JPH0826073B2 - Purified polypeptide chain constituting UsnRNP complex, method for producing the same, and method for measuring anti-UsnRNP antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a purified UsnRNP complex-constituting polypeptide chain which is effectively used for predicting and diagnosing autoimmune diseases, autoimmune phenomena, and the like. The present invention relates to a production method and a method for measuring anti-UsnRNP antibody.

[Conventional technology]

The small ribonucleoprotein (UsnRNP) complex is a complex particle composed of one RNA chain and 6 to 9 polypeptide chains, and U ₁ , U depending on the type of constituent RNA chain and constituent polypeptide chain. ₂ , U ₄ , U ₅ , and U ₆ RNP. In addition, these UsnRNPs are ubiquitous in the cell nucleus of animals and their antigenicity is common across species. Usually, a healthy person does not have an antibody group against the complex, but an autoimmune disease,
Systemic lupus erythematosus (SLE), mixed connective tissue disease (MCT)
The patient of D) has a group of antibodies against the complex in serum.

Therefore, the clinical significance of detecting the antibody group in the serum of patients lies in the prediction, diagnosis or follow-up of autoimmune diseases and autoimmune phenomena.

BACKGROUND ART Conventionally, an anti-RNP antibody group and an anti-Sm antibody group are widely known as antibody groups against a ribonucleoprotein (UsnRNP) complex. However, both of these antibody groups are aggregates of antibodies against different antigenic determinants on the corresponding ribonucleoprotein (UsnRNP) complex, and the combination and amount of constituent antibodies vary depending on the patient. However, actually, the antibodies against the ribonucleoprotein (UsnRNP) complex are only detected separately in the two groups of the anti-RNP antibody group and the anti-Sm antibody group. This is detected by, for example, the fusion phenomenon of the appearance sedimentation line by the binary immunodiffusion method in comparison with the reaction pattern of the crude antigen solution and the standard serum containing the anti-RNP antibody group or the anti-Sm antibody group, which is conventionally controlled. It is not necessary that the antigen be purified.

Many methods have been described in the scientific literature for purification and isolation of the ribonucleoprotein (UsnRNP) complex [J. Bio
l. Chem. 258, 2604-2613 (1983), etc.]. In most methods, purification is carried out by ion-exchange chromatography, molecular sieve chromatography or a combination of both chromatographies, and according to the method further by immunoadsorption chromatography. However, in each case, the method involved purification and isolation as a ribonucleoprotein (UsnRNP) complex.

[Problems to be Solved by the Invention]

As mentioned above, small ribonucleoproteins are composed of several polypeptide chains that are strongly non-covalently bound to one RNA chain, but this binding does not dissociate under physiological conditions. Also, some of the constituent polypeptide chains can react with anti-RNP antibody, and some of them can react with anti-Sm antibody. There are also polypeptide chains that react with any antibody.

Therefore, it is difficult to obtain an antigen preparation that reacts only with an anti-RNP antibody by the conventional technique, and to develop a method for detecting an antibody of an autoimmune disease patient using such an antigen preparation. Was also difficult.

Therefore, the inventors of the present invention have conducted extensive studies on a method for isolating a polypeptide chain constituting a low molecular weight ribonucleoprotein without losing its antigenicity, and have completed the present invention.

[Means for solving the problem]

The present invention extracts UsnRNP complexes from animal tissue extracts, and then dissociates the polypeptide chain to be isolated from other UsnRNP complex constituents in the presence of a nonionic surfactant. Ion-exchange chromatography is performed to separate the fraction containing the polypeptide chain, and then the molecular chain chromatography is used to separate the polypeptide chain to be isolated, which reacts specifically with the anti-RNP antibody. A method for producing a polypeptide chain constituting a purified UsnRNP complex (hereinafter referred to as the first invention), a polypeptide chain obtained by the production method, and the polypeptide The present invention relates to a method for measuring an anti-UsnRNP antibody (hereinafter referred to as the second invention), which comprises using a peptide chain as an antigen and detecting or quantifying an antibody against the polypeptide chain.

 First, the first invention will be described in detail.

In the process of extracting a ribonucleoprotein (UsnRNP) complex from an animal cell extract or cell nuclear extract, the complex is solubilized at a dilute salt concentration or a physiological salt concentration,
In order to protect from invasion by all possible proteolytic enzymes that are simultaneously solubilized, it is preferable to perform extraction with an extraction buffer containing an inhibitor for the proteolytic enzymes.
Further, regarding this extraction operation, in order to obtain as many ribonucleoprotein (UsnRNP) complexes as possible, it is preferable to perform extraction from the object to be extracted twice and combine the two extracts.
The detection of the ribonucleoprotein (UsnRNP) complex in the extract is preferably performed by the binary immunodiffusion method because the procedure is simple.

Thereafter, a salting-out agent such as powdery ammonium sulfate is added to the extract to salt out and precipitate the protein. For example, the fractions that precipitate with ammonium sulfate saturation of 30% to 60% are collected by centrifugation. Next, the obtained precipitated protein is dissolved in a starting buffer for ion exchange chromatography as small as possible and dialyzed against the starting buffer for ion exchange chromatography for 2 days. In the present invention, the starting buffer solution for ion exchange chromatography contains a nonionic surfactant. When such a buffer is used, it is considered that a certain polypeptide chain constituting the riboprotein (UsnRNP) complex is dissociated from the complex under the solution environment. This is because, in reality, it is ionically bonded as a dissociated polypeptide chain to the carrier for subsequent ion exchange chromatography. Therefore, the nonionic surfactant is
It is present at the concentration required to allow dissociation.

Nonionic surfactants include polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene heptamethylhexyl ether, polyoxyethylene methyl dodecyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitol. Esters and the like can be used. The concentration is preferably 0.03 to 0.3% (weight / volume).

Subsequently, the dialyzed product is passed through a column packed with a carrier for ion exchange chromatography to ion bond the polypeptide chain to the functional group.

As a carrier for ion exchange chromatography, spherical or fine granular cross-linked agarose having functional groups such as diethylaminoethyl group, diethyl- (α-hydroxy-propyl) aminoethyl group, polyethyleneimine group and amine mixture, Crosslinked dextran, crosslinked cellulose, polymethacrylate resin, styrene / divinylbenzene copolymer, hydrophilic silica and the like can be used.

As such a carrier for ion exchange chromatography, DEAE-Sephadex, DEAE-Sapharose, DEAE-cellulose (Cellulose),
DEAE-Sephacel, DEAE-Biogel (Bio
Gel), Cellulose-DEAE, QAE-Sephadex, Cellulose-TEA
E etc. are commercially available.

Ion-exchange chromatography is an amount within the range of the total ion-exchange capacity of the ion-exchange carrier packed in the column.
150ml / hour or less (0.7l / hour or less for Fast flow),
It is particularly preferable to carry out under the condition of 50 to 100 ml / hour.

Thereafter, the column is washed with an excess amount of the ion exchange chromatography starting buffer to remove other nucleic acid and protein components or polypeptide chains which do not ionically bond with the functional group. After washing the column, the ionic strength in the buffer solution is increased by adding salts, and an ion exchange reaction is carried out with the polypeptide chain captured by the functional group to elute the polypeptide chain. The eluate was collected as fractions, the content of the polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex was detected by the binary immunodiffusion method, and the fractions containing the polypeptide chain were combined to determine the exclusion limit molecular weight. Is 10,000, for example, under an ultrafiltration membrane made of polysulfone, under nitrogen gas pressure,
Concentrate to about 1/50 of the volume of the molecular sieve chromatography column that follows.

Incidentally, as the ultrafiltration membrane, other than polysulfone type,
It is also possible to use poly-n-phenylene isophthalamide type (aromatic polyamide type), a complex of sodium polystyrene sulfonate and polyvinylbenzyltrimethylammonium chloride, a cellulose type and the like.

Thereafter, molecular sieve chromatography was performed by further passing through a column packed with spherical cross-linked agarose having a protein fraction range of 1 × 10 ⁴ to 1.3 × 10 ³ equilibrated with a phosphate buffered saline containing a chelating agent. The polypeptide chains can be separated. As the carrier, in addition to the spherical crosslinked agarose, spherical or fine granular crosslinked dextran having the above protein fraction range, crosslinked cellulose, a crosslinked product of N, N'-methylenebisacrylamide and allyldextran, porous polyacrylamide, etc. Can also be used.

Such carriers include Sephade
x), Sephacryl, Sepharose
arose), Sepharose CL, Cellulofin
e), Bio-Gel P, Bio-Gel A, etc. are commercially available.

As conditions for molecular sieve chromatography, it is preferable to use a sample having a column volume of 1/25 or less, particularly about 1/100 volume, or a column volume of water of 1/200 or less, particularly 1/1000 or less. Preferably, the flow velocity is a flow velocity to obtain a flowing water pressure equal to or lower than the allowable physical strength defined for each carrier, generally the column length.
It is preferable to carry out at a flow rate of not more than 1 hour per 10 cm.

The eluate is collected as fractions, the presence of the polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex is detected by the binary immunodiffusion method, and the fractions containing the polypeptide chain are combined. In this case, it does not matter whether the polypeptide chains that can be molecularly sieved are polymerized to form multimers or bound to contaminating RNA chains. This is because, in the solution environment in which the described molecular sieve chromatography is performed, the form that the polypeptide chain to be isolated can take is always constant, and depending on the property regarding the presumed existence mode of the polypeptide chain, , Because it often increases the separation effect.

Therefore, by combining the above-mentioned ion exchange chromatography in the presence of nonionic surfactant and molecular sieve chromatography under conditions close to physiological buffer environment, ribonucleoprotein (UsnRNP) complex was formed. It becomes more effective to separate and purify the derived polypeptide chain.

The polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex thus obtained is considered to be the B subunit and B'subunit constituting the U ₁ ribonucleoprotein complex (Arthritis Rheum. Vol. 29). Pp. 457-460 (1986)) constitutes the A 1 subunit that constitutes the U ₁ ribonucleoprotein complex and the A'subunit that constitutes the U ₂ ribonucleoprotein complex, U ₁ ribonucleoprotein complex. B
B'subunit constituting a subunit and U ₂ ribonucleoprotein complex, B'subunit constituting a U ₁ ribonucleoprotein complex, and B'subunit constituting a U ₂ ribonucleoprotein complex, or U ₁ ribonucleoprotein complex It may be a combination of B subunit, B'subunit constituting the complex and B "subunit constituting the U ₂ ribonucleoprotein complex. The size of each subunit differs depending on the animal species. This is because, for example, in the case of humans, the B subunit is reported to be about 28,000 and the B'subunit is reported to be about 29,000, but it is considered that the molecular weight is higher in cattle and the like. According to the method of the present invention, depending on the animal species, about 28
A polypeptide chain having a molecular weight of 000 to 34,000 is separated and purified. The above-mentioned polypeptide chain specifically reacts with an anti-RNP antibody conventionally used immunochemically, and therefore can be used as an antigen used as a diagnostic agent for detecting an antibody against a self-component.

Next, the second invention will be described in detail. In the second invention, for example, a polypeptide chain derived from a ribonucleoprotein complex separated and fractionated by a method combining biochemical methods or biochemical means as in the first invention is used as an antigen. The assay method of the present invention may be any assay method as long as the antigen is used. For example,
Enzyme immunoassay, radioimmunoassay, immunoturbidimetric method, immunohistochemistry method, latex agglutination method, hemagglutination method, fluorescent immunoassay method, immunochemophotometric method, dye immunoassay method and the like can be performed. A labeled immunoassay method will be described as a preferred example.

A solid surface, eg polystyrene pores, is covered with the polypeptide chain. Usually, this coating operation has a buffering action in the alkaline region, for example, a polypeptide chain is dissolved in a sodium carbonate buffer solution and used as a 0.01 to 100 μg / ml solution, and it is carried out at low temperature overnight. After that, the polypeptide chains that have not been physically adsorbed on the solid surface are removed by suction together with a buffer solution, and then 0.01 to 1% of a hydrophilic globular protein having no immunochemical cross-linking with the polypeptide chains, such as milk casein, is added. Blocking with (weight / volume) solution at room temperature for about 1 hour. This is a solution to be tested or a labeled secondary antibody solution to be added thereafter by covering a hydrophobic site on the solid surface not coated with the polypeptide chain or on the molecular surface of the polypeptide chain physically adsorbed on the solid surface. This is to prevent non-specific adsorption of protein components therein.

After that, in order to remove the polypeptide chain or protein component that has not been used for coating or blocking from the solid surface, it is thoroughly washed with a neutral washing solution containing a nonionic surfactant. As described above, the polypeptide chain serving as the antigen is immobilized on the carrier, and then the antibody is detected or quantified.

A subject to be appropriately diluted with a physiological buffer containing a nonionic surfactant, the polypeptide chain and a hydrophilic globular protein having no immunochemical cross-linking property, for example, patient serum, is coated with the polypeptide chain. The solid surface is contacted for a sufficient time to complete the antigen-antibody binding reaction.

Thereafter, the solid surface is sufficiently washed with a neutral washing solution containing a nonionic surfactant to complete the antigen-antibody binding reaction of the solid surface with a physiological solution containing an excessive amount of a labeled secondary antibody. Contact for a sufficient period of time. Here, the labeling substance is not particularly limited, and may be an enzyme, a radioisotope, a fluorescent substance or the like, but an enzyme label is particularly preferable.

Then, the solid surface is sufficiently washed with a neutral washing solution containing a nonionic surfactant to detect the presence or amount of the labeled secondary antibody. In the case of enzyme labeling, the solid surface is contacted with a substrate solution specific for the enzyme for a time sufficient to detect the product of the enzymatic reaction. In this case, the amount of the product produced by the enzymatic reaction is proportionally dependent on the amount of the antibody against the antigenic determinant on the polypeptide chain contained in the test subject, and thus indirectly in the test subject. Can be quantified.

(Example) Hereinafter, the present invention will be described in detail with reference to Examples regarding isolation and purification of polypeptide chains having a molecular weight of 33000 and 34000 constituting a ribonucleoprotein (UsnRNP) complex and its use.

Isolation and purification of polypeptide chain Example 1 Sodium chloride 8 g, potassium chloride 0.2 g, in buffer solution A: 11,
Phosphate buffer solution containing 2.7 g of disodium phosphate dodecahydrate and 0.2 g of 1 potassium phosphate.

Buffer B: As a protease inhibitor, ethylene glycol tetraacetic acid (EGTA) 10 ^-3 M, fluorophenylmethylsulphonyl fluoride (PMSF) 10 ^-3 M, leupeptin 0.05% (weight / volume), antipine Buffer A further containing 0.05% (weight / volume), chymostatin 0.05% (weight / volume), pepstatin A 0.05% (weight / volume).

Buffer C: Sodium chloride 0.14M, Nonidet P-40 (Poly
oxyethylene (9) p-tert-octylphenol, nonionic surfactant) 0.05% (volume / volume), ethylene glycol tetraacetic acid (EGTA) 5 × 10 ^-4 M, phenylmethyl sulfonyl fluoride (PMSF) 2 × 10 ^-4 M, dithiothreitol (DT
T) Tris-based buffer containing 2 × 10 ⁻⁴ M 0.02 M × HCl pH
7.4.

Buffer D: Sodium chloride 0.18M, Nonidet P-40 0.05
% (Volume / volume), ethylene glycol tetraacetic acid (EGTA)
5 × 10 ^-4 M, Fluorinated phenylmethylsulphonyl (PMS
F) Tris-based buffer solution containing 2 × 10 ⁻⁴ M and dithiothreitol (DTT) 2 × 10 ⁻⁴ M 0.02 M × HCl pH 7.4.

Buffer E: Ethylene glycol tetraacetic acid (EGTA) 2.5 x 10 ^-4 M
Buffer A further containing

1. Acquisition of used ribonucleoprotein (UsnRNP) complex fraction All the following operations were performed at 4 ° C.

450 ml of buffer solution B was added to 30 g of calf thymus acetone powder (manufactured by Pel-Freeze), and the mixture was dissolved overnight. Then, the suspension was centrifuged at 10,000 × g for 30 minutes, and the supernatant was used as the extract A. In order to carry out a second extraction from this precipitate, 100 ml of buffer solution B was added to the precipitate and stirred for 4 hours. Then, the suspension is added to 10,000 × g at 30
After centrifuging, the supernatant was used as the extract B and the extract A was combined with it to obtain the extract C. The total protein content of the obtained nuclear soluble fraction was 7.04 g as a result of protein quantification by the Lowry method.
Met.

Ammonium sulfate was added to the extract C so that the degree of saturation was 30%, the mixture was stirred for a time sufficient for salting out the protein (4 hours or more), and then centrifuged at 10,000 × g for 20 minutes. Then, 60% saturated ammonium sulfate was added to the supernatant, and the mixture was stirred for a time sufficient for salting out, followed by centrifugation at 10,000 xg for 20 minutes to obtain a ribonucleoprotein (UsnRNP) complex fraction. .

2. Fractionation by ion exchange chromatography The precipitate obtained as a ribonucleoprotein (UsnRNP) complex fraction was dissolved in as little buffer solution C as possible and
Was dialyzed for 2 nights. The dialyzed product was applied to a φ3.2 × 11 cm diethylaminoethyl (DEAE) sepharose (manufactured by Pharmacia) column equilibrated with buffer C at a flow rate of 100 ml / hour. Thereafter, the column was washed with buffer C until the unbound protein was removed (overnight). After washing the column,
The fraction containing the polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex was eluted with buffer D. The fractionation volume was 8.0 ml per fraction, and the protein in each fraction was quantified by the Lowry method. The state of this fractional elution is shown in FIG.

3. Fractionation by molecular sieve chromatography Fractions 135 to 155 were collected as fractions containing the polypeptide chain derived from the obtained ribonucleoprotein (UsnRNP) complex, and the exclusion limit molecular weight was 10.000. The mixture was concentrated to 3 ml under pressure of nitrogen gas using a filtration membrane (Advantech UK-10). The total protein amount after concentration was 92.2 mg. This was equilibrated with buffer E, and φ1.6 × 68 cm Ultrogel AcA44 (LKB
Column). Then, the sample in the column was developed and eluted with buffer D at a flow rate of 10 ml / hour, 1.75 ml was collected, and the absorbance at 280 nm was measured. Of the collected fractions, ribonucleoprotein (Us
Fractions containing the polypeptide chain derived from the (nRNP) complex (fractions 36 to 44) were combined and used as a purified polypeptide chain. Here, the binary immunodiffusion method is to dispense an antigen solution and an antibody solution into predetermined holes in a gel, respectively, and diffuse them to determine the shape of the precipitation line formed by the antigen-antibody reaction. This is a method for determining specificity. In the above example, as the antibody solution,
Serum containing anti-RNP antibody and serum containing anti-Sm antibody were used. The state of separation is shown in FIG. As a result of protein quantification by the Lowry method, this purified sample was 28 mg.

The molecular weight was measured by the SDS-PAGE method. Specifically, according to the method of Laemmli et al., A standard sample was developed in 12.5% acrylamide gel (degree of crosslinking 0.8). Electrophoresis conditions are 40 mA at the start of electrophoresis, 15 mA at concentrated electrophoresis, 20 mA at separation electrophoresis.
And In addition, the sample to be electrophoresed was a buffer containing no reducing agent in advance (312.5mN Tris-HCl, PH6.8, 0.1% BPB, 10% S
0.25 volume of DS, 20% glycerin) was added and the mixture was treated at room temperature for 30 minutes. After electrophoresis, the gel was stained with 0.05% CBB overnight, and the next day, 0.7
Decolorized with% acetic acid. As a result, protein bands with molecular weights of 33000 and 34000 were detected.

Measurement of anti-ribonucleoprotein antibody by ELISA method Example 2 Buffer a: sodium carbonate buffer 50 mM pH 8.85.

Sodium chloride 8g, potassium chloride 0.2 in buffer solution b: 1
g, disodium phosphate dodecahydrate 2.7 g, 1 potassium phosphate 0.2
A phosphate-based buffer solution containing g and 203 ml of Tween.

Sodium chloride 8g, potassium chloride 0.2 in buffer c: 1
g, disodium phosphate dodecahydrate 2.7 g, 1 potassium phosphate 0.2
g, Tween 203 ml, phosphate buffer containing 5% 2% non-fat dry milk solution.

2.5 μg / ml of polypeptide chains separated by the method described in Example 1 in which a polystyrene 96-well ELISA microplate (manufactured by Sumitomo Bakelite Co., Ltd.) was dissolved in the buffer solution a per well
200 μl of the solution was coated overnight at 4 ° C. Then, the next day, the cells were blocked with 400 μl of 0.1% skim milk powder solution at room temperature for 1 hour.

Thereafter, the polystyrene pores were washed 6 times with buffer solution b, and patient serum containing anti-RNP antibody diluted with buffer solution c 2
00 μl was added and reacted at room temperature for 1 hour. Thereafter, the polystyrene pores were further washed 6 times with buffer solution b, and the alkaline phosphatase-labeled anti-human IgG dissolved in buffer solution c was used.
+ IgA + IgM antibody [KPL (enzyme / antibody = 3)] 500ng
200 μl of the / ml solution was added and reacted at room temperature for 1 hour.

Subsequently, following these two steps of antigen-antibody reaction, the polystyrene holes were washed 6 times with buffer solution b, 200 μl of 1 mg / ml alkaline solution of paranitrophenyl phosphate was added, and the enzyme reaction was carried out at room temperature for 1 hour to produce The amount of para nitrophenol is measured by a plate spectrophotometer (Corona Electric Co., Ltd.) as an increase in absorbance at 405 nm. The results are shown in FIG.

Measurement of antibody by immunoblotting method Example 3 The immunochemical cross-reactivity of both obtained polypeptide chains was analyzed by the immunoblotting method.

The protein band after SDS polyacrylamide gel electrophoresis was transferred onto a nitrocellulose membrane according to the method of Towbin et al. The transcription operation was carried out in a 0 ° C water bath to maintain the antigenicity of the transcribed protein. After the transfer is completed, the nitrocellulose membrane is coated with Tris buffered saline containing 2% bovine serum albumin (15 mM Tris-HCl, pH 7.4, 0.2 M sodium chloride).
The untranscribed sites were coated overnight with. The next day, this nitrocellulose membrane was diluted 1/500 with the above-mentioned Tris buffered saline containing 0.2% bovine serum albumin and a secondary antibody similarly diluted (peroxidase-labeled anti-human IgG +
A + M antibody) solution was sequentially dipped and reacted at room temperature for 20 minutes. As the primary antibody solution, anti-RNP antibody, anti-Sm antibody, and normal human serum were used. In addition, unreacted primary antibody and secondary antibody remaining after each antigen-antibody reaction, an excess amount of washing solution (15 mM Tris-HCl, pH7.4, 0.1
It was washed off with 5% Triton X-100, 0.2M sodium chloride) three times for 5 minutes. After completion of these antigen-antibody reactions, the nitrocellulose membrane was transferred to a substrate solution (50 mM Tris-HCl, pH
8.0, 0.9% sodium chloride, 0.05% hydrogen peroxide solution, 0.05
% 3,3 ′ diaminobenzidine tetrahydrochloride) to develop the color.

As the anti-RNP antibody and anti-Sm antibody used in this method, patient sera containing the same type of antibody as American CDC standard serum was used.

As a result, whichever polypeptide chain (33000, 34000)
Both of them strongly reacted with serum containing anti-RNP antibody, and did not react with normal human serum or serum containing anti-Sm antibody at all. From this result, it can be evaluated that these polypeptide chains are specific to the anti-RNP antibody.

〔The invention's effect〕

Conventionally, a cell extract has been used as it is as an antigen used when measuring an antibody against a ribonucleoprotein complex.

However, in recent years, the molecular properties of the elements constituting the complex have been clarified, and various theories have been discussed regarding the relationship between these functions and the functions of antibodies against them that are present in the serum of patients with autoimmune diseases and the etiology. It has become important to recognize the role of each component of the complex.

Therefore, as in the present invention, the establishment of a method for measuring the antibody in the test object corresponding to the polypeptide chain unit that constitutes the complex provides important medical information. Therefore, it is very effective in diagnosing autoimmune diseases and the like.

[Brief description of drawings]

FIG. 1 shows the ribonucleoprotein (UsnRN) in the example of the present invention.
P) a diagram showing fractionated elution by ion exchange chromatography of a fraction containing a polypeptide chain derived from the complex,
FIG. 2 shows the ribonucleoprotein (UsnRN) in the example of the present invention.
It is a figure which shows the classification | segmentation by the molecular sieve chromatography of the polypeptide chain originating in (P) complex. Furthermore, in FIG.
FIG. 2 is a diagram showing the measurement range of a subject containing an antibody against the polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex isolated and purified by the method of the present invention by ELISA.

Claims (3)

[Claims] 1. A UsnRNP complex is extracted from an animal tissue extract, and then the polypeptide chain to be isolated is dissociated from other UsnRNP complex constituents in the presence of a nonionic surfactant. Ion-exchange chromatography in the state, fractions containing the polypeptide chain are separated, and then obtained by separating the polypeptide chain to be isolated by molecular sieve chromatography, and purified Us
A polypeptide chain that constitutes an nRNP complex and specifically reacts with an anti-RNP antibody. 2. A UsnRNP complex is extracted from an animal tissue extract, and then the polypeptide chain to be isolated is dissociated from other UsnRNP complex constituents in the presence of a nonionic surfactant. Ion exchange chromatography in the state, fractions containing the polypeptide chain are fractionated, and then the polypeptide chain to be isolated is fractionated by molecular sieve chromatography, and specifically reacts with the anti-RNP antibody. A method for producing a polypeptide chain constituting a purified UsnRNP complex, which comprises collecting a fraction. 3. A method for measuring an anti-UsnRNP antibody, which comprises using the polypeptide chain according to claim 1 as an antigen and detecting or quantifying an antibody against the polypeptide chain.