JPH0826074B2 - 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 an anti-UsnRNP antibody using this polypeptide chain.

[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 the constituent RNA chain and the constituent polypeptide chain. Classified as ₂ , U ₄ , U ₅ and U ₆ RNPs.
In addition, these UsnRNPs are ubiquitously present in the cell nucleus of animals, their antigenicity is common across species, and they are conserved in the evolution of animals. Normally, healthy people do not have an antibody group against the complex, but in autoimmune diseases, particularly systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), the serum does not contain the complex. Possess antibody group.

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 react with anti-RNP antibody, and some 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 anti-RNP antibody by conventional techniques, and to develop a method for detecting an antibody in 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 extracts UsnRNP in the presence of a non-ionic detergent.
Ion exchange chromatography is performed with part of the polypeptide chains dissociated, and the fraction containing the UsnRNP complex derivative containing the undissociated polypeptide chains is collected, and urea is then added to it. And an anionic surfactant are present to dissociate the polypeptide chain to be isolated, and then molecular sieve chromatography is performed to separate the polypeptide chain to be isolated, which is specific to the anti-RNP antibody. Purified UsnRNP, characterized in that it obtains a fraction that reacts chemically
Method for producing a polypeptide chain constituting a complex (hereinafter referred to as the first
Of the invention), a polypeptide chain obtained by the production method and the polypeptide chain as an antigen,
The present invention relates to a method for measuring an anti-UsnRNP antibody (hereinafter referred to as a second invention), which comprises detecting or quantifying an antibody against the polypeptide chain present in a test subject.

 First, the first invention will be described in detail.

In the process of using a cell extract or a cell nuclear extract as an animal tissue extract and extracting a ribonucleoprotein (UsnRNP) complex from the cell extract, the complex is solubilized at a dilute salt concentration or a physiological salt concentration. , An inhibitor of proteolytic enzyme was added to protect RNA that contributes to the maintenance of the higher-order structure of the complex from the invasion of all possible proteolytic enzymes that are solubilized at the same time. It is preferably extracted with an extraction buffer. Furthermore, regarding this extraction procedure, as many ribonucleoproteins (UsnRNP) as possible
In order to obtain a complex, it is preferable to perform extraction from the object to be extracted twice and combine the extracts twice. Further, the detection of the ribonucleoprotein (UsnRNP) complex in the extract is preferably performed by the binary immunodiffusion method because the operation is simple.

Thereafter, a salting-out agent such as powdery ammonium sulfate is gradually added to the extract to salt-out and precipitate the protein and its complex. For example, ammonium sulfate saturation of 30%
Fractions that precipitate at -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, a nonionic surfactant is contained in the starting buffer solution for ion exchange chromatography. When such a buffer solution is used, there are polypeptide chains that dissociate from the ribonucleoprotein (UsnRNP) complex and those that do not dissociate, and the column packed with the ion exchange carrier dissociates from the dissociated polypeptide chains. A ribonucleoprotein (UsnRNP) complex derivative containing an unmodified polypeptide chain is passed through as a solution. The nonionic surfactant is present at a concentration necessary to dissociate some polypeptide chains.

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 was passed through a column packed with a carrier for ion exchange chromatography, and a ribonucleoprotein (Us containing the polypeptide chain and the undissociated polypeptide chain)
The derivative of the nRNP) complex is ionically bound to the functional groups of the carrier.

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.

After that, the column is added with an excess amount of the above-mentioned starting buffer for ion exchange chromatography and a starting buffer for ion exchange chromatography added with a concentration of a salt that does not elute the substance to be isolated. Nucleic acid and protein components and polypeptide chains eluted from the carrier under the conditions are washed away. After washing the column, the ionic strength in the buffer solution was increased by adding salts, and a derivative of a ribonucleoprotein (UsnRNP) complex containing undissociated polypeptide chains captured by the salts and functional groups, and An ion exchange reaction is performed with the dissociated polypeptide chains to elute them. The eluate was collected as fractions, and the ribonucleoprotein (UsnR) was detected by the two-way immunodiffusion method using an antibody against two or more ribonucleoprotein (UsnRNP) complexes as an antibody to be compared.
NP) having a polypeptide chain derived from a complex and detecting a fraction in which the polypeptide chain is not in a dissociated state, and having an exclusion limit molecular weight of 10,000, for example, an ultrafiltration membrane made of polysulfone It is concentrated under nitrogen gas pressurization to about 1/50 of the volume of the subsequent molecular sieve chromatography column.

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.

Then, urea and an anionic surfactant are added to the obtained fraction, and the mixture is allowed to stand on ice for a certain period of time. In this case, by adding urea and an anionic surfactant, the polypeptide chain to be isolated becomes ribonucleoprotein (UsnRNP).
It is likely to dissociate from the derivative of the complex. As the anionic surfactant, it is preferable to use N-lauroyl sarcosine sodium. The concentration is 0.1
It is preferably set to 1.0% (weight / volume).

The urea concentration is preferably 3 M (molar concentration) or more, and particularly preferably the maximum dissolved concentration that does not cause precipitation at low temperatures (about 4 ° C to 10 ° C).

Then, phosphate buffered saline containing urea and anionic surfactants at the same concentration as the final concentration of urea and anionic surfactants added to the concentrated fraction after ion exchange chromatography, and a chelating agent. 1 equilibrated with
Molecular sieve chromatography can be performed by passing through a column packed with spherical cross-linked agarose or spherical cross-linked acrylamide having a protein fraction range of × 10 ⁴ to 1.3 × 10 ⁵ to separate the polypeptide chain to be isolated.

As the carrier, in addition to the spherical cross-linked agarose and spherical cross-linked acrylamide, spherical or microgranular cross-linked dextran having the above protein fraction range, cross-linked cellulose, cross-linked product of N, N'-methylenebisacrylamide and allyl dextran, porous Polyacrylamide or the like 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 was collected as fractions, dialyzed against a physiological buffer, and the presence of a polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex was detected by the binary immunodiffusion method. Combine the fractions to be used. When molecular sieve chromatography is performed in such a solution environment, the polypeptide chain to be isolated is considered to be in a state of being dissociated from the derivative of the ribonucleoprotein (UsnRNP) complex, but the polypeptide that can be subjected to molecular sieving It does not matter whether the peptide chain is polymerized to form a multimer or is bound to a contaminating RNA chain. This is because, in the solution environment in which the described molecular sieve chromatography is performed, the form of the polypeptide chain to be isolated is constant, and due to the nature of this putative polypeptide chain, it is often separated. This will increase the effect.

In the present invention, a ribonucleoprotein (UsnRNP) complex is obtained by combining the above-mentioned ion exchange chromatography in the presence of a nonionic surfactant and molecular sieve chromatography in the presence of urea and an anionic surfactant. Separation and purification of the derived polypeptide chain is effectively performed.

The polypeptide chain derived from the ribonucleoprotein (UsnRNP) complex thus obtained has the ribonucleoprotein (UsnRNP)
P) A polypeptide chain having a molecular weight of about 67,000 to 80,000 constituting a complex.

It is considered to be the largest polypeptide chain that constitutes U ₁ SnRNP. The range of the molecular weight is as described above because the molecular weight of the polypeptide chain of the ribonucleoprotein complex varies depending on the animal species.

According to the method of the present invention, about 67,0
Polypeptide chains having a molecular weight of 00-80,000 are separated.

Since the above-mentioned polypeptide chain specifically reacts with an anti-RNP antibody which is conventionally used immunochemically, it 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, the polypeptide chain derived from the ribonucleoprotein complex separated and fractionated according to 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, immunochemiluminescence method, dye immunoassay method, etc. can be performed. it can. 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 with the polypeptide chain The coated solid surface is contacted for a time sufficient 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 for isolation and purification of a polypeptide chain having a molecular weight of about 80,000 constituting a ribonucleoprotein (UsnRNP) complex.

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

Buffer solution B: As a proteolytic enzyme inhibitor in buffer solution A, ethylene glycol tetraacetic acid (EGTA) 10 ^-3 M, fluorophenylmethylsulphonyl fluoride (PMSF) 10 ^-3 M, leupeptin 0.05%
(Weight / volume), antipine 0.05% (weight / volume),
Chymostatin 0.05% (weight / volume), pepstatin A0.0
Buffer additionally containing 5% (weight / volume).

Buffer C: Sodium chloride 0.14M, Nonidet P-40 (Pol
yoxyethylene (9) p-tert-octylphenol, nonionic surfactant) 0.05% (volume / volume), ethylene glycol tetraacetic acid (EGTA) 5 × 10 ^-4 M, fluorinated phenylmethyl sulfonyl (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: Sodium chloride 0.3M, Nonidet P-40 0.05
% (Volume / volume), ethylene glycol tetraacetic acid (EGTA)
5 × 10 ^-4 M, Phenylmethylsulfonyl fluoride (PMS
F) Tris-based buffer containing 2 × 10 ⁻⁴ M, dithiothreitol (DTT) 2 × 10 ⁻⁴ M, 0.02 M × HCl pH 7.4.

Buffer F: Buffer A Urea 4.0 M, buffer containing N-lauroylsarcosine sodium 0.5% (weight / volume) and ethylene glycol tetraacetic acid (EGTA) 2.5 × 10 ⁻⁴ M.

Buffer G: Buffer A with ethylene glycol tetraacetic acid (EGTA)
Buffer additionally containing 2.5 × 10 ⁻⁴ M.

(1) Obtaining the used ribonucleoprotein (UsnRNP) complex fraction The following procedure was carried out using molecular sieve chromatography.
Everything was done at 4 ° C except at 0 ° 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
He dialyzed for 2 days and 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 is buffered with buffer D until the proteins, nucleic acids and polypeptide chains bound to the ion exchange carrier in the unbonded or buffer C solution environment are removed (1
Washed). After washing the column, a fraction containing a derivative of a ribonucleoprotein (UsnRNP) complex having the polypeptide chain to be isolated as one component was eluted with buffer E. 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 separation and elution is shown in FIG.

(3) Fractionation by molecular sieve chromatography The fractions 215 to 230 containing the obtained derivative of the ribonucleoprotein (UsnRNP) complex are excluded. An ultrafiltration membrane (Advantech UK-10 ) Under nitrogen gas pressurization 2.
It was concentrated to 4 ml. The total protein amount after concentration was 100.8 mg. To this, a final concentration of 4M urea and a final concentration of 0.5% N-
Lauroyl sarcosine sodium was added and dissolved, and the mixture was allowed to stand on ice for 1 hour. Next, φ1 equilibrated with buffer F.
It was applied to a 6 × 68 cm Ultrogel AcA44 (manufactured by LKB) column. Then, the sample in the column is treated with buffer F at 10 ml /
It was developed and eluted at a flow rate of time, and 1.65 ml each was collected as fractions. The collected fractions were dialyzed against the buffer G for each fraction, and the polyisotope derived from the ribonucleoprotein (UsnRNP) complex-derived complex was analyzed by the binary immunodiffusion method. Fractions in which peptide chains were detected (32nd to 35th fractions) were combined to give purified polypeptide chains. 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 Examples, the serum containing anti-RNP antibody and the serum containing anti-Sm antibody were used as the antibody solution. The state of separation is shown in FIG. The yield of the obtained purified polypeptide chain was 3.0 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 (crosslinking degree 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, it was detected as a protein band having a molecular weight of 80,000.

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 similarly diluted secondary antibody (peroxidase-labeled anti-human IgG + I).
gA + IgM antibody) solution and then reacted for 20 minutes at room temperature. 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 NaCl for 3 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%
It was immersed in sodium chloride, 0.05% hydrogen peroxide solution, 0.05% DAB) 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, the purified polypeptide chain having a molecular weight of 80,000 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 biological functional properties of the elements constituting the complex have been clarified, and there are various theories about the relationship between these functions and the role of antibodies against them present in the serum of patients with autoimmune diseases and the etiology. Has been discussed, and recognition of physiological functions in constituent units of the complex has become important.

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 constituting the complex is one that provides important medical information. Therefore, the present invention 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.

Claims (3)

[Claims] 1. UsnRNP complex is extracted from an animal tissue extract, and then UsnRNP is present in the presence of a nonionic surfactant.
Ion exchange chromatography is performed with part of the polypeptide chains dissociated, and the fraction containing the UsnRNP complex derivative containing the undissociated polypeptide chains is collected, and urea is then added to it. And purified by the presence of an anionic surfactant to dissociate the polypeptide chain to be isolated, followed by molecular sieve chromatography to fractionate the polypeptide chain to be isolated, purified UsnRNP A polypeptide chain that constitutes a complex and specifically reacts with an anti-RNP antibody. 2. UsnRNP complex is extracted from an animal tissue extract, and then UsnRNP is present in the presence of a nonionic surfactant.
Ion exchange chromatography is performed with part of the polypeptide chains dissociated, and the fraction containing the UsnRNP complex derivative containing the undissociated polypeptide chains is collected, and urea is then added to it. And an anionic surfactant are present to dissociate the polypeptide chain to be isolated, and then molecular sieve chromatography is performed to separate the polypeptide chain to be isolated and specifically react with the anti-RNP antibody. Purified UsnRNP, characterized in that
A method for producing a polypeptide chain constituting a complex. 3. An anti-UsnRNP characterized by using the polypeptide chain according to claim 1 as an antigen to detect or quantify an antibody against the polypeptide chain present in a test subject.
Antibody measurement method.