JP2508915B2 - Anti-SSA / Ro and SSB / La antibody measuring antigen, method for producing the same, and anti-SSA / Ro and SSB / La antibody measuring method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is directed to the prediction, diagnosis, and diagnosis of autoimmune diseases and autoimmune phenomena.
The present invention relates to an antigen for measuring anti-SSA / Ro and SSB / La antibodies used in an autoantibody test performed as part of follow-up observation, a method for producing the same, and a method for measuring anti-SSA / Ro and SSB / La antibodies.

[Conventional technology]

In a systemic autoimmune disease, an antibody against cell components of self is produced by some mechanism, which causes lesions associated with various autoimmune phenomena such as inflammation, ulcer, skin rash, and dryness. To do. Furthermore, the self-constituting components corresponding to the antibodies appearing in the sera of patients with autoimmune diseases vary among the disease groups, and the appearance frequency and appearance profile of the antibodies tend to be epidemiologically statistical among the autoimmune diseases. Is known to show. Moreover, in the course of a specific autoimmune disease, it has been established that there is a relationship between the clinical picture or the disease form on a certain day of the disease and the circulating autoantibodies in the serum of the affected day. is there.

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

In autoimmune diseases, proteins that bind to small RNAs derived from cytoplasm, such as SSA, are one of the cellular constituents of self that can be recognized as an antigen by the immune system of the living body.
There are / Ro protein and SSB / La protein, but as an antigen reagent conventionally used to detect an antibody group against the protein, an extract from human cultured cells or mammalian cells or processed products thereof or The cells themselves have been used as antigens. This is because, since the antigenicity of the protein is common across species, any mammal-derived antibody can be used as an antigen for detecting an antibody against an autocomponent found in the serum of an autoimmune disease patient, that is, an autoantibody. Furthermore, the detection of the antibody group can be carried out by, for example, a two-way immunodiffusion method in accordance with the reaction mode of the conventionally administered standard anti-SSA / Ro or standard anti-SSB / La serum and the above-mentioned antigen solution. It is based on the fact that the antigen reagent did not have to be purified because it was only detected by the fusion phenomenon of the qualitative appearance sedimentation line.

In recent years, under the background of sophistication of medical level, it is necessary to sensitively quantify the autoantibodies existing in the serum of the patient even in the diagnostic area of autoimmune diseases.

[Problems to be Solved by the Invention]

Antigen reagents used in conventional qualitative detection methods for anti-SSA / Ro and SSB / La antibodies are soluble proteins extracted from mammals or cultured cells or their processed products under physiological solution environment. Is used as is.
The SSA / Ro and SSB / La proteins present in such a solution are bound to low-molecular-weight RNA due to the physiological properties of the protein, so that immunochemical reactivity as an antigen is suppressed to some extent. It is possible that

As a method for recovering the suppressed antigen activity, a method of exposing the antigen to a certain solution environment to dissociate the antigen protein from the RNA chain may be considered.However, depending on the extent to which the protein can be separated from the bound RNA chain, the antigenicity disappears. Or it may be reduced, and the method should be carefully considered.

As a result of diligent studies on the above-described method, the present inventors have found that the RNA chain degradation and removal by a ribonucleolytic enzyme is conserved without undergoing enzymatic digestion of the RNA difference at the protein binding site, and only the RNA chain responsible for the crosslinkable structure is preserved. The present inventors have completed the present invention by finding that the antigenicity of the protein, which has been impaired by the cross-linking RNA construct, is higher than the antigenicity because the antigenicity is not reduced due to the degradation.

[Means for solving the problem]

That is, the present invention provides anti-SSA / Ro and SSB lacking at least a part of the constituent RAN chains, which is obtained by treating an RNA-binding protein containing SSA / Ro protein and SSB / La protein with a ribonucleolytic enzyme. Anti-SSA / Ro and SSB / La antibodies for detecting or quantifying anti-SSA / Ro and SSB / La antibodies in a test sample using the anti-SSA / Ro and SSB / La antibody measuring antigens The present invention relates to a method for measuring SSB / La antibody.

First, the antigen for measuring anti-SSA / Ro and SSB / La antibodies and the method for producing the same will be described in detail.

First, a diluted salt-soluble fraction containing an RNA-binding protein is extracted as an antigen solution from animal tissues, cultured cells, or processed products thereof. In this process, it is preferable to perform extraction with an extraction buffer containing an inhibitor for the proteolytic enzyme in order to protect it from invasion by all possible proteolytic enzymes that are simultaneously solubilized. Further, regarding this extraction operation, in order to obtain as much soluble protein as possible, it is preferable to perform extraction from the object to be extracted twice and combine the extracts twice. 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 nuclear histones are not dissolved,
Preference is given to carrying out at physiological salt concentrations.

Then, the obtained extract is sufficiently dialyzed against a ribonucleolytic enzyme reaction buffer, and a ribonucleic acid enzyme solution is added to start the enzymatic reaction. The addition amount at this time is 100
It is preferable that it is a solution having an enzyme concentration of about 1 mg / ml per 1 volume of the extract. Enzyme reaction temperature is SSA / Ro protein and SS
The temperature is not particularly limited as long as the antigen activity of the B / La protein is not lost and the enzyme activity is expressed, but it is preferably 37 ° C or lower. The enzyme reaction time depends on the enzyme reaction temperature, but it is necessary to perform the reaction within a time that does not impair the antigen activity of SSA / Ro and SSB / La proteins. Is preferable, and when performed at 25 ° C., it is preferably 2 hours or less.

The SSA / Ro and SSB / La antigens thus treated will lack at least part of the constituent RNA chains.

The SSA / Ro and SSB / La antigens obtained by the above steps can be used as they are as antigens used in some immunoassays such as the Octaloni method. Furthermore, highly purified SSA / Ro and SSB / La antigen preparations can be obtained by combining at least one step with operations such as molecular sieve chromatography, ion exchange chromatography, adsorption chromatography, and certain group-specific chromatography. The preparation can be used as an antigen for use as a diagnostic agent for measuring anti-SSA / Ro and SSB / La antibodies whose immunochemical reaction is the measurement principle.

The antigen thus obtained can be used for detecting or quantifying anti-SSA / Ro and SSB / La antibodies in a test sample. Next, this measuring method will be described.

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, immunoturbidimetric method, immunonephelometric method, latex agglutination method, hemagglutination method, fluorescent immunoassay method, immunochemiluminescence method, dye immunoassay method and the like can be performed. .
As a preferred example, an immunoassay using a labeled secondary antibody will be described.

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. Thereafter, the polypeptide chain not physically adsorbed on the solid surface is removed by suction together with a buffer solution, and then a hydrophilic globular protein having no immunochemical cross-reactivity with the polypeptide chain, for example, 0.01 to 1 of milk casein or the like.
% (Weight / volume) solution at room temperature for about 1 hour. This is achieved by covering the solid surface not coated with the polypeptide chain or the easily adsorbable site on the molecular surface of the polypeptide chain physically adsorbed on the solid surface, so that the test object solution or the labeled secondary antibody to be added subsequently This is for preventing the protein component in the solution from being non-specifically adsorbed.

Thereafter, in order to remove the polypeptide chains or protein components that have not been used for coating or blocking from the solid surface, the substrate is sufficiently 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 and a hydrophilic globular protein having no immunochemical crossability, for example, a patient surface and a solid surface coated with the polypeptide chain and an antigen Contact for a sufficient time to complete the 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 sample, and thus indirectly determines the amount of the antibody in the test sample. It can be quantified.

The antigenic activity of the antigen of the present invention is more than 4-fold higher than that of the untreated one, for example, by the enzyme immunocompetitive inhibition assay method.

〔Example〕

Hereinafter, the method for producing the anti-SSA / Ro and SSB / La antibody measuring antigens of the present invention will be described in detail with reference to Examples.

Buffer A: 11 in sodium chloride 8g, potassium chloride 0.2
g, disodium phosphate dodecahydrate 2.7 g, 1 potassium phosphate 0.2
Phosphate buffer containing g.

Buffer B: as proteolytic enzyme inhibitors, ethylene glycol -00'- bis (2-aminomethyl) -NNN'N'-4 acetate (EDTA) 10 ^-3 M, fluoride phenylmethylsulfonyl (PMSF) 10 ^- Buffer A further containing ³ M, leupeptin 0.05% (weight / volume), antipain 0.05% (weight / volume), chymostatin 0.05% (weight / volume), pepstatin A 0.05% (weight / volume).

Buffer C: Tris buffer 10 mM x HCl pH 8.0.

Buffer D: Ribonucleolytic enzyme (Biozyme)
Manufactured by the company) was dissolved in 1 mg / ml, magnesium chloride 4 mM,
Buffer C further containing 10% glycerin (weight / volume).

Buffer E: A carbonate type buffer containing 0.21 g of sodium chloride and 4.03 g of sodium hydrogen carbonate in 11

Buffer F: 11, sodium chloride 8g, chloride (cleaning solution) potassium 0.2g, disodium phosphate dodecahydrate
Phosphate buffer containing 2.7 g, 0.2 g of potassium monophosphate, and Tween-801 g.

Buffer solution G: Buffer solution F further containing non-fat dry milk (manufactured by KPL) 0.1% (weight / volume).

Note) The relationship between "volume" and "weight" shown below is 1
When the volume is 1 ml, 1 weight is 1 g.

1) Acquisition of Tissue Extract Containing RNA Binding Protein Used The following operations were all performed at 4 ° C.

Rabbit thymus acetone powder (Pel-Freez
e) (manufactured by the company), 10 volumes of buffer solution B was added to 1 weight thereof, and the mixture was dissolved overnight. After that, add 10,000 x g of the suspension.
After centrifuging for 30 minutes, the supernatant was designated as Extract A. In order to carry out the second extraction from this precipitate, the buffer B2 volume was added to the precipitate and the mixture was stirred for 4 hours. Then, the suspension is
After centrifuging at 30 g for 30 minutes, the supernatant was used as the extract B and combined with the extract A to give a crude antigen solution.

2) Treatment of SSA / Ro and SSB / La crude antigen solutions with ribonucleic acid degrading enzyme To 100 volumes of crude antigen solution obtained in 1), add 1 volume of enzyme solution D and perform enzyme treatment at 37 ° C for 30 minutes SSA / Ro and SSB /
It was used as the La antigen reagent.

3) Calculation of degree of affinity increase of SSA / Ro and SSB / La antigen proteins treated with ribonucleic acid degrading enzyme 1) The untreated SSA / Ro and SSB / La antigens obtained in 1) were buffer E
Diluted to 1/500 with 200μ in each well of 96-well EIA plate.
Each was dispensed and coated overnight at 4 ° C. After that, a cleaning solution,
Uncoated antigen was removed by washing 3 times with 400μ. Next, a blocking solution containing 0.2% skim milk powder (KPL
(Manufactured by K.K.), the unreacted surface of each hole and the easily adsorbed site on the surface of the coated antigen protein were blocked at room temperature for 2 hours to prepare an antigen-immobilized plate.

Then, it was prepared by diluting with buffer G 1)
(Untreated with enzyme) and 2) (treated with enzyme) (SSA / R
o) and 50 μl of SSB / La antigen reagent dilution series were added to the wells, and diluted to 1/5000 with buffer G.
200μ of La antiserum was added to the above-mentioned antigen-immobilized plate, and the untreated antigen coated with the enzyme-treated antigen in the liquid phase was allowed to competitively react with a fixed amount of the reference anti-SSB / La antigen for 2 hours at room temperature. After that, it was washed 5 times with the washing liquid F400μ. Subsequently, 200 μm of phosphatase-labeled anti-human IgG, A, M antibody (manufactured by KPL) diluted 1/1000 with buffer G was dispensed into each hole, reacted for 2 hours at room temperature, and washed 5 times with washing liquid F400 μm. . After that, 200 μm of the enzyme phosphatase substrate p-nitrophenyl phosphate (PNPP) (manufactured by KPL) was added to each hole, and the released amount of p-nitrophenol after 1 hour was measured.
It was measured by the increase in absorbance at 405 nm in the visible region measured by a spectrophotometer. The results are shown in FIG. 1, and the inhibition curve for the anti-SSB / La antibody was shifted to the low antigen concentration side by treating SSA / Ro and SSB / La antigens with ribonucleolytic enzyme. From this result, it was found that the predetermined amount of the antigen necessary for neutralizing the anti-SSB / La antibody activity at a predetermined concentration was reduced to 1/4 or less by the treatment with the ribonucleolytic enzyme.

4) Measurement of anti-SSB / La antibody The SSA / Ro and SSB / La antigen reagents obtained in 1) and 2) were diluted 1/500 with buffer E, and 200μ was added to each well of a 96-well EIA plate. Dispensed and coated overnight at 4 ° C. Then, the washing solution F400μ was washed three times to remove the uncoated antigen. Next, the unreacted surface of each hole and the easily adsorbed sites on the surface of the coated antigen protein were blocked with 400 μm of a blocking solution containing 0.2% non-fat dry milk (KPL) to prepare an antigen-immobilized plate. .

After that, the standard anti-SS diluted to 1/1000 with buffer G
200 μm of B / La antiserum was added to the above-mentioned antigen-immobilized plate, reacted at room temperature for 2 hours, and then washed 5 times with washing liquid F400 μm. Then, 200μ of phosphatase-labeled anti-human IgG, A, M antibody (manufactured by KPL) diluted 1/1000 with buffer G
Was dispensed into each hole, reacted at room temperature for 2 hours, and then washed with F400μ
It was washed 5 times. After that, the substrate of the enzyme phosphatase p-nitrophenyl phosphate (PNPP) (manufactured by KPL) 200
μ was added to each hole, and the amount of released p-nitrophenol after 1 hour was measured by a spectrophotometer with an increase in absorbance at 405 nm in the visible region.

The results are shown in Table 1, and the antigenic activity of SSB / La is increased by about 20% by the treatment with ribonucleolytic enzyme.

[Effects of the Invention] Anti-SSA / Ro and SSB / La antibody measuring antigens according to the present invention are used as constituent reagents used in clinical tests performed for the diagnosis and follow-up of autoimmune diseases, or for the prediction of autoimmune phenomena. It becomes a reagent with high performance, and
The method for measuring SSA / Ro and SSB / La antibodies has high measurement sensitivity.

[Brief description of drawings]

FIG. 1 shows the anti-reactivity of SSB / La antigen protein obtained by the present invention.
It is a diagram comparing the reactivity against SSB / La antibody before and after treatment with ribonucleolytic enzyme by enzyme immunocompetitive inhibition assay,
The vertical axis shows the absorbance (wavelength 405 nm), and the horizontal axis shows the amount of antigen used.

Claims (3)

(57) [Claims] 1. An anti-SSA obtained by treating an RNA-binding protein containing an SSA / Ro protein and an SSB / La protein with a ribonucleolytic enzyme, which lacks at least a part of its constituent RNA chains.
/ Ro and SSB / La antibody measurement antigens. 2. A method for extracting an RNA-binding protein from an animal tissue, a cultured cell or a processed product thereof, and treating the resulting extract with a ribonucleolytic enzyme so as to lack at least a part of the constituent RNA chains. Anti-SSA / Ro and SSB / La
Method for producing antigen for antibody measurement. 3. An anti-SSA / Ro and SSB / La in a test sample using the anti-SSA / Ro and SSB / La antibody measuring antigens according to claim 1.
Anti-SSA / Ro and SSB / La antibody assay to detect or quantify antibodies.