JPH04198199A - Production of purified rna-binding protein and method for measuring anti-rna-binding protein antibody - Google Patents

Abstract

PURPOSE:To specifically and efficiently purify the subject protein useful as an antigenic reagent for autoantibody tests by extracting an RNA-binding protein from an animal tissue, etc., and subjecting the resultant RNA-binding protein-containing extract solution to special affinity gel chromatography. CONSTITUTION:An RNA-binding protein is initially extracted from an animal tissue, cultured cell or its processed substance and the resultant extract is then brought into contact with an affinity gel comprising a support containing polyuridylic acid polymer (e.g. polyuridylic acid-agarose) as a ligand under an environment of a salt-containing solution (preferably a 0.1M magnesium chloride solution). The RNA-binding protein adsorbed on the aforementioned affinity gel is eluted by a method (e.g. a gradient method) for increasing the salt concentration in the solution containing the aforementioned support suspended therein. Furthermore, the RNA-binding protein is preferably extracted by using an extracting buffer solution prepared by adding an inhibitor for proteolytic enzymes thereto at a physiological salt concentration.

Description

[Detailed description of the invention] [Industrial application field] The present invention is for predicting autoimmune diseases and autoimmune phenomena.

The present invention relates to a method for producing RNA binding proteins and a method for measuring anti-RNA binding protein antibodies, which are effectively used as antigen reagents for autoantibody tests performed as part of diagnosis and follow-up.

[Conventional technology]

Proteins that specifically bind to small RNA include a group of proteins that bind to small RNA in the cell nucleus and contribute to RNA splicing, and small RNA in the cytoplasm.
A group of proteins that bind to RNA and control RNA metabolism are known.

In systemic autoimmune diseases, the RNA is
Antibodies against the binding protein are produced, leading to inflammation and ulcers, for example.

Lesions associated with various autoimmune phenomena, such as skin rash and dryness, develop. Furthermore, the RNA-binding proteins that correspond to antibodies that appear in the serum of patients with autoimmune diseases vary among disease groups; for example, in systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), Antibodies are produced at high frequency against RNA binding proteins in the cytoplasm in sicca syndrome (SjS), as opposed to nuclear RNA binding proteins.

Therefore, the essential clinical significance of detecting various autoantibodies appearing in the serum of patients with these autoimmune diseases lies in the diagnosis, follow-up, and prediction of autoimmune phenomena of the diseases.

Conventionally, as antigens used to detect this antibody group, cultured human cells, mammalian cell extracts, or cells themselves have been used as crude antigens. this is,
Since the antigenicity of the protein is common across species, proteins derived from any mammal can be used as antigens for detecting antibodies against self components found in the serum of patients with autoimmune diseases, that is, autoantibodies. Anti-ribonucleoprotein antibodies and anti-Smith antibodies are anti-ribonucleoprotein antibodies and anti-Smith antibodies are anti-nuclear RNA-binding proteins, and anti-S S A /
The Ro antibody group and the anti-SSB/La antibody group are widely known, and the detection of these antibody groups is carried out by contrasting the reaction pattern between the conventionally controlled reference serum and the crude antigen solution described above. For example, antigens are detected simply by the fusion phenomenon of appearing sedimentation lines by binary immunodiffusion method or by interpretation of stained images of cultured cells by fluorescent antibody method, and there is no need for the antigen to be purified or pickled6. As the properties and functions of antigens for these antibody groups have been clarified through molecular biology and protein chemistry, it has become necessary to establish sensitive and quantitative clinical testing methods that use purified antigens as detection reagents. There is.

Separation and purification methods for RNA-binding proteins have been described in many scientific literature (C1ir+ Exp, Immu
nol, *54.731-738. (1983), J.
Biol.

Chew, 258.2604-2613 (1983)
Such). In most methods, purification is performed using biochemical separation means based on the physicochemical properties of the protein to be purified, such as ion exchange chromatography and molecular sieve chromatography. In addition, depending on the method, purification is performed by adsorption chromatography using an adsorbent such as a dye as a carrier (5cand.

J. Immunol. 15, 1-7. (1982))
.

[Problem to be solved by the invention]

As mentioned above, traditional methods that separate RNA-binding proteins to be isolated based on differences in protein molecular weight, charge state, or adsorption to acid substances result in a considerable amount of contamination from other proteins. is unavoidable, and a dramatic increase in the purification ratio cannot be expected. This is because proteins that exhibit the same physicochemical behavior exist in biological materials. Therefore, when a specific protein is separated by the method described above, it is necessary to increase the purification ratio by combining a plurality of separation means, and the number of days for purification is extended.

On the other hand, there is a method that uses biological affinity to significantly increase the purification ratio in the -step operation. For example, a substance that has biological affinity for the target to be isolated is immobilized as a ligand. There is an affinity chromatography method using a carrier that is

Therefore, the present inventors have completed the present invention as a result of intensive studies regarding a method for significantly increasing the purification ratio in such a step.

[Means to solve the problem]

That is, 1 the present invention extracts RNA-binding proteins from animal tissues, cultured cells, or processed products thereof, and then puts the obtained extract in a solution environment containing salt or chaotropic ions, using a uridylic acid polymer as a ligand. A purified RNA characterized by comprising a step of bringing the RNA into contact with an affinity gel containing a carrier, and then eluting the RNA binding protein adsorbed to the affinity gel by increasing the salt concentration or chaotropic ion concentration in the solution. A method for producing a binding protein, and an anti-R method for detecting or quantifying antibodies present in a test object using the purified RNA binding protein obtained by the method as an antigen.
This invention relates to a method for measuring NA-binding protein antibodies.

RNA from animal tissue, cultured cells, or their processed products
In the process of extracting the dilute salt-soluble fraction containing the bound protein, it is extracted with an extraction buffer supplemented with inhibitors for proteolytic enzymes to protect it from attack by all possible proteolytic enzymes that may be simultaneously solubilized. It is preferable to Furthermore, regarding this extraction operation, as many RNs as possible
In order to obtain A-binding protein, extraction from the target material is carried out in 2 steps.
It is preferable to carry out the extraction twice and to combine the two extracts. The salt concentration of the extraction buffer used in this extraction procedure is not particularly limited as long as it easily dissolves the proteins of the globulin fraction and does not dissolve nuclear histones, but it is preferable to use a physiological salt concentration. . Furthermore, since the detection of RNA-binding proteins in the extract is easy to perform, it is preferable to perform the detection by a two-way immunodiffusion method using an antiserum that specifically recognizes the RNA-binding protein of interest.

Next, the extract (unpurified fraction) extracted by the method described above is sufficiently dialyzed against a weak alkaline buffer containing a predetermined concentration of salt or chaotropic ions. The chaotropic ion is an anion with a large ionic radius, and the ion acts to increase the water solubility of hydrophobic molecules and weaken the hydrophobic bond between the carrier and the object to be separated. Chaotropic ions include 5CN-, I-, C4○4-
9No, -, Br-, C1-, CH, Go, -1p-
.

so, "-, etc., and the order of the magnitude of their effect (chaotropicity) is the same as the above order.

Compounds containing salts or chaotropic ions include:
Specifically, highly soluble monovalent or divalent metal chlorides such as sodium chloride, potassium chloride dimagnesium chloride, and metal thiocyanate compounds such as sodium thiocyanate and potassium thiocyanate can be mentioned. Magnesium is preferred. Its concentration varies depending on the type of salt; for example, in the case of magnesium chloride, it is about 0.
Approximately 1M is preferable. Then, the uridylic acid polymer equilibrated in advance with the buffer is brought into contact with a carrier whose ligand is a carrier, and the RNA-binding protein in the unpurified fraction is bound to the carrier. As the carrier, polyuridylic acid agarose (commercially available), affinity gel in which polyuridylic acid is coupled to Sepharose gel using polyuridylic acid as a ligand, etc. are used. The amount of carrier to be contacted is such that the total protein concentration is 10 mg.
/mQ per 1 ml of extract liquid is preferably 5 mQ or more. Furthermore, after that, the carrier is washed with the weak alkaline buffer solution used to equilibrate the carrier having the above-mentioned uridylic acid polymer as a ligand. The RNA-binding proteins are then eluted by increasing the concentration of salt or chaotropic ions in the solution in which the carrier is suspended from the time of contact between the extract and the carrier. The elution and recovery method is not particularly limited and may be any method, such as chromatography.

A batch method etc. can be used. Furthermore, there is no particular restriction on the method of increasing the salt or chaotropic ion concentration of the solution in which the carrier having the uridylic acid polymer bound to the RNA binding protein as a ligand is suspended, and a gradient method, stepwise method, etc. can be adopted. .

Through the steps described above, the ratio of RNA-binding protein to be separated and purified to the total protein amount can be dramatically increased. That is, most of the other fibrous proteins can be removed.

Here, specific RNA-binding proteins that can dramatically increase the purification ratio by this method include SSB/La protein, SSA, which constitutes the YRNA complex protein.
Examples include proteins of various molecular weights constituting /Ro protein and U sn RNA binding protein.

The RNA binding protein obtained by the above steps can be used as it is as an antigen for some immunoassay methods such as the ophthaloni method.

Furthermore, by combining at least one step of operations such as molecular sieve chromatography, ion-exchange chromatography, adsorption chromatography, and certain group-specific chromatography, it is possible to obtain a more highly purified RNA-binding protein specimen. It can also be used as an antigen for any diagnostic agent for measuring autoantibodies whose measurement principle is immunochemical reaction.

Next, the obtained RNA binding protein is used as an antigen to detect or quantify antibodies present in the test object.Anti-RNA
The method for measuring binding protein antibodies will be explained.

The measuring method of the present invention may be any measuring method as long as the above-mentioned antigen is used. For example, enzyme immunoassay, radioimmunoassay, immunoturbidimetry.

Immunofluorescence method, latex agglutination method, hemagglutination method.

Fluorescence immunoassay, immunochemiluminescence, seven-dye immunoassay, etc. can be performed. As a preferred example, an immunoassay method using a labeled secondary antibody will be described.

A solid surface, for example a polystyrene pore, is covered with the polypeptide chains. Usually, this coating operation has a buffering effect in the alkaline region. For example, a polypeptide chain is dissolved in a sodium carbonate buffer and used as a 0.01 to 100 μg/m Q solution, and the reaction is carried out overnight at a low temperature. Thereafter, the polypeptide chains that have not been physically adsorbed to the solid surface are removed by suction together with a buffer solution, and then a hydrophilic globular protein that does not have immunochemical cross-reactivity with the polypeptide chains, such as milk casein, etc. In a 1% (wt/vol) solution.

Blocking is performed at room temperature for about 1 hour. By covering easily adsorbable sites on the molecular surface of solid surfaces that are not covered with polypeptide chains or of polypeptide chains that are physically adsorbed to the solid surface, the test object solution or labeled secondary antibody that is added subsequently is coated. This is to prevent non-specific adsorption of protein components in the solution.

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

A solid surface coated with the polypeptide chain of a test object, such as patient serum, appropriately diluted with a physiological buffer containing a nonionic surfactant and a hydrophilic globular protein with no immunochemical cross-reactivity, and an antigen. Contact is allowed for a sufficient time for the antibody binding reaction to be completed.

Thereafter, the solid surface is thoroughly washed with a neutral washing solution containing a nonionic surfactant, and the solid surface is immersed in a physiological solution containing an excess amount of labeled secondary antibody to complete the antigen-antibody binding reaction. contact for a sufficient period of time. Here, the labeling substance may be an enzyme, a radioactive isotope, a fluorescent substance, or the like, but is not particularly limited, but an enzyme label is particularly preferred.

Subsequently, the solid surface is thoroughly washed with a neutral washing solution containing a nonionic surfactant, and the presence or amount of the labeled secondary antibody is detected. In the case of enzyme labeling, the solid surface is contacted with a specific substrate solution for the enzyme for a sufficient time so that the products of the enzymatic reaction are detected. In this case, the amount of product produced by the enzymatic reaction is proportionally dependent on the amount of antibody against the antigenic determinant on the polypeptide chain contained in the test object, and thus indirectly Antibodies can be quantified.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to Examples regarding a method for separating and purifying SSB/La protein among RNA binding proteins.

Example Buffer A: In 11, 8 g of sodium chloride, 0.2 g of potassium chloride, 2.
7 g, phosphate buffer containing 0.2 g of monopotassium phosphate.

Buffer B: As a protease inhibitor, ethylene glycol-XX'-bis(2-aminomethyl)NNN'N'tetraacetate (EDTA
) 10-3M, phenylmethylsulfonyl fluoride (PMSF) 10-3M,
A buffer solution further containing leupeptin 0.05% (wt/vol), antipain 0.05% (wt/vol), chymostatin 0.05% (wt/vol), pepstatin A 0.05% (wt/vol) A.

Buffer C Nidris buffer 10mMXHCl2 pH 8,0
゜Buffer D: Buffer C0 containing 0.1M magnesium chloride Eluent E1: Buffer C0 containing 0.2M magnesium chloride Eluent E2: Buffer C0 containing 0.3M magnesium chloride Eluent E3: Buffer C0 containing 0.4M magnesium chloride Eluent E4: Buffer C0 containing 0.5M magnesium chloride All operations shown below were performed at 4°C.

1) Obtaining a tissue extract containing RNA binding proteins 300 mA of buffer B was added to 30 g of rabbit thymus acetone powder (manufactured by Pel Freeze), and the mixture was dissolved overnight. Thereafter, the suspension was centrifuged at 10,000 x g for 30 minutes, and the supernatant was designated as extract A. To perform a second extraction from this precipitate, 50 mQ of buffer B was added to the precipitate and stirred for 4 hours. Thereafter, the suspension was mixed with 10. 30 in OOOXg
After centrifugation for a minute, the supernatant liquid was designated as extract B, which was combined with extract A to create extract C.

2) Fractionation using a carrier having a uridylic acid polymer as a ligand The extract C obtained in step 1) was dialyzed against a buffer solution for two days and nights, and then 0.1 volume of the extract was mixed with buffer D0.
9 volumes and 1 volume of a carrier having a uridylic acid polymer as a ligand (polyuridylic acid agarose, manufactured by Sigma), equilibrated in advance with a buffer solution, were added, and the mixture was slowly mixed by inversion for 15 minutes or more. Subsequently, the supernatant was removed by centrifugation, and 1 volume of buffer solution D was added to wash the carrier by centrifugation. Subsequently, 11 volumes of eluent E was added and centrifuged, and the RNA-binding protein eluted in the supernatant was recovered. Similarly, eluent E2. Similar operations were performed using E3 and E4 in order, and the RNA-binding proteins in each eluate were recovered.

3) Measurement of SSB/La protein in each eluent by enzyme immunoassay Inject SSB/La protein solution diluted to 1/500 (for each eluate) into a 96-well ELISA microplate. 200 μQ of liquid) was added and allowed to adsorb overnight at 4°C. For dilution, O.1M sodium carbonate buffer, pH 8.6, was used. The next day, the plate was blocked with 400 μQ of 0.2% milk solution at room temperature for 1 hour to cover unreacted sites on the plate and easily adsorbed sites on the surface of the adsorbed protein. Next, the primary antibody solution (anti-SSB/La serum was added to 0
．． 1:1,000 in Dulbets cholate-buffered saline containing 1% milk and 0.1% Tween 20.
200 μQ (diluted to 20%) was added and reacted at room temperature for 2 hours to bind the anti-SSB/La antibody to the coated antigen. 1
After the next antibody reaction, wash the plate using a washing buffer.
Washing 5 times for 3 minutes with Dulbets choline buffered saline containing 0.1% Tween 20), secondary antibody solution (
Dilute phosphatase-labeled anti-human IgG・+A 10M antibody serum (manufactured by KPL) to 1/1,000 with Dulbets choline buffered saline containing 0.1% milk and 0.1% Tween 20) and add 2oμQ. Then, the reaction was further carried out at room temperature for 2 hours to allow the secondary antibody to bind to the primary antibody (anti-SSB/La antibody) on the plate. Following the secondary antibody reaction, the plate was washed in the same manner as above, and a substrate solution (1 mg/mΩ p-ditrophenyl phosphate, 1
Add 200 μQ of M jetanolamine buffer) and
The enzyme activity of the labeled secondary antibody captured by the secondary antibody was determined by measuring absorbance at a wavelength of 405 nm using a spectrophotometer. This enzyme activity is determined by the S S B on the plate.
/La Since there is a proportional relationship with the amount of antigen, the amount of antigen in the sample can be measured based on the magnitude of enzyme activity.

The results obtained are shown in FIG. In addition, the protein amount of each eluent was determined by the Lowry method, and the SSB/La antigen activity of each eluent (MgCQ 20.2M, 0.3M, and 0.4M) per protein amount was significantly higher. It was increasing.

4) Analysis by SDS polyacrylamide gel electrophoresis method Each eluate was developed as an electrophoresis sample in a 12.5% acrylamide gel (crosslinking degree 0.8) according to the method of Laemmli et al. The electrophoresis conditions were 40 mA at the start of electrophoresis, 4 V/am during concentration electrophoresis, and 8 V/am during separation electrophoresis. In addition, the electrophoresis sample was prepared in advance with a sample buffer (312.4mM Tris-HCl, pH 6,8,0,
25% by volume of 1% bromophenol blue, 10% sodium dodecyl sulfate, 20% glycerin) was added and treated at room temperature for 30 minutes. The gel after electrophoresis was stained with 0.05% Coomassie Brilliant Blue overnight, and the next day it was destained with 0.7% acetic acid. In addition, as a molecular weight marker, Dalton is 92.5.

Dalton in 66.2, 45. OK Dalton, 31. OK
Dalton, BI○-RAD molecular weight markers having Dalton markers at 21.5 and Dalton at 14.4 were used.

As a result, the magnesium chloride concentration was 0.2M.

At 0.3M and 0.4M, the ratio of the density of the Dalton band to the total stained protein band was increased to approximately 50.

〔Effect of the invention〕

Conventionally, cell extracts have been used as they are as antigens for measuring antibodies against RNA binding proteins.

However, in recent years, R
The molecular properties of NA-binding proteins are being clarified,
These functions occur in the serum of people suffering from autoimmune diseases. Various theories have been discussed regarding the function of antibodies against these and their relationship with the pathogenesis, or the relationship between autoimmune phenomena observed during the course of the disease and fluctuations in serum antibody titer, or the relationship with the date of illness. It is becoming important to understand the contribution of binding protein antibodies and the relationship with clinical symptoms.

Therefore, as in the present invention, specific efficiency <RNA>
Establishing a method to separate binding proteins from other proteins and components is an effective method for preparing reagent components used in clinical tests for diagnosing and monitoring the progress of autoimmune diseases or predicting autoimmune phenomena. It is something. The method for measuring anti-RNA binding protein antibodies of the present invention using the obtained RNA binding protein is clinically very effective.

[Brief explanation of the drawing]

FIG. 1 shows the RNA binding protein (
This figure shows the elution of SSB/La protein) from polyuridylic acid agarose, and the vertical axis shows the elution of SSB/La protein in each supernatant.
The abscissa represents the absorbance at a wavelength of 405 nm when the amount of B/La antigen was quantified by enzyme immunoassay, and the concentration of magnesium chloride used is shown on the horizontal axis.

Claims (1)

[Claims] 1. RN from animal tissues, cultured cells, or processed products thereof
The A-binding protein is extracted, and the resulting extract is brought into contact with an affinity gel containing a carrier having a polyuridylic acid polymer as a ligand in a salt-containing solution environment, and then the RNA-binding protein adsorbed on the affinity gel is A method for producing a purified RNA-binding protein, comprising the step of elution by increasing the salt concentration in the solution. 2. RN from animal tissue, cultured cells, or their processed products
The A-binding protein is extracted, and then the resulting extract is brought into contact with an affinity gel containing a carrier having a polyuridylic acid polymer as a ligand in a solution environment containing chaotropic ions, and then the RNA-binding protein adsorbed on the affinity gel is extracted. 1. A method for producing a purified RNA-binding protein, the method comprising the step of eluting a chaotropic ion by increasing the concentration of chaotropic ions in a solution. 3. The method for producing a purified RNA binding protein according to claim 1 or 2, wherein the RNA binding protein to be purified is SSB/La protein. 4. The method for producing a purified RNA binding protein according to claim 1 or 2, wherein the RNA binding protein to be purified is SSA/Ro protein. 5. Using the purified RNA binding protein obtained by the production method according to claim 1, 2, 3 or 4 as an antigen,
Anti-RN for detecting or quantifying antibodies present in a test object
Method for measuring A-binding protein antibodies.