positive rate of the RA test was 60.0% (40.0% false negative), indicating that sensitivity is RP compared to RA test
-Ab method was clearly higher. Also, healthy people,
In patients with renal failure, liver disease, and cancer, the RP-Ab method had no false positives, whereas the RA test had false positives of 6.7% to 33.3%, depending on the disease. , R.A.
The RP-Ab method was clearly more specific than the test. From the above results, compared to the conventional method of measuring rheumatoid factor, RP
The rheumatism diagnosis method using the -Ab method is clearly superior, and it has been shown that RP-Ab is very useful for routine tests in rheumatism diagnosis.

[Brief explanation of drawings]

Figure 1 is a two-dimensional electrophoretic image of purified RP, Figure 2 is a two-dimensional electrophoretic image of rheumatoid arthritis patient plasma, Figure 3 is a two-dimensional electrophoretic image of healthy human plasma, and Figure 4 is RP-Ab. It is a sedimentation spot.

Claims (1)

[Claims] 1. Approx.
Shows a molecular weight of 170,000 and does not use protein denaturants2
The isoelectric point in dimensional electrophoresis is 7.3-7.8, and the mobility (assuming the mobility of the leading edge of albumin is 1.0)
A rheumatoid-specific protein obtained from rheumatoid plasma and having an amino acid composition in the range of 0.40 to 0.55 as shown below. Amino acid composition (unit: mole%) Lys 6.6, His 1.9, Arg 4.0, Asp
7.0, Thr 8.5, Ser10.0, Glu 10.4, Pro
7.9, Gly10.4, Ala 4.7, Cys/2 1.0, Val
7.5, Met 1.6, Ile 2.9, Leu 8.3, Tyr
4.5，Phe 2.9