JPH07325086A - Method for measuring anti-rna combined protein antibody - Google Patents

Abstract

PURPOSE:To provide a method for measuring anti-RNA combined protein antibody, which can efficiently separate the RNA connective protein from other proteins and components. CONSTITUTION:The RNA connective protein is extracted from the animal organization, cultured cell and the processed material thereof, and this obtained extract is made to contact with the affinity gel, which includes the carrier having the polyuridylic acid polymer as a ligand, under the liquid environment including the salt, and thereafter, the RNA connective protein adsorbed by the affinity gel is eluted by increasing the condensation of salt in the solvent so as to obtain the refined RNA combined protein, and this re fined RNA combined protein is used as an antigen so as to detect or measure the quantity of the antibody, which exists in the objective material to be tested.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for assaying anti-RNA binding protein antibody which is effectively used as an antigen reagent for autoantibody test performed as part of the prediction, diagnosis and follow-up of autoimmune diseases and autoimmune phenomena. Regarding

[0002]

2. Description of the Related Art As a protein that specifically binds to low molecular weight RNA, it binds to low molecular weight RNA in the cell nucleus, a group of proteins that contribute to RNA splicing, and low molecular weight RNA in the cytoplasm, and RNA metabolism A group of proteins that control regulation are known. In a systemic autoimmune disease, an antibody against the RNA-binding protein is produced by some mechanism, which causes lesions associated with various autoimmune phenomena such as inflammation, ulcer, skin eruption, and dryness. . Furthermore, RNA-binding proteins corresponding to antibodies appearing in the sera of patients with autoimmune diseases are diverse among the disease groups, and for example, systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are nuclear. In spite of the internal RNA-binding protein, in the dry syndrome (SjS), antibodies are frequently produced against the RNA-binding protein in the cytoplasm. Therefore, the clinical essential significance of detecting various autoantibodies appearing in the sera of patients with these autoimmune diseases is the diagnosis of the diseases.
It is in follow-up and prediction of autoimmune phenomena.

Conventionally, as an antigen used to detect the antibody group, an extract of human cultured cells or mammalian cells or the cells themselves have been used as a crude antigen. This is because, since the antigenicity of the protein is common across species, any mammal-derived antigen can be used as an antigen for detecting an antibody against an autologous component found in the serum of an autoimmune disease patient, that is, an autoantibody. Is. Antibodies to the RNA-binding protein in the cell nucleus include anti-ribonucleoprotein antibody group and anti-Smith antibody group, and antibodies to the RNA-binding protein in cytoplasm include anti-S protein group.
The SA / Ro antibody group and the anti-SSB / La antibody group are widely known, and the detection of these antibody groups is carried out in comparison with the reaction mode between the conventionally controlled standard serum and the crude antigen solution described above.
For example, it was detected only by the phenomenon of fusion of appearance sedimentation lines by the binary immunodiffusion method or by reading the stained image of the cultured cells by the fluorescent antibody method, and it was not necessary that the antigen was purified.

In recent years, the properties and functions of antigens against the antibody group have been elucidated by molecular biology or protein chemistry, and it is necessary to establish a sensitive and quantitative clinical test method using purified antigen as a detection reagent. Sexuality is occurring. RNA
Many scientific literatures describe methods for separating and purifying binding proteins (Clin Exp. Immunol., 54, 731-738, (198
3), J. Biol. Chem., 258, 2604-2613 (1983)). In most methods, purification is carried out by biochemical separation means such as ion exchange chromatography and molecular sieve chromatography based on the physicochemical properties of the protein to be purified. Further, depending on the method, purification is performed by adsorption chromatography using an adsorbent such as a dye as a carrier (Scand.
J. Immunol., 15, 1-7, (1982)).

[0005]

As described above, the conventional method for separating the RNA-binding protein to be isolated based on the difference in the molecular weight of the protein, the difference in the charge state, or the difference in the adsorptivity to the substance. Therefore, it is inevitable that a considerable amount of other proteins will be contaminated, and a dramatic increase in the purification rate cannot be expected. This is because there are proteins that exhibit the same physicochemical behavior in biomaterials. Therefore, in general, when a specific protein is separated by the above-mentioned method, it is necessary to increase the purification rate by combining a plurality of separation means, which results in a long purification time. On the other hand, as a method of greatly increasing the purification rate in a single step operation, there is a method of utilizing a biological affinity. For example, a substance having a biological affinity for an isolation target is immobilized as a ligand. There is an affinity chromatography method using the prepared carrier. Therefore, the present inventors have completed the present invention as a result of intensive studies on a method of significantly increasing the purification rate in such a single step.

[0006]

[Means for Solving the Problems] That is, according to the present invention, an RNA-binding protein is extracted from an animal tissue, a cultured cell, or a processed product thereof, and the obtained extract is then subjected to a solution environment containing a salt or a chaotropic ion. To the affinity gel containing a carrier having a uridylic acid polymer as a ligand, and then the RNA-binding protein adsorbed to the affinity gel is eluted by increasing the salt concentration or chaotropic ion concentration in the solution. The present invention relates to a method for measuring an anti-RNA binding protein antibody, which comprises using the purified RNA binding protein as an antigen and detecting or quantifying an antibody present in a test subject.

During the process of extracting the dilute salt-soluble fraction containing RNA-binding protein from animal tissues, cultured cells or their processed products, the protein is protected 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 degrading enzyme. Further, regarding this extraction operation, in order to obtain as much RNA-binding protein as possible, it is preferable to perform extraction from the substance to be extracted twice and combine the two extracts. The salt concentration of the extraction buffer used in this extraction operation is not particularly limited as long as the protein of the globulin fraction is easily dissolved and the histones in the nucleus are not dissolved, but it is preferable to perform it at a physiological salt concentration. . Further, the detection of the RNA-binding protein in the extract is easy to perform, so that the target R
It is preferable to carry out by a two-way immunodiffusion method using an antiserum which specifically recognizes an NA-binding protein.

Next, the extract (unpurified fraction) extracted by the above-mentioned method is sufficiently dialyzed against a weak alkaline buffer solution containing a predetermined concentration of salt or chaotropic ion. The chaotropic ion is an anion having a large ionic radius, which increases water solubility of the hydrophobic molecule and weakens the hydrophobic bond between the carrier and the separation target. Chaotropic as the ion SCN ^-, I ^-,
ClO ₄ ⁻ , NO ₃ ⁻ , Br ⁻ , Cl ⁻ , CH ₃ CO ₂ ⁻ , F ⁻ ,
SO ₄ ^{2− and the} like can be mentioned, and the order of the magnitude of its action (chaotropy) is the same as the above order.

Specific examples of salts or compounds containing chaotropic ions include monovalent or divalent salts having high solubility such as sodium chloride, potassium chloride and magnesium chloride.
Examples thereof include valent metal chlorides, metal thiocyanate compounds such as sodium thiocyanate and potassium thiocyanate, and among them, magnesium chloride is particularly preferable. The concentration varies depending on the type of salt, and for example, in the case of magnesium chloride, it is preferably about 0.1M. Then, the RNA-binding protein in the unpurified fraction is bound to the carrier by bringing the uridylic acid polymer equilibrated with the buffer solution into contact with the carrier as a ligand. As the carrier, polyuridylate agarose (commercially available), affinity gel obtained by coupling polyuridylic acid as a ligand to a sepharose gel, and the like are used. The amount of the carrier to be contacted is preferably 5 ml or more per 1 ml of the extract having a total protein concentration of 10 mg / ml. After that, the carrier is washed with the weak alkaline buffer solution used for equilibrating the carrier having the uridylic acid polymer as a ligand. Subsequently, the RNA-binding protein is eluted by increasing the concentration of salt or chaotropic ion in the solution in which the carrier is suspended more than when the extract is in contact with the carrier. The method of eluting and collecting is not particularly limited, and for example, a chromatography method, a batch method and the like can be used. Further, the method of increasing the salt or chaotropic ion concentration of the solution in which the carrier having the uridylic acid polymer bound with the RNA binding protein as the ligand is suspended is not particularly limited, and the gradient method or the stepwise method can be employed.

By the steps described above, the ratio of the RNA-binding protein to be separated and purified to the total amount of protein can be dramatically increased. That is, most of other contaminant proteins can be removed. Here, as a specific RNA-binding protein capable of dramatically increasing the purification rate by this method, SSB constituting a YRNA complex protein
/ La protein, SSA / Ro protein, and protein of each molecular weight that constitutes UsnRNA binding protein. The RNA-binding protein obtained by the above steps can be used as it is as an antigen used in some immunoassays such as the Octaloni method. Furthermore, a highly purified RNA-binding protein preparation can be obtained by combining at least one step of operations such as molecular sieve chromatography, ion exchange chromatography, adsorption chromatography, and certain group-specific chromatography. It can be used as an antigen for use in any diagnostic agent for measuring autoantibodies whose immunochemical reaction is the measurement principle.

Next, a method for measuring an anti-RNA binding protein antibody for detecting or quantifying an antibody present in a test subject using the obtained RNA binding protein as an antigen will be described. The assay method of the present invention, as long as the above antigen is used,
Any measuring method may be used. For example, enzyme immunoassay, radioimmunoassay, immunoturbidimetric method, immunoparasitic method,
A latex agglutination method, a hemagglutination method, a fluorescent immunoassay method, an immunochemiluminescence method, a dye immunoassay method and the like can be performed. As a preferred example, an immunoassay method using a labeled secondary antibody will be described.

The solid surface, eg polystyrene pores, is covered with the polypeptide chain. Usually, this coating operation has a buffering effect in the alkaline range. For example, the polypeptide chain is dissolved in a sodium carbonate buffer solution and used as a 0.01 to 100 μg / ml solution, which is performed overnight at low temperature. After that, the polypeptide chains not physically adsorbed on the solid surface are removed by suction together with a buffer solution, and subsequently, a hydrophilic globular protein having no immunochemical cross-reactivity with the polypeptide chains, for example, 0.01 to 0.01 of milk casein or the like is used. Blocking is performed with a 1% (weight / volume) solution at room temperature for about 1 hour. This is a solution of a test object or a labeled secondary antibody to be added thereafter by covering an easily adsorbable site on the surface of a solid that is not coated with a polypeptide chain or on the surface of a polypeptide physically adsorbed on the surface of a solid. 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 not 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.

[0013] A test subject appropriately diluted with a physiological buffer containing a nonionic surfactant and a hydrophilic globular protein having no immunochemical cross-linking property, for example, a solid obtained by coating patient serum with the polypeptide chain. The surface is contacted for a time sufficient to complete the antigen-antibody binding reaction. Thereafter, the solid surface is thoroughly washed with a neutral washing solution containing a nonionic surfactant, and the antigen-antibody binding reaction is completed on the solid surface in a physiological solution containing an excess amount of labeled secondary antibody. For sufficient time to allow 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, subsequently, 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 specific substrate solution for the enzyme for a time sufficient to detect the products of the enzymatic reaction. In this case, the amount of product produced by the enzymatic reaction is proportionally dependent on the amount of antibody to the antigenic determinants on the polypeptide chain contained in the test object, and thus indirectly in the test object. The antibody can be quantified.

[0014]

Examples Among the RNA binding proteins, SSB /
The present invention will be described in detail with reference to examples of a method for separating and purifying La protein. Example Buffer A: A phosphate buffer containing 8 g of sodium chloride, 0.2 g of potassium chloride, 2.7 g of disodium phosphate 12-hydrate, and 0.2 g of 1 potassium phosphate in 1 L. Buffer B: ethylene glycol-OO'-bis (2aminomethyl) -NNN'N 'as a protease inhibitor
-4 Acetate (EDTA) 10 ^-3 M, Fluorinated Phenylmethylsulfonyl (PMSF) 10 ^-3 M, Leupeptin 0.0
5% (weight / volume), antipine 0.05% (weight / volume)
Buffer A, further containing 0.05% chymostatin (weight / volume), 0.05% peptantine A (weight / volume). Buffer C: Tris buffer 10 mM x HCl pH 8.0. Buffer D: Buffer C containing 0.1 M magnesium chloride. Eluent E1: Buffer C containing 0.2 M magnesium chloride. Eluent E2: Buffer C containing 0.3 M magnesium chloride. Eluent E3: Buffer C containing 0.4M magnesium chloride. Eluent E4: Buffer C containing 0.5 M magnesium chloride.

All the following operations were carried out at 4 ° C. 1) Acquisition of tissue extract containing RNA-binding protein 300 ml of buffer B was added to 30 g of rabbit thymus acetone powder (Pel Freeze) and the mixture was dissolved overnight. Then, add 10,000 × to the suspension.
After centrifugation at g for 30 minutes, the supernatant was designated as Extract A. In order to carry out a second extraction from this precipitate, 50 ml of buffer solution B was added to the precipitate and stirred for 4 hours. Then, the suspension was centrifuged at 10,000 × g for 30 minutes, the supernatant was used as the extract B, and the extract A was combined with the extract A to obtain the extract C.

2) Fractionation with carrier having uridylic acid polymer as a ligand The extract C obtained in 1) is dialyzed against the buffer D for 2 days and night, and then 0.1 volume of the extract is buffered. Liquid D0.9
And 1 volume of a carrier (polyuridylic acid agarose, manufactured by Sigma) whose ligand is uridylic acid polymer previously equilibrated with buffer solution D was added, and the mixture was gently mixed by inversion for 15 minutes or more. Subsequently, the supernatant was removed by centrifugation and the buffer D1 was added to wash the carrier by centrifugation. Further on, the eluent E
The RNA-binding protein was collected by adding 11 volumes and spun down and eluting in the supernatant. Similarly, eluents E2, E3, and E4 were used in this order, and the same operation was performed to recover the RNA-binding protein in each eluent.

3) Measurement of SSB / La protein in each eluent by enzyme immunoassay: SSB / La protein solution (each eluent) diluted to 1/500 in a 96-well ELISA microplate.
μl was added and adsorption was carried out at 4 ° C. overnight. 0.1 for dilution
M sodium carbonate buffer, pH 8.6 was used. The next day, it was blocked with 400 μl of 0.2% milk solution at room temperature for 1 hour to coat unreacted sites on the plate and easily adsorbed sites on the surface of the adsorbed protein. Then, the primary antibody solution (anti-SSB / La serum was added to 0.1% milk and 0.1%
200 μl of Dulbecco's phosphate buffered saline containing 20% Tween 20) was added and reacted at room temperature for 2 hours to bind the anti-SSB / La antibody to the coated antigen. After the primary antibody reaction, the plate was washed (using a Dulbecco's phosphate buffered saline containing 0.1% Tween 20 as a washing buffer for 5 minutes for 3 minutes),
Secondary antibody solution (phosphatase-labeled anti-human IgG + A
+ M antibody serum (manufactured by KPL) was diluted with Dulbecco's phosphate-buffered physiological saline containing 0.1% milk and 0.1% Tween 20 at a ratio of 1/1000), and 20 μl thereof was added. After reacting for a time, the secondary antibody was bound to the primary antibody (anti-SSB / La antibody) on the plate. Two
Following the next antibody reaction, the plate was washed in the same manner as above, and the substrate solution (1 mg / ml p-nitrophenyl phosphate,
200 μl of 1M diethanolamine buffer),
The enzyme activity of the labeled secondary antibody captured by the primary antibody was determined by measuring the absorbance at a wavelength of 405 nm with a spectrophotometer. This enzymatic activity is due to SSB / La on the plate.
Since there is a proportional relationship with the amount of antigen, the amount of antigen in the sample can be measured by the magnitude of the enzyme activity.

The results obtained are shown in FIG. In addition, Lowry
The amount of protein in each eluent was determined by the method. Each eluent per amount of protein (MgCl ₂ 0.2M, 0.3M and 0.4M
The activity of SSB / La antigen of (1) was significantly increased.

4) Analysis by SDS polyacrylamide gel electrophoresis According to the method of Laemmli et al., Each eluent was developed as a migration sample in 12.5% acrylamide gel (degree of crosslinking 0.8). Electrophoresis conditions are 40 mA at the start of electrophoresis and 4 at the time of concentrated electrophoresis.
V / cm, 8 V / cm at the time of separation and migration. In addition, the sample to be electrophoresed was a sample buffer solution (312.4 mM Tris-hydrochloric acid, pH 6.8, 0.1% bromphenol blue, containing no reducing agent in advance,
25% by volume of 10% sodium dodecyl sulfate and 20% glycerin) was added, and the mixture was treated at room temperature for 30 minutes. After the electrophoresis, the gel was stained with 0.05% Coomassie Brilliant Blue overnight, and the next day, it was destained with 0.7% acetic acid. As molecular weight markers, 92.5K Dalton, 66.2K Dalton, 4
BI with markers of 5.0K daltons, 31.0K daltons, 21.5K daltons and 14.4K daltons
A molecular weight marker manufactured by O-RAD was used. as a result,
Magnesium chloride concentration 0.2M, 0.3M and 0.4
M has a molecular weight of about 50K for all stained protein bands.
The density ratio of the Dalton band was increasing.

[0020]

EFFECT OF THE INVENTION Conventionally, a cell extract has been used as it is as an antigen used when measuring an antibody against an RNA-binding protein. However, in recent years, the molecular properties of RNA-binding proteins have been clarified as cell constituents that can be self-antigens, and these functions and the function and etiology of antibodies to them appearing in the serum of autoimmune disease sufferers have been investigated. Theories have also been discussed in relation to the relationship between E.coli, the autoimmune phenomenon observed during the course of the disease, and the change in serum antibody titer or the disease day, and the contribution of anti-RNA binding protein antibody as a causative agent and clinical features. Understanding the relationship with is becoming important. Therefore, as in the present invention, a method for specifically and efficiently separating an RNA-binding protein from other proteins or components is established by a clinical test for the diagnosis or follow-up of an autoimmune disease or prediction of an autoimmune phenomenon. It is effective as a method for preparing the reagent constituents used in. The method for measuring the anti-RNA binding protein antibody of the present invention using the obtained RNA binding protein is clinically very effective.

[Brief description of drawings]

FIG. 1 shows an RNA-binding protein (S
(SB / La protein) is a diagram showing elution from polyuridylate agarose, and the vertical axis represents SSB / La in each supernatant.
Wavelength of 405nm when the amount of antigens of spleen is quantified by enzyme immunoassay
And the horizontal axis represents the concentration of magnesium chloride used.

Claims (6)

[Claims] 1. An affinity gel comprising a carrier having a polyuridylic acid polymer as a ligand, which is obtained by extracting an RNA-binding protein from an animal tissue, a cultured cell or a processed product thereof, and then subjecting the resulting extract to a solution environment containing a salt. The purified RNA-binding protein obtained by the step of eluting the RNA-binding protein adsorbed on the affinity gel by increasing the salt concentration in the solution is used as an antigen, A method for measuring an anti-RNA binding protein antibody, which comprises detecting or quantifying an existing antibody. 2. The RNA-binding protein to be purified is SSB / L
The method for measuring an anti-RNA binding protein antibody according to claim 1, which is a protein. 3. The RNA-binding protein to be purified is SSA / R
The method for measuring an anti-RNA binding protein antibody according to claim 1, which is an o protein. 4. An RNA binding protein is extracted from an animal tissue, a cultured cell or a processed product thereof, and the resulting extract is then subjected to a solution environment containing a chaotropic ion and containing a carrier having a polyuridylic acid polymer as a ligand. The purified RNA-binding protein obtained by the step of contacting with a gel and then eluting the RNA-binding protein adsorbed on the affinity gel by increasing the concentration of chaotropic ions in the solution is used as an antigen, A method for measuring an anti-RNA binding protein antibody, which comprises detecting or quantifying an antibody present therein. 5. The RNA-binding protein to be purified is SSB / L
The method for measuring an anti-RNA binding protein antibody according to claim 4, which is a protein. 6. The RNA-binding protein to be purified is SSA / R
The method for measuring an anti-RNA binding protein antibody according to claim 4, which is an o protein.