JPH0826075B2 - Purified polypeptide chain constituting ribonucleoprotein complex, method for producing the same, and method for measuring anti-Smith antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to the prediction, diagnosis, and diagnosis of autoimmune diseases and autoimmune phenomena
The present invention relates to a polypeptide chain constituting a purified ribonucleoprotein complex which is effectively used for follow-up observation, a method for producing the same, and a method for measuring an anti-Smith antibody using this polypeptide chain.

[Conventional technology]

The ribonucleoprotein complex has a structure in which several polypeptide chains are non-covalently bound to uridine-rich low molecular weight nuclear RNA, and is universally present in the nucleus of animals. Therefore, the immune system of a healthy organism does not have the opportunity to recognize the complex, and even if such an opportunity occurs, the cells that should produce the antibody against the complex do not become a prohibited clone and never produce the antibody. . However, in autoimmune diseases, especially systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD),
Antibodies are produced against the complex by some mechanism,
Due to this, lesions associated with various autoimmune phenomena such as inflammation, ulcer, and skin rash are developed.

Therefore, the essential clinical significance of detecting the antibody group appearing in the sera of patients suffering from these autoimmune diseases is
In predicting, diagnosing, or observing autoimmune diseases and phenomena.

Hitherto, as an antigen used for detecting the antibody group, an extract of human cultured cells or mammalian cells or the cells themselves have been used. This is because since the antigenicity of the complex is common across species, any mammal-derived antibody can be used as an antigen for detecting an antibody against an autologous component found in the serum of an autoimmune disease-affected person, that is, an autoantibody. Because you can.

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

Many methods have been described in the scientific literature for the purification and isolation of ribonucleoprotein complexes [J. Biol. Chem.
8: 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 all cases, the methods involved purification and isolation as a ribonucleoprotein complex.

Further, since purification of the complex by immunoadsorption chromatography uses a patient serum-derived one as an immobilized ligand, it is difficult to obtain a considerable amount of the serum and the antibody composition contained in the serum is not uniform. It was unsuitable as a means for refining for industrial purposes because it was not like that.

[Problems to be Solved by the Invention]

As mentioned above, the ribonucleoprotein complex is 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-ribonucleoprotein antibodies and some with anti-Smith antibodies. There are also polypeptide chains that react with any antibody.

Therefore, it is difficult to obtain an antigen preparation that reacts only with anti-Smith antibody by the conventional techniques, and a method for detecting an antibody of a patient suffering from an autoimmune disease using such an antigen preparation is developed. It was also difficult.

Therefore, the present inventors have conducted extensive studies on a method for isolating a polypeptide chain that constitutes a ribonucleoprotein that reacts with an anti-Smith antibody without losing its antigenicity,
The present invention has been completed.

[Means for solving the problem]

The present invention extracts a ribonucleoprotein complex from an animal tissue extract, and then ionizes it in the state where a part of the polypeptide chain of the ribonucleoprotein complex is dissociated in the presence of a nonionic surfactant. After exchange chromatography, fractions containing ribonucleoprotein complex derivatives containing undissociated polypeptide chains are collected, and then subjected to molecular sieve chromatography in a physiological solution environment to confirm that they are not dissociated. A fraction containing a ribonucleoprotein complex derivative containing a polypeptide chain is fractionated, and further, an anionic surfactant and a protein higher order structure modifier are present therein to isolate the poly The purification is characterized by dissociating the peptide chains, followed by molecular sieve chromatography to separate the polypeptide chains to be isolated, and to obtain a fraction that specifically reacts with the anti-Smith antibody. For producing a polypeptide chain constituting the ribonucleoprotein complex (hereinafter referred to as the first invention), the polypeptide chain obtained by the production method, and the purification of the nuclear complex separated and fractionated by the above production method The present invention relates to an anti-Smith antibody assay method (hereinafter, referred to as a second invention) for detecting or quantifying an anti-Smith antibody present in a test subject using the constituted polypeptide chain as an antigen.

 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 complex from the cell extract, the complex is solubilized at a dilute salt concentration or a physiological salt concentration. It is preferably extracted with an extraction buffer supplemented with an inhibitor against the proteolytic enzyme in order to protect it from invasion by all possible proteolytic enzymes to be solubilized. Further, regarding this extraction operation, in order to obtain as many ribonucleoprotein complexes as possible, it is preferable to perform extraction from the substance to be extracted twice and combine the extracts twice. The detection of the ribonucleoprotein 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, most of the polypeptide chains to be isolated are not dissociated, but some polypeptide chains are considered to be dissociated from the nuclear complex under the solution environment. Therefore, the operation of dissolving the obtained precipitated protein in the starting buffer for ion exchange chromatography, and dialyzing against the starting buffer for ion exchange chromatography, and the purpose of the subsequent ion exchange chromatography are as follows: The derivative of the ribonucleoprotein complex to which the polypeptide chain is bound is separated from some other dissociated polypeptide chain. The nonionic surfactant is present at a concentration necessary to dissociate some polypeptide chains.

Examples of nonionic surfactants include polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene methylhexyl ether, polyoxyethylene methyl dodecyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitol ester. Etc. 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, and the derivative of the ribonucleoprotein complex containing the undissociated polypeptide chain is ionically bonded to the functional group of the carrier.

As the carrier for ion exchange chromatography, diethylaminoethyl group, diethyl- (α-hydroxy-
Propyl) aminoethyl group, polyethyleneimine group, spherical or microgranular crosslinked agarose, crosslinked dextran, crosslinked cellulose having functional groups such as amine mixture,
Polymethacrylate resin, styrene / divinylbenzene copolymer, hydrophilic silica, etc. can be used.

As such a carrier for ion exchange chromatography, DEAE-Sephadex, DEAE-Sapharose, DEAE-cellulose (Cellulose),
DEAE-Bio Gel, Cellulose
e) -DEAE, QAE-Sephadex, cellulose (Cellulose) -TEAE and the like are commercially available.

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

Then, 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 are not ionically bound to the functional group. After washing the column, the ionic strength in the buffer solution was increased by adding salts, and an ion exchange reaction was performed with a derivative of the ribonucleoprotein complex containing the undissociated polypeptide chain captured by the functional group. And elute them. The eluate was collected as fractions, and the fraction containing the derivative of the ribonucleoprotein complex was detected by the binary immunodiffusion method, and the eluate was combined and the exclusion limit molecular weight was 10,000, for example, polysulfone ultrafine material. It is concentrated to about 1/50 of the column volume of molecular sieve chromatography under a nitrogen gas pressurization, followed by a physiological solution environment with a filtration membrane.

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, molecular sieve chromatography is performed in a physiological solution environment. Specifically, spherical cross-linking having a protein fraction range of 1 × 10 ⁴ to 1.3 × 10 ⁵ equilibrated with a phosphate buffered saline containing a chelating agent is performed. Molecular sieve chromatography can be performed by passing through a column packed with agarose. Thus, the polypeptide chain to be isolated is separated as a ribonucleoprotein complex derivative.

As the carrier, in addition to the spherical agarose, spherical or fine-granular crosslinked dextran having the protein fractionation range, crosslinked cellulose, a crosslinked product of N, N′-methylenebisacrylamide and allyldextran, porous polyacrylamide, etc. It can also be used.

Such carriers include Sephadex.
x), Sephacryl, Sepharose
arose), Sepharose CL, Cellulofi
ne), Bio-Gel P, Bio-Gel A, etc. are commercially available.

As the conditions of this molecular sieve chromatography, 1/25 volume or less of the column volume, particularly 1/100 volume of the sample, or 1/200 or less of the water weight of the column volume, particularly preferably 1/1000 or less of the sample, The flow velocity is preferably a flow velocity for obtaining a flowing water pressure equal to or lower than the permissible physical strength defined for each carrier, and generally a flow velocity of 1 hour or less per 10 cm of column length.

The eluate is collected as fractions, and the fractions that react with the anti-Smith antibody are collected by the binary immunodiffusion method and combined to form an exclusion limit molecular weight of 10,000. For example, an ultrafiltration membrane made of polysulfone is used under nitrogen gas pressure, Concentrate to approximately 1/50 of the volume of the subsequent molecular sieve chromatography column.

In addition, as the ultrafiltration membrane, other than polysulfone type,
It is also possible to use poly-n-phenylene isophthalic acid amide type (aromatic polyamide type), a composite of polystyrene sulfonic acid sodium soda and polyvinylbenzyltrimethylammonium chloride, and a cellulose type.

Further, subsequently, an anionic surfactant and a protein conformational modifier are added to this concentrated solution to dissociate the polypeptide chain to be isolated from the derivative of the ribonucleoprotein complex. As the anionic surfactant, it is preferable to use N-lauroylsarcosine sodium. The concentration is preferably 0.1 to 1.0% (weight / volume).

Further, it is preferable to use urea as a protein structural denaturant. 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). And then
It has a protein fraction range of 1 × 10 ⁴ to 1.3 × 10 ⁵ equilibrated with a phosphate buffered saline containing such an anionic surfactant, a protein higher order structure modifier and a chelating agent. Molecular sieve chromatography can be carried out by passing through a column packed with spherical cross-linked agarose to separate the polypeptide chain to be isolated.

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. It can also be used.

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

As conditions for this molecular sieve chromatography, use a sample of 1/25 volume or less of the column volume, particularly about 1/100 volume, or 1/200 or less of the water weight of the column volume, especially 1/1000 or less. The flow rate is preferably such that a flow pressure below the permissible physical strength determined for each carrier is obtained, generally, a flow rate of 1 hour or less per 10 cm of column length. The temperature at which the chromatography is performed is 10 ° C or lower,
The temperature is preferably a temperature at which the added protein structural denaturant does not precipitate.

The eluate is collected as fractions, subjected to SDS polyacrylamide gel (15% gel concentration, crosslinking degree 0.8), and the protein band is transferred to a nitrocellulose membrane for immunoblotting analysis. Fractions containing a polypeptide chain that reacts with the anti-Smith antibody were detected by the analysis, and these fractions were combined to obtain a purified polypeptide chain sample bearing the Smith antigen.

The polypeptide chain derived from the ribonucleoprotein complex thus obtained is a polypeptide chain having a molecular weight of about 15,000 to 18,000 (SDS-PAGE method, the migration distance of a molecular weight marker as a comparison control, and the migration of the standard sample). It is the value calculated from the comparison with the distance).

It is considered to be the D subunit that constitutes the ribonucleoprotein complex. The reason why the molecular weight varies as described above is that the molecular weight of the polypeptide chain varies depending on the animal species.

According to the method of the present invention, about 15,0
Polypeptide chains having a molecular weight of 00 to 18,000 are separated.

Since the above-mentioned polypeptide chain specifically reacts with an anti-Smith antibody conventionally used immunochemically, it can be used as an antibody against an autologous cell component, that is, an antigen used for a diagnostic agent for detecting autoantibodies.

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 to be added thereafter by covering an easily adsorbable site on the molecular surface of the solid surface which is not coated with the polypeptide chain or on the solid surface which is physically adsorbed on the solid surface. This is to prevent non-specific adsorption of protein components in the solution.

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) The molecular weight of the ribonucleoprotein complex of about 16,0 is shown below.
The present invention will be described in detail by way of Examples with respect to isolation and purification of the polypeptide chain of 00.

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% (weight / 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
% (Weight / 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
% (Weight / 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: Ethylene glycol tetraacetic acid (EGTA) in buffer A
Buffer additionally containing 2.5 × 10 ⁻⁴ M.

Buffer G: Urea 4.0M in buffer A, N-lauroylsarcosine 0.5% (weight / volume), ethylene glycol tetraacetic acid (E
GTA) A buffer solution further containing 2.5 × 10 ⁻⁴ M.

All the operations described below were performed at 4 ° C. except that molecular sieve chromatography in the presence of an anionic surfactant and urea was performed at 10 ° C.

(1) Acquisition of Tissue Extract Containing Ribonucleoprotein Complex Used The buffer B450 ml 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 perform the 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 Extract C so that the degree of saturation was 30%, the mixture was stirred for a time sufficient for salting out proteins (4 hours or more), and then centrifuged at 10,000 × g for 20 minutes. Then, add 60% saturated ammonium sulfate to the supernatant, stir for sufficient time for salting out, and centrifuge at 10,000 xg for 20 minutes to remove the precipitate, which is a ribonucleoprotein complex fraction. Obtained (2) Fractionation by ion exchange chromatography The precipitate obtained as a ribonucleoprotein complex fraction was dissolved in as little buffer solution C as possible and
I dialyzed at night and lunch. Φ3.2 × 1 equilibrated dialysate with buffer C
A 1 cm diethylaminoethyl (DEAE) Sepharose (Farmasia) column was loaded at a flow rate of 100 ml / hour. Then, the column was washed with buffer D under a solution environment of unbound or buffer C until the protein, nucleic acid and polypeptide chains bound to the ion exchange carrier were removed (overnight). After washing the column, a fraction containing a derivative of a ribonucleoprotein complex having the polypeptide chain to be isolated as one component was eluted with buffer solution 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 in a physiological solution environment 215 containing a derivative of the obtained ribonucleoprotein complex
The 230th fraction was concentrated to 2.4 ml under pressure of nitrogen gas with an ultrafiltration membrane (Advantech UK-10) having an exclusion molecular weight of 10,000. The total protein amount after concentration was 100.8 mg.

Then, it was applied to a φ1.6 × 68 cm Ultrogel AcA44 (manufactured by LKB) column equilibrated with the buffer solution F. Thereafter, the sample in the column was developed and eluted with the buffer F at a flow rate of 11 ml / hour, 1.8 ml each was collected, and the absorbance at 280 nm was measured. Of the collected fractions, from the 34th fraction containing the ribonucleoprotein complex to which the polypeptide chain carrying Smith antigen derived from the ribonucleoprotein complex has been added by the binary immunodiffusion method Excluding the 42nd, molecular weight exclusion limit 10,000
It was concentrated to 2 ml under pressure of nitrogen gas using an ultrafiltration membrane (Advantech UK-10).

 The state of separation is shown in FIG.

(4) Fractionation by molecular sieve chromatography in a solution environment containing an anionic surfactant and a protein higher order structure denaturing agent The fraction containing the concentrated ribonucleoprotein complex was added to the anionic surfactant. N-lauroyl sarcosine as 0.
5% (weight / volume), as a protein structural denaturant, was added to urea G at a concentration of 4.0 M to pre-equilibrate with a buffer G to a φ1.6 × 68 cm Ultrogel AcA44 (LKB) column. Hung Then, the sample in the column was developed and eluted with buffer G at a flow rate of 10 ml / hour, 1.65 ml was collected, and the absorbance at 280 nm was measured. Of the collected fractions, the immunoblotting analysis method was performed to combine and purify the 44th to 47th fractions containing the polypeptide chain that carries the Smith antigen derived from the ribonucleoprotein complex. It was used as a polypeptide chain preparation.

 The state of separation is shown in FIG.

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 sample buffer solution (312.5 mM 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, the purified polypeptide chain migrated to about 16,000 positions calculated from the migration position of the molecular weight marker.

Measurement of antibody by immunoblotting method Example 3 The immunochemical crossability of the obtained polypeptide chain 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 the primary antibody (anti-Smith antibody) solution and the similarly diluted secondary antibody (peroxidase-labeled anti-human). IgG + IgA + IgM antibody) solution was sequentially dipped and reacted at room temperature for 30 minutes. In addition, unreacted primary antibody and secondary antibody remaining after each antigen-antibody reaction was treated with an excessive amount of washing solution (15 mM Tris-HCl, pH7.4, 0.15).
% Triton X-100, 0.2M NaCl) and washed off 3 times for 5 minutes. After completion of these antigen-antibody reactions, the nitrocellulose membrane was immersed in a substrate solution (50 mM Tris-hydrochloric acid, pH 8.0, 0.9% sodium chloride, 0.05% hydrogen peroxide solution, 0.05% DAB) for color development.

As the anti-Smith 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 about 16,000 reacted strongly with the serum containing anti-Smith antibody. Since this polypeptide chain specifically reacts with the anti-Smith antibody, it does not react with 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 that appear 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 for diagnosing and observing autoimmune diseases and the like, or for predicting autoimmune phenomena.

[Brief description of drawings]

FIG. 1 shows the fractional elution by ion exchange chromatography of the fraction containing the polypeptide chain derived from the ribonucleoprotein complex in the example of the present invention, and FIG. 2 shows the ribonucleoprotein in the example of the present invention. FIG. 3 is a diagram showing the separation of the polypeptide chains derived from the complex by molecular sieve chromatography in a physiological solution environment. FIG. 3 is the anionic interface of the peripeptide chains derived from the ribonucleoprotein complex in the example of the present invention. It is a figure which shows the classification | category by the molecular sieve chromatography in the presence of a higher order structure denaturing agent of an active agent and a protein.

Claims (3)

[Claims] 1. A ribonucleoprotein complex is extracted from an animal tissue extract, and then a part of the polypeptide chain of the ribonucleoprotein complex is dissociated in the presence of a nonionic surfactant. Fractions containing ribonucleoprotein complex derivatives containing undissociated polypeptide chains were collected by ion exchange chromatography and then subjected to molecular sieve chromatography in a physiological solution environment to dissociate the fractions. A fraction containing a ribonucleoprotein complex derivative containing a non-polypeptide chain is fractionated, and an anionic surfactant and a protein conformational modifier are present therein for isolation. A purified ribonucleoprotein complex is obtained by dissociating the polypeptide chains and then performing molecular sieve chromatography to fractionate the polypeptide chains to be isolated. , Anti-Smith antibodies specifically reactive with the polypeptide chain which. 2. A method in which a ribonucleoprotein complex is extracted from an animal tissue extract, and then a part of the polypeptide chain of the ribonucleoprotein complex is dissociated in the presence of a nonionic surfactant. Fractions containing ribonucleoprotein complex derivatives containing undissociated polypeptide chains were collected by ion exchange chromatography and then subjected to molecular sieve chromatography in a physiological solution environment to dissociate the fractions. A fraction containing a ribonucleoprotein complex derivative containing a non-polypeptide chain is fractionated, and an anionic surfactant and a protein conformational modifier are present therein for isolation. Dissociating the polypeptide chain, followed by molecular sieve chromatography to separate the polypeptide chain to be isolated, and obtain a fraction that specifically reacts with the anti-Smith antibody Preparation of a polypeptide chain constituting purified ribonucleoprotein complex. 3. A method for measuring an anti-Smith antibody, which comprises using the polypeptide chain according to claim 1 as an antigen and detecting or quantifying an antibody against the polypeptide chain present in a test subject.