JPH1194836A - Use as a marker for rheumatoid arthritis and an immunological reagent for the diagnosis of rheumatoid arthritis - Google Patents

Abstract

(57) [Problem] To provide an antibody that can be used as a novel marker for rheumatoid arthritis in the field of clinical examination, and to provide an immunoreagent for diagnosing rheumatoid arthritis simply and accurately. SOLUTION: The use of an anti-aldolase A antibody as a marker for rheumatoid arthritis and an immunoreagent for measuring the anti-aldolase A antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the use of an antibody against a specific protein as a marker for rheumatoid arthritis and to a reagent for diagnosing rheumatoid arthritis.

[0002]

2. Description of the Related Art Rheumatoid arthritis (hereinafter sometimes abbreviated as RA) is an unexplained systemic autoimmune disease affecting about 1% of the total population. A characteristic of the condition is the destruction of the affected joint accompanied by proliferation of the synovial membrane. Without proper treatment, pain and motor dysfunction interfere with daily life. The diagnosis of RA is made comprehensively, taking into account blood markers, X-ray findings, histological findings, and clinical courses, referring to the criteria of the American College of Rheumatology (revised in 1987).

[0003] The measurement of blood markers is automated,
It can be used to quickly and accurately test multiple samples, which is a method suitable for clinical testing. Rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) are frequently measured as blood markers for RA. RF is an antibody against denatured immunoglobulin G (IgG) and
It is found in 70-80%, but also appears in other autoimmune diseases, and 2-5% of healthy people are also positive. ESR and CRP are markers of inflammation, and are high in diseases with inflammation other than RA. To date, there has been no blood marker specific for RA, so it was not possible to diagnose RA with blood markers.

[0004] The X-ray findings and histological findings are for observing an X-ray photograph of a joint or a section of a joint tissue to determine a disease state, and the examination requires skill and time. In addition, there is a possibility that the accuracy may vary due to the subjectivity of the inspector.

[0005]

As described above, the conventional R
The diagnostic method of A is complicated and time-consuming, and has a problem in accuracy. Therefore, an object of the present invention is to provide a new RA diagnostic marker that solves the above problems. It is still another object of the present invention to develop a reagent for detecting the RA diagnostic marker which is present in a body fluid used as a subject in a clinical test and exhibits some significant difference between a patient with RA and a healthy subject.

[0006]

Means for Solving the Problems The present inventors believe that RA destroys the joints and releases cellular components from osteoblasts, and that antibodies against these components are specifically present in RA patients. After intensive studies, they have found that an anti-aldolase A antibody is specifically present in RA patients, and have completed the present invention.

That is, the present invention is the use of an anti-aldolase A antibody as a marker for rheumatoid arthritis.

Another invention is an immunoreagent for diagnosing rheumatoid arthritis, comprising aldolase A or denatured aldolase A, and detecting an anti-aldolase A antibody present in a subject. .

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, rheumatoid arthritis (R) is used.
The anti-aldolase A antibody used as a marker for A) is aldolase A, ie, D-fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate lyase A, which is a glycolytic enzyme. The antibody is not particularly limited as long as it is an antibody against (EC 4.1.2.13) and has the property of recognizing and binding aldolase A and denatured aldolase A.

[0010] Such anti-aldolase A antibody can be a marker for RA since it has been confirmed by the present inventors that it is specifically present in the body fluid of RA patients as shown in Examples described later. Anti-aldolase A antibodies present in body fluids of RA patients include all classes, ie, immunoglobulin G (IgG), immunoglobulin M (IgG).
M), immunoglobulin A (IgA), immunoglobulin D (Ig
D) and immunoglobulin E (IgE), and IgG having a high blood concentration is preferably used.

As will be shown in the embodiments described later, RA
Since the amount of anti-aldolase A antibody present in the body fluid of the patient was significantly higher than that of healthy subjects and other autoimmune diseases and diseases presenting with arthritis, anti-aldolase A antibody was used Can be a marker.
That is, anti-aldolase A in body fluids, especially serum or plasma
Measuring the amount of antibody allows the diagnosis of RA. The anti-aldolase A antibody in the body fluid can be measured by a known method utilizing the antigen-antibody reaction between aldolase A and the anti-aldolase A antibody described below.

Further, by using an anti-aldolase A antibody as a marker for RA, an RA diagnostic reagent or R
These performances and effects can be evaluated in the course of the development of a drug for A treatment.

Next, the RA diagnostic immunoreagent of the present invention will be described.

The immunoreagent for diagnosing RA of the present invention utilizes the fact that aldolase A or denatured aldolase A causes an antigen-antibody reaction with an anti-aldolase A antibody present in the body fluid of RA patients. The method is not particularly limited as long as the method can detect the aldolase A, but is preferably a form in which aldolase A or denatured aldolase A is supported on an insoluble carrier.

At this time, the insoluble carrier that can be used is a carrier that is insoluble in a solvent in the reaction system of the antigen-antibody reaction.
The material and shape are not particularly limited, and known insoluble carriers can be used. That is, the shape of the insoluble carrier may be appropriately selected according to the purpose of use, and examples thereof include a test plate, a bead, a sphere, a disk, a tube, and a filter. Further, as the material thereof, those used as carriers for ordinary immunoassays, for example, propylene, styrene, acrylamide,
Synthetic resins such as acrylonitrile, or those into which reactive functional groups such as sulfone groups and amino groups have been introduced by known methods, glass, polysaccharides or derivatives thereof, silica gel, porous ceramics, metal oxides, red blood cells, etc. Can be exemplified.

The method for immobilizing aldolase A or denatured aldolase A on an insoluble carrier includes physical adsorption,
Known methods such as a covalent bonding method, an ion bonding method, and a crosslinking method can be used without any limitation.

To illustrate a specific embodiment of the immunoreagent of the present invention in which such an aldolase A or a denatured aldolase A is supported on an insoluble carrier, the qualitative reagents include a latex agglutinating reagent and a microfluidic reagent. Examples of titer reagents and quantitative reagents include a radioimmunoassay reagent, an enzyme immunoassay reagent, a fluorescent immunoassay reagent, a chemiluminescence immunoassay reagent, and a latex immunoassay reagent. In any of these embodiments, RA diagnosis can be performed by bringing the immunological reagent for RA diagnosis of the present invention into contact with a subject and detecting an antigen-antibody reaction between the anti-aldolase A antibody and the carried aldolase A.

The aldolase A contained in the immunoreagent of the present invention is not particularly limited as long as it can recognize and bind to an anti-aldolase A antibody present in the body fluid of RA patients. Aldolase A produced by a replacement technique and the like and denatured versions thereof can be used. Examples of the method for producing aldolase A include extraction and purification from animal tissues and cultured cells, synthesis using microorganisms and cultured cells by genetic recombination techniques, and chemical synthesis. In addition to the aldolase A protein, a partial peptide of aldolase A synthesized by genetic recombination technology or chemical synthesis, as well as an aldolase A protein, as long as it can recognize and bind to the anti-aldolase A antibody present in the body fluid of RA patients. A partial peptide of aldolase A obtained by chemically and enzymatically cleaving A can also be used. From the viewpoint of availability and reactivity, human tissues or cultured cells can be used, for example, from Achieves of Biochemistry and Biophysics (Arc).
h. Biochem. Biophys.), 228, 342-352 (1984).
Year), human aldolase A extracted and purified according to the method described in US Pat.

The anti-aldolase A antibody present in the body fluid of RA patients recognizes aldolase A regardless of whether it is denatured or undenatured. However, since it has the property of reacting more strongly with denatured aldolase A, it has been modified. When aldolase A is used, an anti-aldolase A antibody can be detected with higher sensitivity.

Examples of a method for denaturing aldolase A include a known method for disrupting the three-dimensional structure of an enzyme protein,
That is, it can be carried out by treatment with heat, an acid, an alkali, an ionic surfactant, a denaturant (urea, guanidine hydrochloride, or the like) or the like. Each of these denaturation methods will be specifically described. For example, the heat denaturation method can be performed by heating aldolase A at about 40 to 100 ° C. for about 1 to 60 minutes. In the case of denaturation with an acid or an alkali, for example, aldolase A is left standing for about 10 minutes to 5 hours in an aqueous solution of about 0.01 to 1 M hydrochloric acid or an aqueous solution of about 0.01 to 1 M sodium hydroxide, respectively. It can be carried out. When an ionic surfactant or a denaturing agent is used, for example, an aqueous solution of about 0.1 to 10% by weight of sodium dodecyl sulfate (SDS),
Aldolase A can be left in an aqueous solution of ９9 M urea or an aqueous solution of guanidine hydrochloride of about 1 to 8 M for about 10 minutes to 5 hours. These modification methods may be performed alone or in combination of a plurality of methods.

Aldolase A modified by these methods
When loading on an insoluble carrier, the pH of the solution at the time of loading is controlled to around neutral so as not to hinder the adsorption to the carrier, and excess ionic surfactants and denaturants are removed by dialysis, gel filtration, etc. It is preferable to carry out after removing. Also,
In order to uniformly carry the denatured aldolase A on the insoluble carrier, it is important that the denatured aldolase A is not insolubilized at the time of loading, and therefore, a reducing agent and / or It is preferable to add about 0.0001 to 1% by weight of a nonionic surfactant. The reducing agent used at this time is 2-
Mercaptoethanol, dithiothreitol, and the like. Nonionic surfactants include Triton X-10.
0, Tween 20, and the like.

When the immunoreagent of the present invention obtained as described above is brought into contact with a subject, if the subject contains an anti-aldolase A antibody, the anti-aldolase A antibody can be detected. it can.

Here, the subject to be used is not particularly limited as long as it may contain an anti-aldolase A antibody, but a body fluid such as serum or plasma is preferably used.

As a method for detecting an anti-aldolase A antibody in a subject using the immunoreagent of the present invention, an anti-aldolase A antibody in the subject and aldolase A or denatured aldolase contained in the immunoreagent of the present invention are used. A method capable of detecting a change in physical quantity based on an antigen-antibody reaction with A can be adopted without any limitation. For example, a method for detecting the above-mentioned antigen-antibody reaction as color development, luminescence, or fluorescence using a labeling substance as performed with a so-called labeled immunoassay reagent, an insoluble carrier as performed with a so-called immunological agglutination reagent A detection method generally used in known immunological diagnostic reagents utilizing an antigen-antibody reaction, such as a method of detecting the above-mentioned antigen-antibody reaction by visual observation or turbidity as agglutination, can be employed.

Specifically, when the immunoreagent of the present invention is a labeled immunoassay reagent, a radioisotope such as radioiodine or radiocarbon or a fluorescent dye such as fluorescein isothiocyanate or tetramethylrhodamine is used as a labeling agent. It may be used to measure radioactivity or fluorescence intensity, or to use an enzyme such as alkaline phosphatase or peroxidase as a labeling agent to measure color development, fluorescence or chemiluminescence based on an enzyme reaction. As the substrate for the enzymatic reaction, a known compound generally used can be used without any particular limitation. For example, when alkaline phosphatase is used as a labeling enzyme, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate,
A dioxetane derivative or the like can be used, and the amount of the labeled enzyme can be measured by color development (colorimetry), fluorescence, and chemiluminescence by the enzyme reaction, respectively. When peroxidase is used as the labeling enzyme, for example, 2,2′-azino-bis (3-ethylbenzo-thiazoline-6-
Sulfonic acid), a luminol derivative or the like can be used as a substrate, and the amount of the labeled enzyme can be measured by colorimetry or chemiluminescence, respectively.

When the immunoreagent of the present invention is a labeled immunoassay, the method of measurement using the reagent may be a conventional radioimmunoassay (RIA), an enzyme immunoassay such as an ELISA, a Western blotting, or a dot blotting. The measurement (EIA) can be performed, for example, as follows. That is, aldolase A is added to the insoluble carrier.
Is carried at a rate of 0.01 to 1000 μg / cm ² , and then a subject is brought into contact therewith to bind an anti-aldolase A antibody by an antigen-antibody reaction. Subsequently, 0.001 to 1000 μg of a labeled anti-human immunoglobulin antibody is allowed to act, and the bound anti-aldolase A antibody can be measured by radioactivity, colorimetry, fluorescence, chemiluminescence, or the like. Further, by using an anti-human immunoglobulin antibody conjugated with biotin and labeled avidin in place of the labeled anti-human immunoglobulin antibody, measurement can be performed with even higher sensitivity. in this case,
After contacting 0.001 to 1000 μg of biotin-conjugated anti-human immunoglobulin antibody, and then contacting 0.001 to 1000 μg of labeled avidin, similarly, an anti-aldolase A antibody bound by radioactivity, colorimetry, fluorescence, chemiluminescence, etc. Can be measured.

When the immunoreagent of the present invention is an immunological agglutination reagent, the measurement using the reagent can be carried out by ordinary hemagglutination, passive agglutination, immunocolorimetry, immunoturbidimetry. Similarly to the above, for example, it can be performed as follows. That is, 0.001 to 1000 mg per 1 g of the particulate insoluble carrier.
The particles carrying aldolase A (hereinafter abbreviated as sensitizing particles) may be dispersed in an aqueous medium so as to have a concentration of 0.001 to 15% by weight and used as an active ingredient of an immunoreagent. The particle size of the antibody carrying aldolase A is preferably an insoluble carrier having an average particle size of 0.05 to 10 μm from the viewpoint of the ease of aggregation after the antigen-antibody reaction and the ease of discrimination of aggregation. is there. The sensitized particles prepared by such a method are contacted with an anti-aldolase A antibody in a subject, and the degree of aggregation of the sensitized particles is measured. Conventional methods such as visual observation and optical measurement can be used for the degree of particle aggregation without any limitation.

[0028]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) In RA, bone destruction progresses at joints, and cell components are released from osteoblasts. Antibodies to these components are R
A, an osteoblastic cell line
63 (ATCC CRL1427) was used to search for antibodies specifically present in RA patient serum as follows.

In MG63 (1 × 10 ⁷ cells), 1 ml of a cell lysate (5
0 mM Tris-HCl buffer pH 7.4, 150 mM NaCl, 1% NP-4
0) added and left on ice for 30 minutes. After centrifugation, the supernatant was taken as a cell extract. After separating the cell lysate by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% gel, the protein is electrotransferred to a nitrocellulose membrane, and the MG63 cell components are recognized using RA patient serum. Antibody search was performed. The protein-transferred nitrocellulose membrane was immersed in PBS containing 5% skim milk (10 mM phosphate buffer pH 7.0, 150 mM NaCl) for 1 hour at room temperature to perform blocking. Washing solution 1 (PBS containing 0.1% Tween 20)
After washing three times for 15 minutes each, the nitrocellulose membrane was divided, and each membrane was immersed in RA patient serum (4 samples) and healthy subject serum (5 samples) diluted 100-fold with Washing Solution 1, and allowed to stand at room temperature.
Shake for 1 hour. After washing 3 times for 15 minutes each with washing solution 1,
The cells were permeated for 1 hour at room temperature in a peroxidase-labeled anti-human IgG antibody (Kappel) diluted 10,000 times with Washing Solution 1.
After washing three times with washing solution 1 for 15 minutes each, an antibody bound to the antigen protein was detected by an ECL western blotting detection reagent (Amersham). FIG. 1 shows a photograph of each membrane after the Western blotting method. Arrows in the figure indicate the position of the 40 kDa protein. In the membrane immersed in the serum of the RA patient, a band can be clearly seen in the arrow portion, whereas in the membrane immersed in the serum of a healthy individual, no band can be seen in this portion. From this, it became clear that an antibody recognizing the 40 kDa protein was specifically present in the serum of RA patients.

In addition, high performance liquid chromatography (HPL)
As a result of purifying the 40 kDa protein from the MG63 cell extract by C) and examining the N-terminal amino acid sequence of 8 residues, it was found that the 8 residues were PYQYPALT. When the homology was searched for in the database, it was completely identical to human aldolase A.

(2) Next, the reaction between RA patient serum and commercially available rabbit aldolase A was examined. In addition, rabbit aldolase A has 96% homology with human aldolase A. RA patient serum and commercial rabbit aldolase A
(Wako Pure Chemical) was examined for reactivity. Rabbit aldolase A was transferred to a nitrocellulose membrane after SDS-PAGE and subjected to an antigen-antibody reaction in the same manner as in the above (1) except that a commercially available rabbit aldolase A was used. . FIG. 2 shows a photograph of each membrane after the Western blotting method. The band indicated by the arrow in the figure represents rabbit aldolase A. A strong band can be confirmed at the arrow portion in the membrane immersed in the serum of RA patient, whereas no band can be confirmed in the membrane immersed in the serum of a healthy individual. From this, it became clear that an antibody recognizing rabbit aldolase A was specifically present in RA patient serum.

(3) Further, an absorption test of serum from RA patients with commercially available rabbit aldolase A was performed as follows. That is, when the MG63 cell extract and the RA patient serum were reacted according to the above (1), an absorption test using commercially available rabbit aldolase A was performed. RA diluted 100 times with washing solution 1
Commercial rabbit aldolase A was added to patient serum at 100 μg / ml or 10 μg / ml, and left at room temperature for 1 hour. As a control, 100 μg / ml bovine serum albumin (BS
Absorption by A) was also performed. After the absorption, an antigen-antibody reaction was performed in the same manner as in (1), and detection was performed using an ECL western blotting detection reagent. FIG. 3 shows a photograph of each membrane after the Western blotting method. The arrow in the figure is 40 kDa
, Ie, human aldolase A. The intensity of the aldolase A band was not changed by the absorption by BSA, but the band disappeared by the absorption by the commercially available rabbit aldolase A. From this, the specific antibody present in the serum of RA patients recognizes a 40 kDa protein,
The Da protein was confirmed to be human aldolase A.

From these results, it was clarified that the 40 kDa protein was aldolase A, and an antibody recognizing aldolase A was present in the serum of RA patients. Further, it was revealed that the antibody recognizing aldolase A present in the serum of RA patients is an anti-aldolase A antibody recognizing human aldolase A and rabbit aldolase A.

Example 2 [Measurement of Anti-Aldolase A Antibody in Subject by ELISA] Commercially available rabbit aldolase A was suspended in an 8M urea solution and denatured by allowing it to stand at room temperature for 1 hour. 0.4% by weight of the solution after denaturation 2-mercaptoethanol, 0.1% by weight
Excess urea was removed by dialysis against PBS containing SDS and 0.1% by weight of Tween 20 at room temperature to prepare a denatured aldolase A solution (3 mg / ml). A denatured aldolase A solution was added to Wash Solution 2 (PBS containing 0.05% Tween 20) (20 μg / ml) to obtain an antigen-sensitized solution. 100 μl of the antigen sensitizing solution was dispensed into each well of a 96-well microplate and left at 4 ° C. overnight. After washing three times with Washing Solution 2, 250 μl of a blocking solution (1% skim milk, 1% Tween 20, PBS) was dispensed and allowed to stand at room temperature for 3 hours. Wash 3 times with cleaning solution 2, 100
μl serum dilution (1% casein, 1% skim milk, 2
% Emulgit 25, PBS) was dispensed into each well. 10 μl
Was dispensed and diluted 11-fold in the wells. The plate was covered with a plate seal, and allowed to stand at 37 ° C. for 1 hour to perform a primary antibody reaction. The serum used was RA patient serum, osteoarthritis (OA) patient serum, systemic lupus erythematosus (SLE) patient serum, gout patient serum, and healthy serum without arthritis. Wash three times with Wash Solution 2 and dilute the peroxidase-labeled anti-human IgG antibody 1000-fold with an antibody diluent (1% BSA, 0.05% Tween 20, PBS).
Each 100 μl was dispensed into each well. The plate was covered with a plate seal, and allowed to stand at 37 ° C. for 1 hour to perform a secondary antibody reaction. Wash 3 times with washing solution, 100μl of 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS) as substrate solution
And a mixture of equal amounts of H ₂ O ₂ solution (KPL) was dispensed into each well. The mixture was allowed to stand at room temperature for 30 minutes while protected from light, and the reaction was stopped by adding 100 µl of an SDS solution. The absorbance at 405 nm of each well was measured (FIG. 4).

As shown in FIG. 4, the absorbance of all the samples other than RA was 0.3 or less, and the anti-aldolase A antibody was detected specifically for RA. These results revealed that the anti-aldolase A antibody was an excellent marker for RA.

Example 3 [Evaluation of the effect of denaturing aldolase A on the reactivity of anti-aldolase A antibody] In the measurement of anti-aldolase A antibody by ELISA, absorption by denatured aldolase A and native aldolase A A test was performed to determine the reactivity of anti-aldolase A antibodies present in RA patients.

That is, 20 μg / ml denatured aldolase A
Alternatively, ELIS was performed using RA patient serum in the same manner as in Example 2 except that a serum diluent containing native aldolase A was used.
Method A was performed. The result is shown in FIG. As shown in FIG. 5, the anti-aldolase A antibody present in the serum of the RA patient was more strongly reacted with denatured aldolase A than with unmodified aldolase A because the antibody was strongly inhibited by absorption by denatured aldolase A. It became clear.

[0039]

The present inventors have found that the amount of anti-aldolase A antibody present in the body fluid of RA patients is significantly higher than that of healthy subjects and other diseases exhibiting autoimmune diseases and arthritis. This indicates that the anti-aldolase A antibody can be effectively used as a novel clinical marker for diagnosing RA. In particular, anti-aldolase A antibodies were
When used as a marker for the test, the incidence of false positives was extremely low (specifically, the number of samples that showed positive as measured in 45 healthy subjects and other subjects with other diseases was 0). The use of the diagnostic reagent of the present invention containing aldolase A enables highly accurate diagnosis of RA.

[Brief description of the drawings]

FIG. 1 is a photograph of each nitrocellulose membrane after Western blotting using an osteoblast cell line (MG63) extract and serum from RA patients or normal subjects.

FIG. 2 is a photograph of each nitrocellulose membrane after Western blotting using commercially available rabbit aldolase A, serum from RA patient, and serum from healthy volunteers.

FIG. 3 shows each nitrocellulose membrane after Western blotting using RA patient serum or RA patient serum absorbed with commercial rabbit aldolase A or BSA and an osteoblast cell line (MG63) extract. It is a photograph of.

FIG. 4 is a graph showing the relationship between serum anti-aldolase antibodies of RA patients, healthy subjects, and patients with arthritis other than RA.
It is a figure showing the result checked by ELISA method.

FIG. 5 This figure shows denatured aldolase A in the ELISA method.
FIG. 4 shows the inhibitory effect of native aldolase A and native aldolase A.

Claims (3)

[Claims] 1. Use of an anti-aldolase A antibody as a marker for rheumatoid arthritis. 2. An immunological reagent for diagnosing rheumatoid arthritis, comprising an aldolase A and detecting an anti-aldolase A antibody present in a subject. 3. An immunological reagent for rheumatoid arthritis diagnosis, comprising denatured aldolase A and detecting an anti-aldolase A antibody present in a subject.