KR100380147B1 - Process for the detection of rheumatoid autoimmune antibodies and kit for the detection thereof - Google Patents

Abstract

The present invention relates to a method and kit for the simultaneous detection of several autoantibodies specific for rheumatoid autoimmune antigens present in plasma or serum, which are easier and less convenient than conventional methods for detecting only one antibody in a single test. Since a positive sample is required, it can be used as a very useful tool for the detection and study of rheumatoid autoimmune antibodies.

Description

Process for the detection of rheumatoid autoimmune antibodies and kit for the detection among others

The present invention relates to the detection of rheumatoid autoimmune antibodies, and more specifically, to a variety of autoantibodies specific to rheumatoid autoimmune antigens present in plasma or serum. The present invention relates to a method for simultaneous detection and a detection kit for rheumatoid autoimmune antibodies.

Rheumatoid disease causes pain in the whole body such as joints, bones, and muscles, and is one of 100 types of autoimmune diseases including arthritis (rheumatoid arthritis) and systemic lupus erythematosus (SLE). Rheumatoid disease is more common in women than in men, the cause of which is not yet clear, but only genetic factors, drugs, environmental pollutants, etc. are supposed to be caused. As such, the cause of the disease is unclear, and it is very difficult to cure a state in which a special therapeutic agent is not yet developed. In addition, since the symptoms of the disease are various and it is not easy to judge the disease simply by the symptoms, it is important to accurately diagnose the patient for early detection and treatment.

Usually, a variety of test methods are used in the diagnosis of rheumatic diseases, among which immunological tests for markers are used as useful references. The markers for rheumatic diseases include rheumatoid factor (RF), anti-nuclear antibody (ANA), antibodies to extractable nuclear antigen (ENA), and Jo-1 protein. have. RF testing is a quantitative method of detecting antibodies to human IgG F _c fragments (IgG, IgM and IgA), with 70-80% of patients positive and some of the normal persons positive. The ANA test is a method of detecting antibodies generated against nucleic acids such as DNA or RNA. ENA testing is also a method of detecting RNA-protein complexes and some nucleic acid-related proteins extractable from the nucleus. Dual RNP (ribonucleoprotein) antigens are complexes of U1 snRNAs and proteins, and are known as marker antigens such as mixed connective tissue diseases, systemic lupus erythematosus or progressive systemic sclerosis. RNP antigens are usually complexed with Sm (Smith) antigens and used together without separation. Sm antigen, a non-histone glycoprotein, is complexed with U1, U2, U4, U5, and U6 snRNA and is a very useful marker for systemic lupus erythematosus. Meanwhile, SS-A and SS-B, which are markers of Sjogren's syndrome, which cause inflammation of the tear glands or salivary glands and inhibit the secretion of tears or saliva, are RNA-protein complexes. It has an antibody against this antigen. Antigens as described above are commonly termed "ENA" as RNA-protein complexes extractable from the nucleus. Scl-70 is a topoisomerase I found not only in the nucleus but also in the cytoplasm and used to diagnose skin lesions (scleroderma). Jo-1 is a histidyl tRNA synthetase and is used for the diagnosis of chronic myositis, such as polymyositis or dermatomyositis.

Such labels are mainly detected by serological methods and provide a very useful basis for the diagnosis of disease. For example, in most systemic lupus erythematosus, ANA is detected and the frequency of RNP antigen, Sm antigen, SS-A and SS-B antigen is also high. In addition, for Sjogren's syndrome, SS-A and SS-B antigens are detected. Therefore, testing only against a single antigen can lead to inaccurate results, and therefore, an accurate diagnosis can usually be made only by testing against multiple antigens.

On the other hand, as an immunological detection method of rheumatoid autoimmune antibodies, enzyme immunoassay, double immunodiffusion, immunofluorescence and the like are known. However, according to this conventional method, since the test must be performed separately for each autoimmune antigen for accurate diagnosis, it is troublesome to use and requires a large amount of samples. Therefore, there has been a demand for the development of a new method capable of simultaneously detecting autoantibodies against several rheumatic autoimmune antigens in one trial.

The inventors have carried out ongoing studies to develop immunoassays that can simultaneously detect autoantibodies against several rheumatic autoimmune antigens in just one test. As a result, it was confirmed that by using a fixture such as a nitrocellulose membrane to which several rheumatic autoimmune antigens were independently attached to each other, it was possible to simultaneously detect several autoantibodies present in the blood of rheumatoid autoimmune patients. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for simultaneously detecting various autoantibodies present in blood using markers of various rheumatic autoimmune diseases and a detection kit for rheumatoid autoimmune antibodies.

1 shows a strip attached with rheumatoid autoimmune antigens and criteria for determination;

Figure 2 shows the results of immunoblot autoimmune test using plasma of rheumatoid autoimmune patients; And,

Figure 3 is a diagram showing the results of immunoblot autoimmunity test using plasma of a normal person.

The present invention is to detect two or more autoantibodies in a sample simultaneously by adding a sample to a fixture to which two or more rheumatoid autoimmune antigens are independently attached to each other and binding an autoantibody present in the sample to the antigen. The present invention relates to a method for detecting autoimmune antibodies. In the method according to the invention, the rheumatic autoimmune antigen is preferably at least one selected from the group consisting of rheumatoid factor, nuclear antigen and ENA, more preferably RNP / Sm, Sm, SS- A, SS-B, Scl-70 and Jo-1. In addition, the autoantibody is preferably human IgG, the fixture is preferably a nitrocellulose membrane, and more preferably, the nitrocellulose membrane is prepared in a strip form.

For example, the method of the present invention includes the following steps:

Iii) adding a plasma or serum sample to a nitrocellulose strip to which two or more rheumatoid autoimmune antigens are independently attached to each other to bind autoantibodies present in the sample with the antigens;

Ii) adding a labeled antibody conjugate to the autoantibody to bind to the autoantibody attached to the strip;

Iii) inducing color development using the label of the conjugated label antibody conjugate; And,

Iii) comparing the degree of color development with a control band.

In this method, the labeling antibody conjugate of step iii) is preferably an antibody that specifically binds to human IgG, and in step iv) it is preferred to use a control at a concentration of at least two.

The present invention also provides a display comprising: a) a fixture wherein two or more rheumatoid autoimmune antigens are independently attached to each other; Ii) labeling antibody conjugates; And iii) a diagnostic kit for a rheumatoid autoimmune specific antibody comprising a substrate solution containing a color developer. In the kit of the invention, the rheumatoid autoimmune antigen is preferably at least one selected from the group consisting of rheumatoid factor, nuclear antigen and ENA, more preferably RNP / Sm, Sm, SS-A, SS-B , Scl-70 and Jo-1. It is preferred that the fixture is a nitrocellulose membrane, in particular a nitrocellulose membrane made in strip form, more preferably two or more concentrations of control attached. It is also preferred that the label antibody conjugate specifically binds to human IgG.

Hereinafter, the present invention will be described in detail.

First, the present invention provides an immunoassay for detecting autoantibodies using a label of a rheumatoid autoimmune disease as an antigen. Specifically, the present invention provides an immunoblot assay for simultaneously detecting autoantibodies present in blood using a fixture to which labeled antigens of various rheumatic autoimmune diseases are independently attached to each other. To this end, the present invention may use nitrocellulose membranes prepared in strip form as fixtures. For example, the immunoblot measurement method according to the present invention

a) reacting the nitrocellulose strip to which the rheumatoid autoimmune antigen is attached by adding serum, a plasma sample, or a sample diluent;

b) washing the strip;

c) reacting the autoimmune antibody with a labeled antibody conjugate thereto;

d) washing the strips;

e) coloring by adding a substrate solution containing a coloring agent and a buffer solution; And,

f) reading the colored strip.

In the method according to the present invention, when serum or plasma of a patient to be diagnosed is added to a nitrocellulose strip to which a rheumatic autoimmune antigen is attached, autoantibodies present in the serum or plasma bind to an autoimmune antigen attached to the nitrocellulose strip. When a label antibody conjugate is added thereto, the conjugate recognizes and binds to the autoantibody attached to the strip. The conjugate thus bound is colored using a colorant to detect autoantibodies.

Secondly, the present invention provides a detection kit for rheumatoid autoimmune antibodies comprising a substrate solution containing two or more rheumatoid autoimmune antigen-attached fixtures, a labeled antibody conjugate, and a coloring agent. The detection kit of the present invention includes a label antibody conjugate, preferably a horsereddish peroxidase (HRP), a basic alkaline phosphatase, a colloidal gold, a fluorescein, a dye, and the like. Labels can be used. In the present invention, for example, goat anti-human IgG-HRP conjugates are used. In addition, the detection kit of the present invention includes a substrate solution that induces a color reaction by reacting with an enzyme-labeled antibody conjugate. Preferably, 4-chloro-1 reacts with a label of the enzyme-labeled antibody conjugate to cause a color deposit. -Use a substrate solution containing a colorant such as naphthol (4-chloro-1-naphtol) and a buffer solution. A color developing agent such as 4-chloro-1-naphthol in the substrate solution was decomposed by HRP used as a label of the enzyme-labeled antibody conjugate to generate a color deposit, and visually confirm the degree of deposition of the color deposit. Detects the presence or absence.

Rheumatoid autoimmune antigens used in the methods and kits according to the invention are rheumatoid factor, DNA, RNA, nuclear antigens such as histones, ENAs, etc. For example, RNP / Sm, Sm, SS-A, SS-B, Scl-70 and Jo-1. The autoimmune antigens are derived from animals such as rabbits and pigs, and preferably those purified by high purity by immunoaffinity chromatography are used.

In addition, in the present invention, human IgG can be used as a control to indicate the intensity of the color reaction according to the amount of autoantibodies. Preferably, human IgG is attached to the nitrocellulose membrane at a concentration of 2 or more to improve the degree of reaction. Use as an indicator to indicate. More specifically, the response is stronger than that of the highest concentration of IgG, which is strongly positive, the weaker than the highest IgG, and the neutral of the weaker than the highest IgG. Allow the quantity to be determined. When three concentrations of IgG are used, the same or stronger than the lowest IgG and lower than the medium IgG, the reaction is treated as negative as the sample that does not occur, but the amount of antibody is recognized as having a slight reaction. Allow for judging by small or nonspecific reactions.

In general, a method of attaching a protein to a nitrocellulose membrane may include simply dropping a protein solution and then drying it, spraying the solution using an injector, or using a mold connected to a decompression device. Bands and small circles are used as proteins to attach to nitrocellulose membranes. In the present invention, the autoimmune antigen is attached to the nitrocellulose membrane in a band shape using, for example, a mold connected to a pressure reducing device.

The methods and kits of the present invention were applied to the plasma of rheumatoid autoimmune patients and normal subjects. As a result, 53 of 55 autoimmune patients responded to autoimmune antigen and only 2 of 52 normal individuals responded weakly. Thus, the methods and kits according to the present invention can be used as very useful tools for the detection and study of rheumatoid autoimmune antibodies.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples are only for the understanding of the present invention, and the scope of the present invention is not limited to these Examples.

Example 1 Immunoblot Measurement for Rheumatic Autoimmune Patients and Normal Subjects

In this example, RNP / Sm, Sm, SS-A, SS-B, Scl-70 and Jo-1 antigens (Biodesign, Affinity Tablet, USA) were used as marker antigens for rheumatoid autoimmune diseases. Used. In addition, human IgG was used as a control to analyze the intensity of the response through the criteria as well as qualitative measurement of autoantibodies (FIG. 1).

Strips to which autoimmune antigens were attached were prepared as follows. Insert the filter paper and the nitrocellulose membrane into the slot mold connected to the decompression device, and assemble them. Then, each concentration of human IgG antibody and autoimmune antigen and control were adjusted to 1-10 mg / ml in phosphate buffer (pH 7.0). After dilution, 2 ml was added to each slot mold. Once the solution had entered, the mold was dismantled and the nitrocellulose membrane was removed and dried at room temperature for 3 to 4 hours. After leaving at room temperature for 2 hours in phosphate buffer containing 1% casein, the nitrocellulose membrane was taken out and dried for 18 hours. After the drying was completed, the nitrocellulose membrane was attached to the adhesive support, and then cut into strips having a length of about 0.3 cm and about 4 cm.

Put the strips attached with autoimmune antigens one by one on a plate with a width of 1 cm and 10 cm, and then use phosphate buffer solution containing 1.0% casein and 0.1-1.0% Triton X-100 as sample dilution solution. 1 ml each was added to the strip and 20 µl each sample was mixed well, followed by reaction for 2 hours with stirring at room temperature. After the reaction was completed, the strip was washed five times using phosphate buffer containing 0.05% Tween 20. A conjugate of goat-derived anti-human IgG-HRP (Cappel, IgG F _c specific, affinity purified, USA) was prepared using phosphate buffer (pH 7.0) containing 30% FBS and 0.1% Tween 20. After diluting at a ratio of 1: 500 to 1: 2000 using a diluent, 1 ml of each enzyme-labeled antibody conjugate was added to the strip and reacted at room temperature for 30 minutes. After completion of the reaction, the well plate was washed five times with phosphate buffer solution containing 0.05% Tween 20, and 15-20 mM 4-chloro-1-naphthol was washed with 0.015% hydrogen peroxide, 0.28% triethanolamine and 0.15 M sodium chloride. 1 ml of the diluted 1: 5 ratio was added to the constructed substrate buffer (pH 7.5) and developed for 20 minutes. After proper color development, the strip was washed twice with distilled water to terminate the reaction. The autoantibody detection test was performed on the plasma samples of the rheumatoid autoimmune patients and the plasma samples of the normal subjects in 52 and the plasma samples of the autoimmune patients in Table 1 and FIG. 2. Test results using the plasma of normal persons are shown in FIG. 3.

As shown in Table 1 and FIGS. 2 and 3, 53 out of 55 plasma samples of autoimmune patients were determined to be positive, and 50 out of 52 plasma samples of normal individuals were determined to be negative.

Example 2 Preparation of a Detection Kit for Rheumatoid Autoimmune Antibodies

Each of the autoimmune antigen-attached nitrocellulose strips, goat-derived anti-human IgG-HRP conjugate solution and 4-chloro-1-naphthol containing substrate buffer solution prepared according to the method described in Example 1 above was placed in a pharmaceutically acceptable container. The package was prepared for the detection kit of rheumatoid autoimmune antibody.

According to the present invention, since it is possible to detect several rheumatoid autoimmune antibodies at the same time in one test, it is easier to use compared to the conventional method of detecting only one antibody in one test. It can be used as a very useful tool for the detection and study of rheumatoid autoimmune antibodies.

Claims (16)

At least two autoantibodies in the sample are simultaneously detected by adding a sample to a fixture to which two or more rheumatoid autoimmune antigens are independently attached to each other and binding an autoantibody present in the sample to the antigen. Method for detecting rheumatic autoimmune antibody. The method of claim 1, wherein the rheumatoid autoimmune antigen is at least one selected from the group consisting of rheumaid factor, nuclear antigen, and extractable nuclear antigen. The method of claim 2, wherein the rheumatoid autoimmune antigens are RNP / Sm, Sm, SS-A, SS-B, Scl-70 and Jo-1. The method of claim 1, wherein the autoantibody is human IgG. The method according to any one of claims 1 to 4, wherein the fixture is a nitrocellulose membrane. The method of claim 5, wherein the nitrocellulose membrane is made and used in the form of a strip. The method of claim 6, Iii) adding a plasma or serum sample to a nitrocellulose strip to which two or more rheumatoid autoimmune antigens are independently attached to each other to bind autoantibodies present in the sample with the antigen; Ii) adding a labeled antibody conjugate to the autoantibody to bind to the autoantibody attached to the strip; Iii) inducing color development using the label of the conjugated label antibody conjugate; Iii) comparing the degree of color development with a control band. 8. The method of claim 7, wherein the labeling antibody conjugate of step ii) specifically binds to human IgG. 8. The method of claim 7, wherein in step iii) the control is used at a concentration of at least two. Iii) a fixture having two or more rheumatoid autoimmune antigens attached independently of one another; Ii) labeling antibody conjugates; And iii) a detection kit for rheumatoid autoimmune antibody comprising a substrate solution containing a color developer. The kit of claim 10, wherein the rheumatoid autoimmune antigen is at least one selected from the group consisting of rheumatoid factor, nuclear antigen and ENA. 12. The kit of claim 11, wherein the rheumatoid autoimmune antigens are RNP / Sm, Sm, SS-A, SS-B, Scl-70 and Jo-1. The kit according to any one of claims 10 to 12, wherein the fixture is a nitrocellulose membrane. The kit of claim 13, wherein the nitrocellulose membrane is made in strip form. The kit of claim 10, wherein at least two concentrations of the control further comprise a fixture to which it is attached. The kit of claim 10, wherein the labeling antibody conjugate specifically binds to human IgG.