JPH03218464A - Antibody to rheumatism-specific protein - Google Patents

Abstract

PURPOSE:To detect the rheumatism-specific protein (RP) in blood plasma by forming an antigen to the RP by the RP, in which the isoelectric point and mobility in two-dimensional electrophoresis without using a protein modifying agent is within a specific range. CONSTITUTION:The rheumatism blood plasma is first subjected to the isoelectric point electrophoresis by a polyacryl amide gel and in succession, the RP is separated on the two-dimensional electrophoresis gel by two-dimensional electro phoresis method to make gel electrophoresis of concn. gradient. This RP is in the ranges of about 7.3 to 7.8 isoelectric point and about 0.40 to 0.55 mobility on the two-dimentional electric gel. The RP is divided from above the gel and is extracted and isolated by a buffer soln. etc. The mol. wt. of the RP of this time is about 17X10<4>. An antiserum is taken from an animal immunized with the RP and is refined to conjugate the RP which is an antigen with the gel by ligand, such as cyanide bromide. The antiserum is passed through a column packed with such gel. The antibody RP-Ab specific to the RP is separated from the column after washing. The RP in the blood plasma is easily measured by using the Rp-Ab if a double immune diffusion method, etc., are executed by using the gel such as, for example, agar.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antibody (hereinafter referred to as RI"Ab) that is specific to rheumatoid arthritis-specific protein (hereinafter referred to as RP) isolated from rheumatoid plasma such as rheumatoid arthritis. be.

Diagnosis of rheumatism currently relies largely on the doctor's findings and the patient's chief complaint, and the reality is that objective, quantitative diagnostic methods are 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, rheumatoid factor detection is widely performed as a routine test.

By the way, rheumatoid factor is found at a high rate in the plasma of rheumatoid patients (although it varies depending on the report,
Rheumatoid factor is detected in 0-70% of cases), and is frequently detected in the plasma of patients with non-rheumatoid conditions such as patients with liver disease and cancer (more than 50% of patients with cirrhosis and nearly 20% of cancer patients). It has been reported that the patient is rheumatoid factor positive).
It is also recognized in 3-7% of healthy people. As described above, the specificity of the presence of rheumatoid factors in rheumatoid arthritis is low, and rheumatoid arthritis cannot be immediately diagnosed based on the presence of rheumatoid factors. 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 rheumatoid arthritis patients, patients with other diseases, and healthy individuals, the present inventors found that a novel protein (RP), which is not found in the plasma of patients with rheumatoid arthritis or other healthy individuals, was detected in the plasma of rheumatoid arthritis patients. ) is expressed at a high rate.
The RP-specific antibody RP- was discovered and successfully isolated using a two-dimensional electrophoresis method that does not use a protein denaturing agent (the following two-dimensional electrophoresis does not use a protein denaturing agent). We were able to obtain Ab. According to the research results of the present inventors, RP was observed in 70 to 80% of rheumatism patients, and was not observed at all in healthy subjects, patients with renal failure, patients with liver disease, and patients with cancer. Compared to the fact that 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 specificity of RP and rheumatoid patients are The positive rates for both cases are clearly high. Therefore, by creating an antibody RP-Ab specific to RP, the RP-Ab
Detection of RP as a routine clinical test using a diagnostic agent using RP can be an effective means for diagnosing rheumatism. The obtained RP is necessary for the creation of this RP-Ab, and therefore RP and RP-A are used in rheumatology diagnosis.
Both b are extremely useful.

When performing two-dimensional electrophoresis of rheumatoid plasma, RP has an isoelectric point (p!) 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. It is a protein with a molecular weight of approximately 170,000 as determined by SDS polyacrylamide gel electrophoresis and is recognized as a spot in the range of 55. In two-dimensional electrophoresis of healthy human plasma, the isoelectric point (pi) is 7.3 to 7.8, and the mobility (assuming the mobility of the leading edge of albumin is 1.0) is 0.40 to 0.5.
No protein was observed in the range of 5, indicating that RP is a completely new protein found in rheumatoid plasma that has not been previously known.

Next, a method for acquiring RP will be described. RP is obtained by a monochromatic method or separated on a two-dimensional electrophoresis gel by a two-dimensional electrophoresis method in which equine plasma is first subjected to isoelectric focusing on a polyacrylamide gel, followed by concentration gradient gel electrophoresis. I can do it. RP has an isoelectric point (pl) of 7.3 to 7.8 on a two-dimensional electrophoresis gel and a mobility (assuming the mobility of the leading edge of albumin to 1.0) of 0.40 to 0.
55, and can be isolated by dividing it on a two-dimensional electrophoresis gel and extracting it with a buffer or the like.

The molecular weight of the RP isolated at this time was approximately 170.
It is 000.

In addition, in order to isolate a large amount of RP, for the precipitate salted out with 25-35% saturated ammonium sulfate from rheumatoid patient plasma, an ion exchange gel such as DEAE-Sephadex A-50 (manufactured by Pharmacia, Sweden) or the like can be used. Ion-exchange chromatography is performed using ion exchange chromatography, and fractions eluted with 0.5M sodium chloride are collected. Furthermore, this fraction can be isolated by performing gel chromatography using a gel such as Sephacryl S-300 (manufactured by Pharmacia, Sweden) and the like, followed by ion exchange chromatography using the above-mentioned ion exchange gel.

When subjected to two-dimensional electrophoresis, the RP isolated at this time had 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, exhibiting a molecular weight of approximately 170,000 as determined by SDS polyacrylamide gel electrophoresis.

RP-specific antibody RP-Ab can be prepared by, for example, converting purified RP to Freund's complete adjuvant 1.
A specific antibody can be obtained by a general method for producing a specific antibody, which involves immunizing an immunizable animal with a specific antibody and collecting and purifying antiserum from the immunized animal. To purify 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 the antiserum is passed through a column filled with the gel. After thorough washing, the antibody can be separated from the column using a glycine buffer or the like using an immunoadsorption method or the like.

The antibody RP-Ab obtained in this way can be confirmed by specifically reacting with purified RP and producing a precipitation line by a general specific antibody confirmation method such as immunoelectrophoresis or immunoprecipitation reaction. .

Measurement of RP in plasma using RP-Ab can be carried out using currently commonly used antigen measurement methods using specific antibodies, such as
Double immunodiffusion using a gel such as agar, various agglutination reactions performed by immobilizing RP-Ab on latex or red blood cells, and adsorption of RP-Ab onto a solid phase such as beads or microplates to perform enzyme or radioactivity reactions. This can be done by immunoassay using isotopes, etc. By such measurement method, RP-A
RP-Ab can be easily measured using RP-Ab, and therefore RP-Ab is important as a routine test method for rheumatism diagnosis.

(Example) <Isolation method of RP> A precipitate precipitated with 25% saturated ammonium sulfate from excreted plasma generated by plasma exchange in a rheumatoid arthritis patient with RP was subjected to 8000×g. The precipitate precipitated with 35% saturated ammonium sulfate was removed by centrifugation for 20 minutes, and further ammonium sulfate was added to the supernatant.
The samples were collected by centrifugation at 000Xg for 20 minutes. This precipitate was dissolved in 0.02M phosphate buffer (pH 7.2, hereinafter PBN).
a), dialyzed against deionized water through a cellophane membrane to remove ammonium sulfate, and then pre-buffered with PBNa, DEAE-Sephadex A-5.
Ion exchange chromatography was performed using a 0 (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, and 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 further purified using Cephacryl S-300.
By performing gel chromatography using a column (manufactured by Pharmacia, Sweden) and collecting fractions corresponding to the fraction positions where RP was confirmed by two-dimensional electrophoresis, RP and molecules mixed in with ion exchange chromatography can be removed. Materials with different sieving behavior were separated. After this, separation based on ionic strength by ion exchange chromatography was performed again using a DEAE-Sephadex A-50 (manufactured by Pharmacia, Sweden) column, and a concentration gradient solution of 0.0M to 0.6M sodium chloride was used. The purified R was eluted and fractions corresponding to the fraction positions where RP was confirmed by two-dimensional electrophoresis were collected.
I got P.

Two-dimensional electrophoresis was performed using this purified RP as a sample and confirmed as follows. 60 ul of purified RP (protein concentration 0.40 mg/II1) was added to ampholine (As+pholine: manufactured by Pharmacia, 6.
25 w/v% (pH3.5-10: pH3
．． 5-5=4:1)) on a polyacrylamide gel containing 0. Using OIM phosphoric acid solution and 0.04M 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.
The electrophoresis was carried out for 0 hours. Next, this electrophoresis gel was mixed with 4% to 17%
The cells were placed on a polyacrylamide slab gel with a concentration gradient of 200 mL and run at constant current (24 mA) for 20 hours. The electrophoresis solution used at this time was a 0.05M Lith-0.384M glycine buffer. After two-dimensional electrophoresis as described above, 0.025% Coomassie Brilliant Blue (Cooa+ersie Brilliant
Blue R 2 5 0 ) +5 0 v /
FIG. 1 shows an electrophoretic image obtained by staining with v% methanol and 7% acetic acid solution for 8 hours and decolorizing with 7% acetic acid and 10% methanol solution for 3 days. The electrophoresis image has an isoelectric point of 7.3-7.8 and a mobility (
(Assuming the mobility of the leading edge of albumin to be 1.0) 0.40
Since it was within the range of ~0.55, it was confirmed that it was RP.

Furthermore, the molecular weight of the RP obtained at this time was determined to be approximately 170.000 by SDS polyacrylamide gel electrophoresis.

For reference, two-dimensional electrophoresis images of plasma from patients with rheumatoid arthritis and plasma from healthy individuals are shown in Figures 2 and 3, respectively. A two-dimensional electrophoretic image of plasma from patients with rheumatoid arthritis shows an isoelectric point of 7.3 to 7.8.
, RP spots are seen within the range of mobility (assuming the mobility of the leading edge of albumin is 1.0) 0.40 to 0.55 (area surrounded by a broken line). However, the two-dimensional electrophoretic image of healthy human plasma shows an isoelectric point of 7.3 to 7.8;
Mobility (assuming the mobility of the leading edge of albumin to be 1.0)
Within the range of 0.40 to 0.55, not only RP but also protein substrates are not observed at all.

Purified RP has a maximum absorption at 280n in the UV spectrum, is ninhydrin positive, and has been tested in the RA test.
The RAHA's Immune Complex test was performed to determine if RP was known to be a rheumatoid factor or!
At the same time, I confirmed that it was not fog mune Con+plex.

Furthermore, the purified RP was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, and then amino acid analysis was performed using an amino acid analyzer (Hitachi KLH-5). The results obtained at this time were as follows (unit: soleX).

Lys 6.6, old s 1.9. Arg4.
0, Asp 7.0, Thr 8.5, Ser
10. Sail Glu 10.4, Pro 7.9,
Gly 10.4, Ala 4.7. Cys
/21.0, Val 7.5. Met 1.6,
lie 2.9, Leu 8.3, Tyr
4.5, Phe 2.9 <Creation of RP-Ab> Using a domestic rabbit as the immunized animal, purified RPIml (
Protein content: 0.32 mg) and Freund's
) A stable water-in-oil emulsion was prepared by mixing complete adjuvant IIjI1 and injected subcutaneously into adult rabbits. After one month, purified RP1d (protein amount 0.40 +
Ag) and Freund's Complete Adjuvant 1- were mixed to form a stable water-in-oil emulsion again and injected subcutaneously into the same rabbits.

After another week, blood was collected to obtain antiserum.

Antibody was purified from antiserum as follows. First, Sepharose 4B (manufactured by Pharmacia, Sweden) 2
0d was pre-rinsed with distilled water on a Buchner funnel, and then 20a of distilled water! 1
! Add cyanogen bromide aqueous solution (2g/40d) to 4N
The pH was immediately adjusted to 11.0 with sodium hydroxide. This was stirred for 8 minutes while keeping the pH at 11.0 to 11.3, and after 8 minutes, the gel was suction filtered using a Buchner funnel, and the gel on the funnel was cooled with 0.1+of sodium carbonate buffer. (pH 9.0) three times, suction filtered, drained and placed in an ice water bath. Separately, purified RP20ad!
was dialyzed against sodium carbonate buffer using a cellophane membrane, cooled on ice, and added to the prepared gel in an ice water bath and stirred for 10 minutes to prepare an antigen-binding gel.

This antigen-binding gel was packed into a column, washed with PBNa, and then an antiserum 10IIi obtained from a domestic rabbit was passed through the column to cause RP-Ab in the antiserum to be adsorbed onto the antigen-binding gel. After thorough washing with PBNa, RP-Ab was collected by flowing a pH 2.3 glycine buffer (0.17M) through the column, and immediately
]0 amount of pH 11.5 glycine buffer (IM) was added to neutralize, and RP-Ab was purified and isolated. Purified R
P-Ab was confirmed as follows. First, purified R
After P was developed by two-dimensional electrophoresis, a mixture of 1% agar and 0.5 d of purified RP-Ab was poured onto the gel that had been developed by two-dimensional electrophoresis, and the agar solidified. Thereafter, the immunoprecipitation reaction was performed after being left at room temperature for one day.

The precipitated spots that appear on the agar after the immunoprecipitation reaction have an isoelectric point of 7.
．． 3 to 7.8, and the mobility (assuming the mobility of the leading edge of albumin to 1.0) was within the range of 0.40 to 0.55.

The sedimentation spots obtained at this time are shown in FIG. It was shown that the RP-specific antibody RP-Ab could be purified.

<Examples of rheumatism diagnosis using RP-Ab> 60 cases of rheumatoid arthritis patients, 30 cases of healthy people, 20 cases of patients with renal failure, confirmed by comprehensive diagnosis results by doctors.
12 patients with liver disease (cirrhosis, etc.) and 40 patients with cancer (gastric cancer, colorectal cancer, etc.) were tested for rheumatoid factors using a double immunodiffusion method using RP-Ab and a commercially available RA test kit (manufactured by Nissui Pharmaceutical Co., Ltd.). Both methods of measurement (hereinafter referred to as RP-Ab)
Diagnosis of rheumatism was made using the RA test. Positive/negative results in the RP-Ab method were determined by the presence or absence of a sedimentation line. At this time, purified RP was used as a positive control, R
Plasma, which had been confirmed to be free of P, 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 (Note) A: Previous history of rheumatoid arthritis B: Healthy subjects C: Patients with renal failure D = Patients with liver disease E: liJ patients The positive rate of the RP-Ab method in patients with rheumatoid arthritis was 76.7% (false negative 23.3%), whereas the positive rate by RA test was 60.0% (40% false negative).
．． 0%), and the degree of confusion was higher than that of the RA test.
The law was clearly higher. Furthermore, in healthy subjects, patients with renal failure, patients with liver disease, and patients with cancer, no false positives were observed with the RI'Ab method, whereas with the RA test, the results ranged from 6.7 to 33.3, although it varied depending on the disease. % of false positives were observed, and the RP-Ab method was clearly more specific than the RA test.

The above results show that the rheumatism diagnosis method using the RP-Ab method is clearly superior to the conventional method of measuring rheumatoid factors, and that RPAb is extremely useful for routine tests in rheumatism diagnosis. Shown.

[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 a two-dimensional electrophoretic image of RP Ab. This is a settling spot.

Claims (1)

[Claims] Approximately 170,0 by SDS polyacrylamide gel electrophoresis
It has a molecular weight of 0.00, an isoelectric point of 7.3 to 7.8 in two-dimensional electrophoresis without using a protein denaturing agent, and a mobility (assuming the mobility of the leading edge of albumin to 1.0) of 0.40 to 7.8. 0
．． 55, an antibody against a rheumatoid-specific protein produced from a rheumatoid-specific protein obtained from rheumatoid plasma.