JPH0582399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for separating and purifying rheumatoid arthritis-specific proteins from rheumatoid plasma such as rheumatoid arthritis.

(Prior art) Rheumatoid arthritis has a large number of patients [according to the American Rheumatism Association's diagnostic criteria described below].
In our country, 0.3% of the population is classical
Or, it is said that the patient has definite rheumatoid arthritis. Furthermore, it is said that probable or possible chronic rheumatoid arthritis patients account for as much as 1 to 3% of the population. ], it is also a disease that progresses to physical disability and is sometimes accompanied by severe organ lesions. Although the disease is characterized by primarily affecting joints with synovial membranes, it is considered to be a systemic disease with systematic inflammatory lesions in connective tissues throughout the body. Among organ lesions, cardiac lesions (pericarditis, myocarditis) are the most severe and often cause death. This is followed by lung lesions (pleurisy, pneumonitis), and many systemic symptoms such as polyneuritis, skin ulcers, and episcleritis. For these reasons, rheumatoid arthritis is an important disease both medically and socially.

However, medical research on rheumatoid arthritis disease has lagged behind, and although it is clear that immunological abnormalities are involved in the onset of the disease, the etiology remains largely unknown, and treatment methods have only recently been developed. It has just begun to develop.

Diagnosis of rheumatoid arthritis disease is generally made according to the diagnostic criteria of the American Rheumatology Association. With the establishment of the American Rheumatology Association's diagnostic criteria, the diagnosis of rheumatoid arthritis disease has become unified. The American Rheumatology Association's diagnostic criteria consist of 11 items.
Diagnosis of rheumatoid arthritis is made based on how many of these items are met, but (1) synovial fluid testing does not always allow collection of synovial fluid, and histopathological testing cannot be easily performed by a doctor. (2) Rheumatoid nodules, rheumatoid factors, or changes on bone and joint radiographs are
It is not always positive from the beginning, and it is difficult to be a decisive factor in early diagnosis. (3) Therefore, items consisting of all clinical symptoms other than 1 and 2 above are important. There are many problems such as the fact that none of these are characteristic of rheumatoid arthritis. Therefore, although the diagnostic criteria of the American Rheumatology Association are currently widely used, diagnosis is not necessarily easy, and there are high expectations for new parameters for diagnosing rheumatoid arthritis.

Rheumatoid arthritis-specific protein was first discovered by Oda et al. (September 1982, 56th Japanese Biochemical Society) in the plasma of patients with rheumatoid arthritis, and its importance in diagnosing rheumatoid arthritis was pointed out. According to Oda et al., rheumatoid arthritis-specific proteins have a molecular weight of 150,000 to 170,000, an isoelectric point of 7.3 to 7.8, and a mobility (in two-dimensional electrophoresis that does not use a protein denaturant, the mobility of the leading edge of albumin is assumed to be 1.0).
It is a protein within the range of 0.30 to 0.45. Furthermore, when rheumatoid-specific proteins are first reduced with a reducing agent such as 2-mercaptoethanol and then measured by SDS polyacrylamide electrophoresis, two types of molecular weights are determined: 25,000-30,000 and 55,000-60,000. This rheumatoid-specific protein is found in approximately 80% of patients with rheumatoid arthritis.
Since it is not seen in healthy people or patients with other diseases (Oda et al.), expectations are high for it to be a new diagnostic method for diagnosing rheumatoid arthritis.

Purification of rheumatism-specific proteins has conventionally been carried out by the following method. First, ammonium sulfate or the like is added to the sample for salting out, and then ion exchange chromatography is performed using an ion exchange gel such as DEAE-Sephadex (Pharmacia, Sweden).
-300 (Pharmacia, Sweden), etc., followed by ion exchange chromatography again, and by collecting fractions in which rheumatism-specific proteins were detected by two-dimensional electrophoresis. It has been done.

(Problems to be Solved by the Invention) However, the above method actually requires dialysis and concentration operations for each step, making it very complicated and requiring a long time to perform all the steps (the present inventor According to their experience, it takes approximately 2 weeks). Furthermore, the purity of the rheumatoid arthritis-specific protein finally obtained is as low as about 50 to 60%, and this cannot be said to be an industrially viable method for purifying rheumatoid arthritis specific protein.

(Means for solving the problem) Rheumatoid-specific protein is a protein that exhibits similar properties to human immunoglobulin G (IgG) and IgG, which has some immunological common antigenicity, according to research by the present inventors. It was revealed that. As a result of further intensive research based on these research results, the present inventors have developed an industrially useful purification method that can easily and quickly obtain a rheumatoid-specific protein of high purity that is superior to the above methods. was successfully established.

That is, electrophoresis or ion exchange chromatography is performed on raw materials (mixture of proteins such as plasma and serum) containing rheumatoid-specific proteins,
Removes gamma globulin, especially IgG. Regarding the fraction from which γ globulin has been removed, for example, by collecting a fraction that has reactivity with protein A, which is an anti-human IgG antibody or a protein derived from Staphylococcus, and which has binding properties with human IgG. ,
This is to purify rheumatism-specific proteins.
More specifically, a raw material such as rheumatism patient plasma is subjected to electrophoresis using polyacrylamide or the like as a support, and a fraction corresponding to the β position is collected. next,
For example, protein components are extracted from the collected fractions using a solution such as a phosphate buffer. Or for example
DEAE-Sephadex A-50 (Pharmacia,
After performing ion-exchange chromatography using an ion-exchange gel (e.g. (Sweden)) and removing fractions eluted at low salt concentrations such as sodium chloride (0.3M or less), Collect fractions eluting at 0.5M. (This purification step using ion-exchange chromatography is aimed at removing gamma globulin, especially the IgG fraction. Rheumatoid-specific protein and gamma globulin
Since globulin, especially IgG, behaves differently in ion exchange chromatography, various ion exchange gels can be used for the purpose of this step, and many elution methods can be selected. Therefore, the types of ion exchange gels and ion exchange chromatography conditions described in this section are not necessarily limited. ) On the other hand, prior to electrophoresis or ion-exchange chromatography, it is possible to first fractionate rheumatism patient plasma, etc. by salting out, etc. to reduce the amount of protein to be subjected to ion-exchange chromatography.
It is effective as used in conventional protein purification. That is, ammonium sulfate is added to rheumatism patient plasma, etc., and a precipitate salted out with 25 to 35% saturated ammonium sulfate is collected.

(Since the primary purpose of this step is to reduce the amount of protein subjected to ion exchange chromatography, the saturated ammonium sulfate concentration to be salted out is
It is not limited to the above 25-35%. For example, a precipitate salted out with 50% saturated ammonium sulfate may be collected. Similarly, other salts commonly used in salting out, such as sodium sulfate, can also be used here. Alternatively, it is also possible to use a commonly used solvent precipitation method using an organic solvent such as ethyl alcohol or acetone. ) Next, for example, anti-human IgG antibody or human IgG, which is a protein derived from Staphylococcus
Protein A is bound to a gel such as Sepharose 4B (Pharmacia, Sweden) by a ligand such as cyanogen bromide. The gel (gel of Sepharose 4B etc. bound to anti-human IgG antibody or protein A derived from Staphylococcus: affinity gel)
A rheumatoid-specific protein crudely purified by ion-exchange chromatography using an anion-exchange gel such as DEAE-Sephadex A-50 is passed through a column (affinity column) packed with 300 ml of rheumatoid arthritis. A method of changing the pH using a glycine buffer, etc. after removing non-bound components of the affinity gel.
Alternatively, rheumatism-specific proteins are separated from the affinity gel by a method normally used in affinity chromatography to elute proteins from the affinity gel, such as a method using a mild protein denaturing effect using urea, etc.
By collecting it, the rheumatism-specific protein can be purified.

(Effect of the invention) The rheumatism-specific protein obtained at this time is subjected to electrophoresis using an electrophoresis support such as polyacrylamide gel, and then subjected to Coomersie Brilliant Blue (Coomersie Brilliant Blue R-
When the support is stained with a protein stain such as 250) and decolorized, the purity is 90% or more when measured using a densitometer or the like. Furthermore, when this rheumatoid-specific protein is subjected to two-dimensional electrophoresis, it has an isoelectric point (pI: the protein's isoelectric point is the pH corresponding to the electrophoresis position of the protein) of 7.3 to 7.8, and a mobility (at the leading edge of albumin). It can be detected as a single spot in the range of 0.30 to 0.45 (assuming a mobility of 1.0) and exhibits a molecular weight of 150,000 to 170,000 when measured by SDS polyacrylamide electrophoresis. In addition, when this rheumatoid arthritis-specific protein is first reduced with a reducing agent such as 2-mercaptoethanol and then the molecular weight is measured by SDS polyacrylamide electrophoresis, the molecular weight is 25,000~
Two types of molecular weights are shown: 30,000 and 55,000-60,000.

(Example) Example 1 Excreted plasma 1 generated by plasma exchange in a rheumatoid arthritis patient with rheumatoid arthritis-specific protein
144 g of ammonium sulfate was added to make a 25% saturated ammonium sulfate solution. After removing the precipitate precipitated with 25% saturated ammonium sulfate by centrifugation at 8000 x g for 20 minutes (refrigerated centrifuge SCR20BA, Hitachi), 61 g of ammonium sulfate was further added to the supernatant (approximately 1 recovery). , a 35% saturated ammonium sulfate solution. 8000×
g, collected by centrifugation for 20 minutes. The collected precipitate was dissolved by adding 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride, and dissolved using a cellophane membrane (manufactured by Visking Company, USA).
Ammonium sulfate was removed by dialysis overnight against a 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride (amount recovered: absorbance at a wavelength of 280 nm, 95.2;
Liquid volume: 90ml). Next, DEAE-Sephadex A-50 (DEAE-Sephadex A-50) equilibrated in advance with 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride.
Ion exchange chromatography was performed using Sephadex A-50 (manufactured by Pharmacia, Sweden). The protein was eluted by increasing the sodium chloride concentration stepwise in 0.02M phosphate buffer (PH7.2). Sodium chloride concentration
After elution with 0.3M 0.02M phosphate buffer (PH7.2), fractions eluted with 0.02M phosphate buffer (PH7.2) with 0.5M sodium chloride concentration were collected (collected amount : Absorbance 0.35 at wavelength 280nm, liquid volume 100ml).

Separately, cyanogen bromide activated Cephalose 4B
(CNBr-activated Sepharose 4B, manufactured by Pharmacia, Sweden), anti-human IgG antibody (goat serum IgG fraction, manufactured by Miles, USA, protein content approximately 9.6 mg, according to the manufacturer's insert, specific antibody)
2.9mg) was reacted to create an antibody-binding gel.
This antibody-binding gel was packed into a column, washed with 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride, and then the fractions collected by ion-exchange chromatography were added to the antibody-binding gel. It was adsorbed. After thoroughly washing the antibody-bound gel with 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride, it was eluted with glycine buffer (0.17M) at PH2.3. The eluted protein fraction was collected and immediately neutralized by adding 1/10 volume of glycine buffer (1M) of pH 11.5. The rheumatoid-specific protein obtained at this time was determined to have a protein content of 2.7 mg using the microbiuret method.
was able to be measured.

Example 1 Salting out and ion exchange chromatography were performed using the same procedure as in Example 1, and the obtained rheumatism-specific protein fraction was used. Specifically, for excreted plasma 1 containing rheumatoid-specific proteins produced by plasma exchange in patients with rheumatoid arthritis, the precipitate precipitated was removed with 25% saturated ammonium sulfate, and ammonium sulfate was further added to the supernatant to give 35% % saturated ammonium sulfate solution, and the deposited precipitate was collected. The collected precipitate
It was dissolved in 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride and dialyzed to remove ammonium sulfate (amount recovered: absorbance at a wavelength of 280 nm).
93.6, liquid volume 85ml). This protein solution was added to DEAE-Sephadex A-50 equilibrated with 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride.
(DEAE-SephadexA-50 manufactured by Pharmacia,
Ion exchange chromatography was performed by passing the mixture through a Swedish column. The sodium chloride concentration in 0.02M phosphate buffer (PH7.2) was increased stepwise to elute the protein, and the protein was eluted in 0.02M phosphate buffer (PH7.2) with a sodium chloride concentration of 0.3M or less.
7.2) was removed, and the fraction eluted with 0.02M phosphate buffer (PH7.2) with a sodium chloride concentration of 0.5M was collected (recovered amount: wavelength
Absorbance at 280nm 0.32, liquid volume 100ml).

The fraction obtained by the above method was purified as follows. Protein A Sepharose CL-4B
(Protein A-Sepharose CL-4B, Pharmacia, Sweden) 0.6 g was swollen (swelled to approximately 2 ml of gel) and packed into a column to prepare a protein A column. After thoroughly washing the Protein A column with 0.1M phosphate buffer (PH8.0), the above fraction was applied to the Protein A column and adsorbed to Protein A. The rheumatism-specific protein adsorbed to Protein A was eluted with 0.1M glycine-hydrochloride buffer (PH3.0), and immediately neutralized by adding 1/10 volume of 0.2M sodium bicarbonate solution. The rheumatoid arthritis-specific protein obtained at this time was determined to have a protein content of 2.1 mg using the microbiuret method.

Example 3 Excreted plasma 200 containing rheumatoid-specific proteins produced by plasmapheresis in patients with rheumatoid arthritis
ml was applied to a DEAE-Sephadex A-50 (DEAE-Sephadex A-50 Pharmacia, Sweden) column equilibrated with 0.02M phosphate buffer (PH7.2) containing 0.05M sodium chloride, and subjected to ion exchange chromatography. I did a graphie. 0.02M phosphate buffer (PH
7.2) was removed, and the fraction eluted with 0.02M phosphate buffer (PH7.2) with a sodium chloride concentration of 0.5M was collected (recovered amount: wavelength
Absorbance at 280nm 0.12, liquid volume 50ml).

The rheumatism-specific protein fraction obtained by the above method was purified as follows. First, the above fractions were thoroughly washed with 0.1M phosphate buffer (PH8.0) using a cellophane membrane (Visking Company, USA), and then the fractions, which had been dialyzed overnight overnight, were
It was applied to a column and adsorbed to protein A. Rheumatoid-specific protein adsorbed to protein A
Elute with 0.1M glycine-HCl buffer (PH3.0),
Immediately, 1/10 volume of 0.2M sodium hydrogen carbonate solution was added to neutralize. The rheumatoid-specific protein obtained at this time was determined by the microbiuret method.
0.22mg, purity determined to be 90% by electrophoresis.

Example 4 Using the rheumatoid arthritis-specific proteins obtained in Example 1 and Example 2 as specimens, the following two
Dimensional electrophoresis was performed (rheumatism-specific protein obtained in Example 1 is referred to as rheumatism-specific protein 1, and rheumatism-specific protein obtained in Example 2 is referred to as rheumatism-specific protein 2).

60 μl of purified rheumatoid-specific protein (protein concentration of rheumatoid-specific protein 1 0.40 mg/ml, protein concentration of rheumatoid-specific protein 2 0.34 mg/ml) was added to Ampholine [Ampholine, manufactured by Pharmacia,
6.25w/v% (PH3.5-10:PH3.5-5=4:1)]
On a polyacrylamide gel containing 0.01 M phosphoric acid solution and 0.04 N 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. did. These running gels were then placed on polyacrylamide slab gels with a concentration gradient of 4% to 17% and run at constant current (27 mA) for 20 hours. A 0.05M Tris-0.384M glycine buffer was used as the electrophoresis solution. After two-dimensional electrophoresis, the gel is
0.025% Coomersie Brilliant Blue R-250, 50v/v
% methanol, 7v/v% acetic acid solution for 8 hours,
It was decolorized for 3 days with a 7v/v% acetic acid solution and a 10v/v% methanol solution, and then observed. (The electrophoretic image of rheumatoid-specific protein 1 is shown in Figure 1. The electrophoretic image of rheumatoid-specific protein 2 is also similar.) Both the electrophoretic images of rheumatoid-specific protein 1 and rheumatoid-specific protein 2 show the isoelectric point The pH corresponding to the position is taken as the isoelectric point of the protein) 7.3 to 7.8, mobility (assuming the mobility of the leading edge of albumin is 1.0)
It was obtained as a single spot within the range of 0.30 to 0.45 (seen in the area surrounded by the broken line in Fig. 1), and from this it was confirmed that it was a rheumatoid-specific protein. At this time, in any case, 2
No protein spots outside the above range were observed on the dimensional electrophoresis gel. Figure 2 shows a two-dimensional electropherogram of rheumatoid patient plasma before purification, and spots of rheumatoid-specific proteins can be seen within the range of isoelectric point 7.3 to 7.8 and mobility 0.30 to 0.45 (dashed line indicates (enclosed part).

Next, add 10 μl of rheumatoid-specific protein 1 (0.40 mg/ml) and rheumatoid-specific protein 2 (0.34 mg/ml),
Electrophoresis was performed through a polyacrylamide slab gel with a concentration gradient of 4-17%, respectively. next,
0.025% Coomersie Brilliant Blue (Coomersie Brilliant Blue R-250),
It was stained with a solution of 50v/v% methanol and 7v/v% acetic acid for 8 hours, and destained for 3 days with a solution of 7v/v% acetic acid and 10v/v% methanol. As a result of measuring each electrophoresis gel with a densitometer (DENSITRON 1M, Jokosha, Japan), the purity of rheumatoid-specific protein 1 was 92%, and the purity of rheumatoid-specific protein 2 was 92%.
I was able to measure it as 94%.

In addition, rheumatoid-specific protein 1 has a molecular weight of approximately 160,000 and 2
- Rheumatoid-specific protein 2 can be determined to have two subunits with molecular weights of approximately 28,000 and 60,000 by SDS polyacrylamide gel electrophoresis performed after reduction with mercaptoethanol, and rheumatoid-specific protein 2 has a molecular weight of approximately 155,000 and 2
SDS polyacrylamide gel electrophoresis after reduction with mercaptoethanol determined two subunits with molecular weights of about 25,000 and about 55,000. Considering measurement errors, it can be said that there is no difference in the measured values between the two rheumatoid arthritis-specific proteins.

[Brief explanation of the drawing]

FIG. 1 is a two-dimensional electropherogram of the rheumatoid-specific protein purified in Example 1, and FIG. 2 is a two-dimensional electropherogram of rheumatoid patient plasma before purification.

Claims (1)

[Claims] 1 A step of separating an IgG fraction and a fraction containing rheumatoid-specific proteins by ion-exchange chromatography, and a step of separating an IgG fraction from a fraction containing rheumatoid arthritis-specific proteins, and an after-treatment step using protein A that has binding properties to anti-human IgG antibodies or human IgG derived from Staphylococcus. A method for separating and purifying rheumatoid arthritis-specific proteins from plasma, the method comprising the step of collecting an adsorbed fraction by initiating chromatography.