WO2010123069A1 - Method for diagnosing autoimmune disease patient, in vitro diagnostic agent and therapeutic agent therefor - Google Patents

Abstract

Disclosed are: a method for diagnosing a patient for whom an antibody therapy and/or endotoxin absorber therapy are efficacious; and an in vitro diagnostic agent to be used in said diagnosis. Also disclosed are: a therapeutic agent which is specifically efficacious for such an autoimmune disease patient as described above; and a therapeutic method therefor. Specifically disclosed are: a method for diagnosing an autoimmune disease patient which is efficacious in an antibody therapy and/or endotoxin absorber therapy, characterized by comprising selecting a patient negative to DR15 at the HLA-DR locus that is expressed in cells of the immune system of the patient; an in vitro diagnostic agent for such an autoimmune disease patient which comprises, as the essential elements, a labeled primer of HLA-DR gene and a stationary phase probe complementary to DR15 gene; a therapeutic agent for an autoimmune disease patient negative to HLA-DR15 that is expressed in cells of the immune system, which comprises immunoglobulin and/or an endotoxin absorber; and a therapeutic method for an autoimmune disease characterized by comprising orally administering said therapeutic agent to such an autoimmune disease patient as described above.

Description

Method for diagnosis of autoimmune disease patient, in vitro diagnostic agent and therapeutic agent thereof

The present invention relates to a method for diagnosing an autoimmune disease patient for distinguishing a patient to whom treatment such as antibody therapy is effective among patients with an autoimmune disease such as rheumatism, an in vitro diagnostic agent used for the diagnosis, and the patient The present invention relates to a therapeutic agent for autoimmune diseases and a therapeutic method thereof.

The immune system recognizes foreign substances that are different from normal self-components, including abnormal cells such as cancer, as well as foreign microorganisms, and maintains the homeostasis of the body by attacking and eliminating these by immune reaction Mechanism. Therefore, it is considered normal that immunity is not established against the self component. However, when the normal immune system loses its balance and is in a state of malfunction, and when it attacks and eliminates self-components, various intractable diseases develop. These diseases are collectively called autoimmune diseases, rheumatoid arthritis, autoimmune hepatitis, autoimmune gastritis, autoimmune nephritis, inner ear autoimmune disease, autoimmune encephalomyelitis, autoimmune thyroiditis, Known are type 1 diabetes, systemic lupus erythematosus, polydermatomyositis, psoriasis, Sjogren's syndrome, ulcerative colitis, Crohn's disease, Guillain-Barre syndrome, and the like.

In addition to the immune response to self, autoimmune diseases can also cause the onset and exacerbation of diseases caused by enterotoxin-derived toxins such as endotoxin and enterotoxin that pass through the gastrointestinal barrier and enter the body. It is said.

Rheumatoid arthritis is the disease with the largest number of patients among autoimmune diseases. In order to contribute to the prevention and treatment of rheumatoid arthritis, the present inventors have elucidated the etiology. The etiology of rheumatoid arthritis is due to the internal factors of the patient's genetic factors and the external factors of the type II collagen from different animals in the diet, and endotoxin and enterotoxin absorption from the gastrointestinal tract I did research based on this idea.

Type II collagen is the collagen that constitutes articular cartilage, and is immunologically different from type I collagen in skin, bone, and blood vessels, and since many antibodies against type II collagen are found in patients with rheumatoid arthritis, It is considered to be an autoimmune antigen in rheumatoid arthritis (see Non-Patent Document 1). The cross-reaction by each antibody is observed between type II collagen such as chicken and cow and human type II collagen, so that the structures are immunologically similar. This suggests that an individual who produces an antibody against ingestion of heterologous type II collagen in food may react with its own articular cartilage and establish autoimmunity.

In order to verify this hypothesis, the arthritis-inducing activity of similar antigens and endotoxin was examined using mice for collagen arthritis in mice. The DBA / 1 mouse is a mouse having a genetic trait that easily develops type II collagen arthritis. When this mouse was orally given chicken-derived type II collagen or heat-denatured, antibodies against mouse type II collagen were produced in the blood of the mouse and arthritis developed. This arthritis was exacerbated by concomitant use of endotoxin (also called “lipopolysaccharide” or “LPS”). Arthritis also developed after long-term administration of endotoxin alone, excluding chicken-derived type II collagen (see Non-Patent Document 2).

Regarding endotoxin being involved in the onset of autoimmune diseases other than rheumatoid arthritis, autoimmune hemolytic anemia (see, for example, Non-patent Document 3), autoimmune cholangitis (see, for example, Non-patent Document 4) ) Etc. That is, it is also supported in the literature that endotoxin is involved in the onset of diseases in common with autoimmune diseases other than rheumatoid arthritis.

Endotoxin, that is, bacterial toxin in the gastrointestinal tract, is one of the components of the outer membrane of Gram-negative bacteria, scientifically composed of lipid A and a polysaccharide portion, and is abbreviated as LPS. LPS is also known as a factor that causes a systemic response known as endotoxin shock. LPS toxicity is known to be in the lipid A moiety. Gram-negative bacteria mainly parasitic on the digestive tract include Escherichia coli, Salmonella, Enterobacter, Serratia, Shigella, Klebsiella, Pseudomonas, Proteus, Yersinia, Hafnia, Morganella, Bacteroides, Bacteroides, Examples include Prevotella, Fusobacterium, Porphyromonas, Leptotricia, Vylophila, Bayonella, Megasfera, Acidominococcus, Campylobacter and Helicobacter.

Macrophages have LPS receptors on the cell surface, are activated by LPS binding, release inflammatory cytokines such as TNF and IL-6 as part of the biological defense function, and have a large pathological condition of endotoxin shock. It is a factor.
Further, as other bacterial toxins exhibiting the same activity as LPS, superantigens represented by staphylococcal enterotoxins are known. Whereas endotoxin is a component of Gram-negative bacterial cells, enterotoxin is a bacterial toxin that is secreted by bacteria and is chemically a protein. Enterotoxin produced by Staphylococcus aureus is well known as a causative toxin for food poisoning.

From the study of the toxic mechanism of Staphylococcus aureus enterotoxin, Staphylococcus aureus enterotoxin has a structure that binds and binds lymphocytes and macrophages, so that many lymphocytes and macrophages are activated simultaneously, and cytokines such as TNF It is known that the simultaneous production of is responsible for the development of toxicity leading to death from poisoning.

In general, all antigens are taken up by macrophages in the process of establishing immunity, and in the process of presenting structural information of antigens to lymphocytes, macrophages and lymphocytes bind and activate both lymphocytes and macrophages. Cytokines are produced, lymphocytes undergo cell division and proliferation, and immunity is established. In this case, since there are very few lymphocytes involved in the immune response, the amount of cytokines produced is small and no physiological abnormality occurs. However, Staphylococcus aureus enterotoxin is also called a superantigen because it has the property of linking many lymphocyte populations to macrophage populations and activating them, unlike antigens. SEA, SEB, and 20 other superantigens are known as enterotoxins produced by Staphylococcus aureus (see, for example, Non-Patent Document 5). As such superantigens, other than Staphylococcus aureus, enterotoxin (SEC) produced by Staphylococcus intermedius, multiple pyrogen toxins (SPEs) produced by group A streptococci, Examples include Yersinia mitogen (YPEM) produced and Mycoplasma mitogen (MAM) produced by mycoplasma.
Since superantigens promote cytokine production in the same way as endotoxin, involvement in rheumatoid arthritis and other autoimmune diseases has been studied (see Non-Patent Document 6).

The present inventors have found that in studies using animal models of rheumatoid arthritis, the effects of endotoxin and enterotoxin in the gastrointestinal tract can be suppressed by orally ingesting immunoglobulin, and prevention, treatment and recurrence of rheumatoid arthritis. It has been disclosed that it has a preventive function (see Patent Document 1). In this specification, the so-called antibody treatment method is a treatment method in which antibodies against bacterial toxins such as endotoxin and enterotoxin, and immunoglobulins against bacteria producing these bacterial toxins are orally ingested as described in Patent Document 1. It is. In addition, for endotoxin in the gastrointestinal tract, we succeeded in suppressing the onset in a mouse rheumatoid arthritis model by orally ingesting an endotoxin-specific absorber, and developed a novel autoimmune disease preventive treatment endotoxin absorber (patent) Reference 2).

JP 2006-151914 A Japanese Patent Application No. 2009-533206 JP 11-335396 A JP 2002-263486 A JP 2002-311029 A JP 2004-292357 A Special Table 2001-512140 Japanese Patent Laid-Open No. 5-192198 JP-A-8-308596

The present inventors conducted clinical trials based on the research on the onset mechanism of autoimmune diseases represented by rheumatoid arthritis as described above and the treatment method thereof. As a result, patients who are effective with such antibody treatment methods. It was found that a patient who does not respond is possible by discriminating the inheritance of the patient.
Based on such new findings, the present invention establishes a diagnostic method for a patient to achieve a more appropriate and effective treatment of a patient with an autoimmune disease, an in-vitro diagnostic agent used for such a diagnosis, and further It aims at providing the therapeutic agent and therapeutic method of an autoimmune disease.

That is, the present invention provides a diagnostic method for discriminating patients in which antibody treatment and / or endotoxin absorber treatment is effective, and a novel in vitro diagnostic agent used for such diagnosis. It is an object to provide a therapeutic agent and a therapeutic method that are particularly effective for patients with autoimmune diseases, particularly patients with rheumatoid arthritis.

The present inventors comprehensively searched for attributes, clinical laboratory test values, and the like for patients in whom oral administration of the antibody is effective in improving rheumatoid arthritis and ineffective patients, and conducted various studies. As a result, it was surprisingly found that DR15 negative patients in the HLA-type DR gene have many effective examples of antibodies, whereas DR15 positive patients are ineffective or have very few effective examples. The present invention has been completed based on these findings.

That is, the present invention has the following contents.
(1) Diagnosis of an autoimmune disease patient effective for antibody therapy and / or endotoxin absorber therapy, wherein a patient who is negative for DR15 of the HLA-type DR gene expressed in the patient's immune system cells is selected Method.
(2) The antibody treatment according to (1) above, wherein a DR15-negative patient is selected using a labeled primer of an HLA-type DR gene and a stationary phase probe complementary to the DR15 gene, and / or A method for diagnosing an autoimmune disease patient effective in endotoxin absorber treatment.
(3) The diagnostic method according to (1) or (2) above, wherein the autoimmune disease is rheumatoid arthritis.
(4) A patient with an autoimmune disease effective for antibody therapy and / or endotoxin absorber therapy, comprising a labeled primer for an HLA-type DR gene and a stationary phase probe complementary to the DR15 gene as essential elements In vitro diagnostics.
(5) comprising as essential elements a labeled primer of an HLA-type DR gene, a stationary phase probe complementary to the DR15 gene, and a stationary phase probe complementary to a gene sequence commonly included in the DR gene An in-vitro diagnostic agent for an autoimmune disease patient effective for the antibody treatment and / or endotoxin absorbent treatment.
(6) The in vitro diagnostic agent according to (4) or (5), wherein the autoimmune disease is rheumatoid arthritis.
(7) A therapeutic agent for an autoimmune disease patient, which contains an immunoglobulin and / or an endotoxin absorber and is negative for DR15 of the HLA-type DR gene expressed in immune system cells.
(8) The therapeutic agent for autoimmune disease according to (7), wherein the autoimmune disease is rheumatoid arthritis.
(9) The therapeutic agent for autoimmune diseases according to (7) or (8) above, wherein the immunoglobulin is derived from an antibacterial toxin antibody.
(10) The above-mentioned (7) to (9), wherein the antibacterial toxin antibody contains any one or more of an anti-endotoxin antibody, an anti-lipid A antibody and an antibody against a bacterial toxin having superantigenicity. The therapeutic agent for autoimmune diseases in any one of.
(11) The therapeutic agent for autoimmune diseases according to any one of (7) to (10), wherein the antibacterial toxin antibody is an antibody derived from milk.
(12) A therapeutic agent containing an immunoglobulin and / or an endotoxin absorber is orally administered to an autoimmune disease patient who is negative for DR15 of an HLA-type DR gene expressed in immune system cells. To treat autoimmune diseases.
(13) The method for treating an autoimmune disease according to (12), wherein the autoimmune disease is rheumatoid arthritis.
(14) The above (12) or (13), wherein the dose of the therapeutic agent is 10 mg to 10 g as human immunoglobulin, 1 g to 100 g as whey protein, or 10 mg to 10 g as endotoxin absorber per person per day. For the treatment of autoimmune diseases.

By using an autoimmune disease patient diagnosis method and an in vitro diagnostic agent effective for the antibody treatment and / or endotoxin absorber treatment of the present invention, DR15 of an HLA-type DR gene of an autoimmune disease patient typified by rheumatoid arthritis is obtained. By pre-diagnosing negative or positive, these patients can be more effectively treated with the therapeutic agent for antibody treatment and / or endotoxin absorber treatment according to the present invention.

Next, the present invention will be described in more detail.
As already stated, based on the onset mechanism that autoimmune diseases represented by rheumatoid arthritis are caused by absorption of type II collagen, endotoxin, and enterotoxin from the gastrointestinal tract, the present inventors A therapeutic agent containing the whey protein concentrate contained as a main ingredient was orally administered to patients with rheumatoid arthritis, and the therapeutic effect was examined, and HLA-DR analysis of the patients was performed. As a result, surprisingly, patients who have HLA-DR15, that is, DR15 positive patients, often do not have an effect, and patients who do not have HLA-DR15, that is, patients who are negative for DR15, have many effective examples. It was found that.

Based on this finding, the present invention establishes an invention relating to a diagnostic method and an in vitro diagnostic agent that can dramatically increase the accuracy of treatment effectiveness by diagnosing the presence or absence of the DR15 gene in patients with autoimmune diseases. It came to. Further, a therapeutic agent and a treatment for orally administering an immunoglobulin and / or an endotoxin absorber as a therapeutic agent to a patient with an autoimmune disease in which DR15 of the HLA-type DR gene expressed in the patient's immune system cells is negative A method is provided.

Human leukocyte antigen (HLA) is one of the most important histocompatibility antigens (MHC). In general, blood type refers to red blood cell antigen types such as A, B, O, and AB types, but HLA type indicates a white blood cell antigen type. However, since HLA exists in addition to leukocytes, it is rarely called by the name of human leukocyte antigen at present and is abbreviated as HLA.

HLA is a gene product governed by a group of genes encoded by the major histocompatibility complex region, abbreviated MHC region, present on chromosome 6. This MHC region consists of an MHC class I (or “HLA-1”) gene region that controls the HLA-A, B, and C antigen systems expressed on the surface of all eukaryotic cell membranes, and lymphocytes. MHC class II gene (or “HLA-2”) that functions in cells that make an immune response, such as macrophages and macrophages, and that is expressed only in immune system cells and controls HLA-DP, DQ, and DR antigen systems It consists of areas etc. The HLA type is a hereditary antigen unique to each person, and there are tens of thousands of combinations.
The HLA test is used for organ donors in organ transplantation, that is, compatibility between donors and organ recipients, that is, recipients, HLA-compatible platelet transfusion, prevention of post-transfusion GVH disease, diagnosis assistance for various diseases, and parent-child testing. It has been implemented.

The functions of HLA-2 in the immune response are as follows. That is, when a pathogen or a foreign substance enters, it is taken up by macrophages and antigen-presenting cells and decomposed. A peptide derived from a foreign substance from the degradation product appears on the cell surface in a state of being bound to the foreign-derived peptide storage groove of the HLA-2 molecule. On the other hand, lymphocytes bind to macrophages via a receptor that fits in the form of an HLA-2 conjugate bound with a peptide derived from a foreign substance, that is, a T cell receptor. Is established.

The HLA-DR gene products contained in the HLA-2 gene region are currently classified into serotypes of HLA-DR1 to HLA-DR18 that are serologically different in antigenicity based on leukocyte antigen analysis. The HLA-DR gene is susceptible to or associated with various diseases. For example, rheumatoid arthritis is associated with HLA-DR4, ulcerative colitis is associated with HLA-DR1, and systemic lupus erythematosus is associated with HLA-. It is known to be more common in DR3. Mycobacterium tuberculosis infection is more common in HLA-DR2.
The presence of specific autoimmune diseases and specific infectious diseases in specific HLA-DR serotypes indicates that the acquisition of immune abnormalities and immune resistance is governed by the HLA-2 gene.

As a result of intensive studies with reference to the relationship between such HLA-DR gene and various diseases, the present inventors have found that patients with autoimmune diseases represented by rheumatoid arthritis are treated with antibodies or endotoxin absorbers. The present inventors have found that such a treatment method works effectively only for patients who do not have HLA-DR15, that is, patients who are negative for HLA-DR15.

Therefore, the present invention provides an antibody therapy or an autoimmune disease patient or an autoimmune disease that can be easily diagnosed by effective determination of DR15 of the patient's own HLA-DR gene. A diagnostic method for autoimmune disease patients effective for endotoxin absorber treatment and an in vitro diagnostic agent for discriminating such patients.

The present invention also relates to an immunoglobulin and / or endotoxin absorber used for autoimmune disease patients effective for antibody therapy or endotoxin absorber treatment because DR15 of such HLA-type DR gene is negative. Is a therapeutic agent for autoimmune diseases negative for HLA-DR15.

Further, the present invention contains an immunoglobulin and / or an endotoxin absorber for an autoimmune disease patient who is effective for antibody therapy or endotoxin absorber treatment because DR15 of such HLA-type DR gene is negative. This is a method of treating an autoimmune disease patient who is negative for HLA-DR15, wherein the therapeutic agent is orally administered.

In the present invention, the HLA type inspection method can be performed by a commonly used method, and the method is not particularly limited.
The HLA type test methods are roughly classified into an antiserum method and a DNA typing method. In the DNA typing method, first, a primer for an HLA gene as a test control is added to grow by the PCR method. The method for determining the antigen type of the proliferated HLA gene is roughly divided into two methods: a method in which DNA base sequence is determined and discriminated (Japanese Patent Laid-Open No. 2004-283165), and a method in which complementary DNA is used as a probe to determine by probe binding. (See Patent Document 9 and Non-Patent Document 7). The DNA typing method, in particular, the method of discriminating the antigen type using a complementary probe as a stationary phase has the advantage that a large number of specimens can be implemented at low cost, and kits for HLA typing reagents are also commercially available.

In the HLA typing reagent kit, DNA of cells collected from blood, oral mucosa, and hair root is extracted, and a primer for the HLA-DR gene is added thereto to amplify the gene by PCR. This primer is subjected to chemical modification such as binding biotin or fluorescent dye in advance, and it is used as a labeled primer. When this is reacted as a stationary phase with a probe having a sequence complementary to each DR gene, The DR gene can be discriminated from the presence or absence of binding of the labeling substance to the stationary phase.

The method for diagnosing an autoimmune disease patient and the in vitro diagnostic agent effective for antibody therapy and / or endotoxin absorber treatment of the present invention are methods including the following processes, and are diagnostic agents used here.
(A) A labeled HLA gene primer is added to the patient's DNA to proliferate the patient's HLA gene labeled by the PCR method.
(B) confirm that the HLA gene is amplified;
(C) In addition to the separately prepared HLA15-specific complementary probe as a stationary phase, those that hybridize with the stationary phase, that is, HLA-DR15 positive, those that do not hybridize, that is, HLA-DR15 negative Using a labeling compound,
It includes a three-stage process. Among these, the process (b) is carried out in the detection of PCR products, and in addition to DNA agarose gel electrophoresis check, the DNA base sequence common to the HLA-DR gene is immobilized as a complementary probe. The object is achieved by preparing the same and performing the same operation as in the case of HLA-DR15 in (c) with or without hybridization.
Therefore, the positive or negative of the DR15 gene indicates that the HLA-DR gene is positive when the reaction of the labeled compound in the DR-15 probe is positive under the condition that HLA-DR gene amplification by PCR reaction is confirmed. When the reaction of the compound is negative, the HLA-DR15 gene is determined to be negative.

Commercially available HLA typing kits use 24 probes to distinguish 13 HLA-DR serotypes, but the present invention can achieve its objective with only DR15.
In the present invention, the DR15 probe sequence used in the in vitro diagnostic kit has been reported in Kawai et al. (See Non-Patent Document 8). That is, as shown in the following Table 1, three kinds of sequence names DRB7011J, sequence name DRB8601, and sequence name DRB8603J and their complementary sequences are known (see SEQ ID NOs: 1 to 6 in the Sequence Listing). Table 1 also shows the reaction specificity of these probes.

DRB7011J alone may be used for selecting the DR15 probe. However, when DDRB8603J is used in combination with two probes, it is possible to select three types of DRB8601. When DRB8601 is included, since DR1 and DR16 also react as shown in Table 1, in order to determine the reaction specificity with DR15, the probes having the sequence names shown in the following Table 2 or these It is necessary to confirm that the reaction is not DR1 or DR16 using the complementary sequence as a probe.

This in-vitro diagnostic agent is premised on that the gene is amplified because the DR gene is amplified by the PCR method and subjected to genetic testing. PCR products are usually detected by agarose gel electrophoresis of DNA, but it is also possible to add DNA detection using a common sequence of DR genes as a probe. The common sequence probe includes TGCAGACACAACTACGGG and its complementary sequence (see SEQ ID NOs: 7 to 8 in the Sequence Listing).

The stationary phase required for the diagnostic method and in vitro diagnostic agent of the present invention is preferably immobilized as a single-stranded nucleic acid on a stationary phase, for example, a microplate, by the method described in Patent Document 8. A sequence containing one or more of the sequences shown in Table 1 or Table 2 in series is prepared, and this is incorporated into a complex vector of M13 phage and plasmid to obtain a single-stranded nucleic acid. It is preferable that the repeat of the sequence introduces 5 to 200 copies.

In the diagnostic method and the in-vitro diagnostic agent of the present invention, if the presence or absence of DR15 can be determined, the purpose is achieved. Therefore, any one or a mixture of three DR15 probes shown in Table 1 and DR1 It is also possible to use any one or a mixture of the seven probes of DR16 as the stationary phase. Of the three types of DR15 probes, when using an in vitro diagnostic agent that does not use DRB8601 that cross-reacts with DR1 or DR16, detection probes for DR1 and DR16 are not required.

The carrier to be immobilized may be either a material capable of non-specifically binding nucleic acid or a material into which a functional group can be introduced and bound to the carrier through the functional group. Actually, a synthetic resin microplate, beads, tubes, or the like can be used.

As a method of fixing to a carrier, a method through a chemical bond can be mentioned (Nucleic Acids Res., 15, 5373-5390 (1987)). For example, there may be mentioned a method of immobilizing an amino group-introduced resin and a nucleic acid amino group by using a cross-linking agent such as glutaraldehyde.

As a method by non-specific adsorption, the nucleic acid can be directly contacted with a carrier and immobilized. For example, it is known that a nucleic acid solution is put in a microplate and efficiently fixed by ultraviolet irradiation (see JP-A-61-219400).

In the present invention, the hybridization reaction conditions between the immobilized probe and the sample DNA are basically the same as those of known hybridisation using a conventional membrane (Hames BD, Higgins SJ, Nicleic Acid). Hybridization, A Practical Approach, IRL Press 1985).

The sample DNA used for the diagnostic method and in vitro diagnostic agent of the present invention is preferably human leukocyte-derived DNA. If the DR15 gene is included, it must be labeled so that it can be detected. Methods of labeling include (a) a method of directly introducing a label into the target nucleic acid, (b) a method of synthesizing a nucleic acid complementary to the target nucleic acid using a labeled oligonucleotide primer, and (c) Examples include synthesis of a nucleic acid complementary to a target nucleic acid using an oligonucleotide primer in the presence of a labeled unit nucleic acid. Among these methods, the method (b) described in Patent Document 9 is preferable because a labeled primer can be obtained as an amplification product by the PCR method.

The labeling substance used here may be either radioactive or non-radioactive as long as it can detect DNA bound to the probe after the hybridization operation. A non-radioactive material is preferable from the viewpoints of easy handling, storage stability, disposal, and the like. Non-radioactive substances include, for example, biotin, 2.4-dinitrophenyl group, digoxigenin group, fluorescein and its derivatives, rhodamine and its derivatives, 4-fluorescein isothiocyanate, dansyl, acridine and the like. When labeling oligonucleotides with these, any of them can be labeled by known means (see JP-A-59-93098).

The labeling of the sample DNA used in the diagnostic method and in vitro diagnostic agent of the present invention is preferably performed simultaneously with amplification by PCR using a labeled oligonucleotide primer. As primers, the sequences TCGTGTCCCCACAGCACGT and CCGTGCACTGTGAAGCTCT are primers for PCR amplification of the DR gene, and biotinylated primers have been reported as sequence names DRBAM-C and DRBAM-B (see Non-Patent Document 8).

In the diagnostic method and in vitro diagnostic agent of the present invention, the operation for detecting the presence or absence of hybridization between a nucleic acid amplified by PCR from a sample DNA using a labeled primer and a probe is appropriately performed according to the label bound to the primer. Can be selected and determined. When the label is a radioisotope, a fluorescent substance, a dye, etc., the detection operation is performed with the labeled nucleic acid bound to the solid phase, or the label is left bound to the nucleic acid, or the label is separated from the nucleic acid. In this state, the sample is released into the solution, and then the detection operation is performed by a method corresponding to the labeling substance. For example, when the label is biotin, a commonly used method is to use a substrate that reacts with enzyme-labeled avidin to bind the enzyme to the hybridizing nucleic acid and develops color by the enzyme reaction. Thus, detection can be performed by colorimetric determination.

Examples of immunoglobulin antibodies used for the treatment of autoimmune diseases in the present invention include those derived from eggs such as chickens and ducks, those derived from the sera of animals such as cows, horses, goats and sheep, cows, goats, sheep and horses. Milk mammals such as buffalo and camel can be used, but milk-derived immunoglobulin that can be easily and safely obtained is preferred. In the case of milk, the milk may be milk collected from a vaccinated mammal or milk collected from a mammal that has not been vaccinated.

For example, antibodies secreted by cows into raw milk can be recovered from whey proteins such as cheese whey, which is a residual solution of cheese manufacture, or acid whey, which is a residue of casein manufacture. That is, since whey protein contains a high concentration of milk-derived immunoglobulin, it can be used as it is as an immunoglobulin in the present invention. Furthermore, whey protein is also sold as a general commercial product and can be used in the form of whey protein concentrate (WPC), whey protein isolate (WPI), desalted whey powder, and the like. In general, among these whey proteins, those having a high immunoglobulin content are preferred. Among the whey proteins, whey protein concentrate (WPC) is particularly preferable because many of them have a high immunoglobulin content.

In the present invention, the immunoglobulin preferably contains one or more antibodies selected from antibacterial toxin antibodies, that is, anti-endotoxin antibodies, anti-lipid A antibodies, and antibodies against superantigen activity. As mentioned above, endotoxin, lipid A, and superantigen, which are bacterial endotoxins, are considered to be involved in the development of rheumatoid arthritis due to abnormalities in the immune system in trace amounts, and contain antibodies against these bacterial toxins. Thus, it is possible to exert a superior therapeutic effect on autoimmune diseases.

In general, various animals have various resident bacteria such as Escherichia coli, or are often immunized naturally by repeated infections, and against these bacterial bodies and toxins produced by the bacteria. Have antibodies. These antibodies are called natural antibodies in the sense that they are produced without artificial immunity such as vaccines. As natural antibodies against bacterial toxins such as endotoxin, lipid A, superantigen, enterotoxin, anti-endotoxin antibody, anti-lipid A antibody, antibody against superantigen activity, anti-enterotoxin antibody, etc. are also detected in blood and milk. Natural antibodies can be used as the antibacterial toxin antibodies of the present invention. When these antibodies bind to the corresponding toxin, the toxin's toxic effect is lost. One method commonly used today to measure the amount of these antibodies is the enzyme antibody method (ELISA) as described in Example 1.

In the present invention, when designing a therapeutic agent for an autoimmune disease patient who is negative for HLA-DR15, when designing an antibody against endotoxin as an index, an antibody against E. coli O-111-derived endotoxin is used for 1 day. In the case of designing with an antibody against superantigen as an index so that it is contained in 4 μg or more in the intake of staphylococcal enterotoxin B, when designing with an antibody against lipid A as an index, Similarly, it is preferable to produce the composition so as to be 2 μg or more in order to obtain a therapeutic effect.

In the present invention, an autoimmune disease patient who is negative for HLA-DR15 is treated by orally administering a therapeutic agent containing an immunoglobulin. The dose is 10 mg to 10 g as an active ingredient immunoglobulin. When a whey protein concentrate is administered as a therapeutic agent containing an immunoglobulin, the daily dose is 1 g to 100 g, more preferably 1 g to 50 g. If the daily dose is less than 1 g, a sufficient effect cannot be obtained, and if it is more than 100 g, the burden on intake increases.

For autoimmune disease patients determined to be negative for HLA-DR15 by the method of the present invention, immunoglobulins and whey protein concentrates containing this antibody may be ingested as they are. It may be ingested as a processed food mixed with raw materials and food additives. As food, there are various forms of food, for example, dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, taste drinks such as fruit juice drinks, vegetable juice drinks, soft drinks, tablet confectionery, candy, chocolate, Examples include cereals, chewing gum and other confectionery, ice cream, pudding, jelly, bavaria and other desserts, cream cheese, mayonnaise, peanuts, condensed milk and other powdered foods, cocoa, powdered green tea powder, and sprinkles. it can. Of these, processed foods that have not been heated for more than 30 minutes at 65 ° C. during the production process are more preferable because more antibodies remain. It may also be taken in the form of a health food containing so-called immunoglobulin antibodies.

In addition to immunoglobulins, endotoxin absorbers that can be safely taken by the human body and can remove endotoxins in the digestive tract are also effective as therapeutic agents for autoimmune disease patients who are negative for HLA-DR15 determined by the method of the present invention It is.

Antibody is difficult to mass-produce in order to be used as a pharmaceutical product, and high production costs are required. Furthermore, since the activity of an antibody is easily lost due to heat and its physical properties are unstable, and it is a protein, oral ingestion has problems such as inactivation of antibody activity by digestive enzymes. The point is big. Therefore, as an alternative to antibodies, the endotoxin absorbent of the present invention is useful as a remover that has stable physical properties, can be mass-produced, is inexpensive, and has no safety problems.

In the manufacture of injections and the like, even if the amount is small, the presence of endotoxin causes side effects such as fever, so it is necessary to remove endotoxin sufficiently. For this purpose, specific endotoxin removers are used industrially. This is a product of synthetic fibers, fabrics, and particles with molecular groups that have a high affinity for endotoxin. By contacting this with a solution in which endotoxin is to be removed, only endotoxin is absorbed specifically, and endotoxin is absorbed. Can be excluded.

The principle of the endotoxin absorbent used in the present invention is the same in principle, and it works by the same mechanism to remove endotoxin in the digestive tract, but it must also be taken with physical properties suitable for oral intake and be safe for the human body. It is. In addition, it is said that there are 100 trillion bacteria inhabiting the digestive tract, and LPS-carrying intestinal bacteria are enormous, so that LPS removal activity is required to be high. The endotoxin absorbent used in the present invention is a novel endotoxin absorbent suitable for human use from the viewpoint of both endotoxin absorption activity and safety in response to these problems.

The toxic activity of endotoxin is a lipid moiety called lipid A in the molecule. The endotoxin absorbent used in the present invention is composed of a lipid A-binding molecule capable of binding to lipid A of endotoxin and fine particles carrying the same. By ingesting this endotoxin absorbent, endotoxins in the digestive tract are adsorbed and excreted in the stool, thereby preventing and treating autoimmune diseases such as rheumatism.

As a lipid A-binding substance in the endotoxin absorbent of the present invention, polymyxin B which is a peptide antibiotic and various peptides having LPS binding properties are known. Polymyxin B is a basic polypeptide antibacterial antibiotic having a molecular weight of 1200 produced by Bacillus polymixer bacteria, and many similar compounds have been reported. Details are described in Patent Documents 3 to 7. Any of these can be used as a lipid A binding substance for the purposes of the present invention.

The carrier carrying the lipid A binding substance is in the form of particles or fine powder suitable for taking, and the carrier material is a polysaccharide such as cellulose, agarose, mannan, glucan, chitin and their derivatives, acrylic, Examples thereof include polystyrene, polypropylene, polyamide, and polyvinyl.

The method for covalently binding the lipid A binding substance to the carrier is not particularly limited, and has a general cross-linking reagent for an immobilized enzyme, for example, ECDI, which is a water-soluble carbodiimide reagent, hexamethylene diisocyanate, and two epoxy groups. Propylene glycol diglycidyl ether and epichlorohydrin can be used.

It is desirable that the combined substance of the lipid A binding substance and the carrier is harmless, does not enter the body, and is not degraded or denatured by microorganisms in the digestive tract as well as digestive enzymes secreted by gastric acid and humans. .

LPS is a polymer having a molecular weight exceeding 10,000, and the adsorption of LPS is considered to be mainly on the surface of carrier particles, and a large surface area is required. For this purpose, the smaller the particle powder, the higher the activity. However, if the particle size is reduced below a certain level, there will be a safety disadvantage of being phagocytosed by the gastrointestinal lymphoid tissue. Therefore, the lower limit of the particle diameter is 5 μm. Furthermore, when taking, it is desirable that the particles are easy to take, and the upper limit is preferably about 50 μm. For the pulverization of the carrier, a general resin pulverization technique may be used. For example, a dry method such as an impact method, a screen method, a grinding method, or a wet method such as a medium agitation method can be used.

The combined substance of a lipid A-binding substance and a carrier is used as a general pharmaceutical preparation used by oral intake, for example, a powder, a powder, a capsule, a tablet, or a liquid for use by humans. These preparations can be made into various preparations together with pharmaceutically acceptable excipients and additives by ordinary methods.
In addition, the dose and frequency of treatment prevention are usually about 10 mg to 1 g per day and can be administered in 1 to 3 divided doses per day.

EXAMPLES Next, although an Example demonstrates this invention in more detail, this invention is not limited at all by these Examples.

In the following examples, immunoglobulin quantification and measurement of anti-bacterial toxin (LPS, SEB, lipid A) antibody by ELISA were performed according to the following method.
Immunoglobulin quantification was performed using the Bovine IgG-NL RID kit from The Binding Site (Birmingham, UK), and the total bovine IgG antibody content was measured by a one-way plate immunodiffusion method.

In an ELISA plate (Nunc 439454), LPS and SEB were each 0.1 mol / L carbonate buffer (pH 9.5), and lipid A was dissolved in a 4: 1 mixture of chloroform and methanol, then diluted with methanol, and the wells. In addition to antigen. Carbonate buffer or methanol was added to wells without antigen. Blocking was performed with goat serum containing trisaminomethane and sodium chloride. Next, a purified anti-E. Coli (E. coli O111) LPS antibody for preparing a calibration curve and a WPC solution as a sample were diluted with a block solution, and added to a well to react. A biotin-labeled anti-bovine IgG goat antibody (Jackson ImmunoResearch) was used as the second antibody. Next, avidin-bound peroxidase (ExtrAvidin-Peroxidase, manufactured by SIGMA) was reacted, and a 3,3 ', 5,5'-tetramethylbenzidine solution was added as an enzyme substrate, followed by reaction at room temperature for 10 minutes. Thereafter, the enzyme reaction was stopped with 3% (v / v) hydrochloric acid, and the absorbance at 450 nm was measured with a microplate reader (Bio Rad, Model 550). The amount of antibody was calculated using the weighing software Microplate Manager III (manufactured by Bio-Rad). The difference in the measured value between the well coated with the antigen and the well coated with no antigen was defined as the antibody concentration at the sample concentration. The antibody content was expressed as the content in 1 g of WPC by multiplying the specimen dilution factor.

(1) Preparation of immunoglobulin standard product As a standard product for the quantitative measurement of antibodies, heat-treated bacteria of E. coli O111: B4 strain were treated with oily adjuvant to immunize pregnant cows, and LPS antigen was obtained from the colostrum. The anti-E. Coli O111 LPS antibody was purified using a column and a protein G column (HiTrap Protein G HP, Amersham Bioscience, Sweden). The antibody solution to be purified was allowed to flow through the column, only the antibody was adsorbed on the column, and the immunoglobulin that was not adsorbed on the column was washed out with a neutral buffer. The anti-LPS antibody bound to the column was eluted with 0.1 M glycine hydrochloride buffer at pH 2.7. The eluate was neutralized and further applied to a protein G column. In order to obtain a purified product, purification with an LPS antigen column and a protein G column was repeated three times. LPS antibody activity in ELISA per protein showed no increase in specific activity after 3 or more purifications. This sample was a single band by acrylamide disk electrophoresis. This purified antibody was used as a standard immunoglobulin.

(2) Preparation of powder composition containing powdery whey protein concentrate For 6 parts by mass of Proliant 8000 (registered trademark, manufactured by James Farrell & Co.), a whey protein concentrate (WPC), Mayoligo, a fructooligosaccharide 3 parts by weight of P (registered trademark, manufactured by Meiji Seika), 0.8 parts by weight of whey calcium (manufactured by Morinaga Milk Industry), 0.2 parts by weight of KC Flock W-300G (registered trademark, manufactured by Nippon Paper Chemicals) Was mixed to prepare a powdery autoimmune disease therapeutic composition of 10 g per packet. This composition contains 243 mg of bovine IgG immunoglobulin per packet. The antibody against the bacterial toxin was examined by the above method. As a result, 77 μg of the antibody against E. coli O-26-derived endotoxin and 77 μg of S. aureus enterotoxin B were found. 11 μg and 6 μg of antibody against lipid A were contained.

(3) Preparation of tablet-type composition The raw material containing whey protein concentrate having the composition shown in Table 3 and water of about 10% of the total composition are mixed in a V-type blender and granulated with a pelleter. Then, it was dried using a shelf dryer until the water content reached 6%. A tablet-type rheumatoid arthritis composition is formed by applying a pressure of 1.5 ton / cm ^{2 with} a rotary tableting machine and compressing it into a circle (diameter 20 mm, thickness 5 mm) to 2 g per tablet. It was a thing. Three tablets of this tablet type therapeutic agent for rheumatoid arthritis contained 4.5 g of whey protein concentrate and 6 μg of antibody against E. coli O-26-derived endotoxin.

(4) Diagnosis of rheumatoid arthritis patients by diagnosis of HLA type DR15 In rheumatology department clinics shown in Table 4, 17 rheumatoid arthritis patients under treatment (1 male, 16 females, age 31-80 years, average 60 years old, First, each patient was determined for HLA-type DR15 as follows, with a medical history of 0.2 to 30 years, an average of 9.7 years).
That is, about 5 mL of patient blood was collected in a tube to which EDTA was added. 1 mL of this was added to a 10 mL plastic container containing 5 mL of Gen TLE solution I (manufactured by Takara Bio) and immediately vortexed and allowed to stand for 10 minutes. Centrifugation was performed at 12,000 times per minute for 10 minutes, the supernatant was removed, 10 mL of Gen TLE solution II was added to the precipitate, mixed by inverting 3 times, and centrifuged at 12,000 times per minute for 5 minutes. The supernatant was removed, 5 mL of Gen TLE solution III was added, vortexed for 20 seconds, and the supernatant was transferred to a new 10 mL container. To this was added 5 mL of isopropanol and mixed by inversion. Centrifugation was performed at 12,000 rpm for 10 minutes, and the supernatant was removed to obtain a DNA precipitate. The DNA was further centrifuged and washed with 70% ethanol and then with 10 mL each of 100% ethanol. It was dissolved in 1 mL of PCR buffer (20 mM KCl, 10 mM pH 8.8 Tris-HCl, 1.5 mM MgCl ₂ , 0.01% Triton X-100), and the absorbance at 260 nm was about 1.

Next, amplification of the HKA antigen gene by PCR was performed as follows using a commercially available kit (manufactured by Yugenaga Pharmaceutical Co., Ltd., MPH-2HLA typing reagent HLA-DR). 5 μL of the extracted DNA is added to a PCR tube containing a total of 95 μL of a mixture of 85 μL of deoxynucleoside triphosphate amplification solution (class II), 9.5 μL of biotin-labeled DR antigen primer solution, and 0.5 μL of DNA polymerase solution. , Set in a PCR device (Takara PCR Thermal Cycler model TP100), after heating at 96 ° C for 5 minutes, one cycle is 96 ° C for 30 seconds, 62 ° C for 1 minute, then 72 ° C for 1 minute, for a total of 40 The cycle was repeated, and finally, the mixture was cooled to 4 ° C. to complete the PCR reaction, and an amplified DNA solution was obtained.

Hybridization and detection with the proliferated HLA-DR gene probe were performed as follows.
In order to denature the propagated DNA into a single strand, 300 μL of an alkaline denaturing solution attached to the kit was placed in a denaturing tube having an internal volume of 1.5 mL. Next, 90 μL of amplified DNA solution after completion of the PCR reaction was added and mixed, and allowed to stand at room temperature for 5 minutes. To this, 900 μL of the neutralizing solution attached to the kit cooled on ice was added and mixed, and left on ice. The kit is a 96-well plate with two HLA-DR antigen DNA probes in 24 wells, HLA-DR plate 1 and HLA-DR plate 2 for each sample. No. 1 to No. 50 μL each was dispensed into 24 wells of 12 wells, sealed to prevent evaporation of the liquid, and then left in an incubator at 37 ° C. for 60 minutes to wait for reaction with the probe. Each well was washed 3 times with 300 μL of a normal temperature washing solution. In order to bind the streptavidin-conjugated HRP enzyme to the biotin label, 50 μL of an enzyme solution preliminarily warmed to 37 ° C. was added and left in an incubator at 37 ° C. for 15 minutes. Each well was washed three times with 300 μL of the washing solution, and 50 μL of the chromogenic substrate ABTS solution previously warmed to 37 ° C. was dispensed into each well and placed in a 37 ° C. incubator for 15 minutes. Subsequently, 50 μL of the reaction stop solution attached to the kit was added, and the absorbance at 415 nm was measured using an ELISA plate reader BioRad model 550. A determination software for the kit was used to determine the HLA-DR antigen type based on the cut-off value of the color absorbance at each probe. The results are shown in Table 5.

(5) Drug administration test to rheumatoid arthritis patients The therapeutic agent composition for rheumatoid arthritis prepared in (2) above for a total of 17 patients including 9 patients with negative DR15 and 8 patients with positive DR15 10 g (6 g as whey protein) per day for 3 months. Number of tender joints (TJC), swollen joints (SJC), acute phase protein (CRP), erythrocyte sedimentation rate (red sediment, ESR), rheumatoid factor, metalloproteinase-3 (MMP3), physical function evaluation mHAQ), patient pain assessment (VAS-1), patient general activity assessment (VAS-2), physician general activity assessment (VAS-3), and Rheumatology Conference Standard Disease Activity DAS28 (ESR) and DAS28 (CRP) were measured, patient complaints were added, and a clinical comprehensive clinical evaluation was performed by a doctor to evaluate the usefulness of whey protein antibodies in patients with rheumatoid arthritis. In addition, blood was collected before ingestion, and leukocyte HLA type test, serum TNF value, serum IL-6 value, articular cartilage collagen antibody, and anti-E. Coli LPS antibody were measured.

In addition, the medical condition of each patient before administering this rheumatic therapeutic composition is as shown in Table 4, and the patient whose DR15 is negative is heavier than the patient whose DR15 is positive, the cytokine level of each patient, The anti-collagen antibody level and the anti-E. Coli antibody level are as shown in Table 5. More patients with DR15 negative had more antibodies against E. coli than those with DR15 positive.

(6) Test results and evaluation The effect of this drug administration test was evaluated according to the following criteria. That is, eight categories of test items that are usually performed for the evaluation of the pathological conditions of rheumatoid arthritis, namely, the number of swollen joints (SJC), the number of painful joints (TJC), the acute phase reactant (CRP), the erythrocyte sedimentation rate (ESR), Over 20% of improvement in each item of joint destruction marker (MMP3), patient general activity evaluation (VAS-2), general activity evaluation by doctor (VAS-3), physical function evaluation (mHAQ) for 3 months The point 1 is determined to be point 1, and the point 1 is determined to be effective when there are three or more inspection items, that is, the point 3 or higher. These results are summarized in Table 6.

In addition, even if the score is 3 or less, the doctor's comprehensive judgment (VAS-3) is improved by 20% or more, and the general health index, anemia, feeling of fatigue, and drug reduction are enabled. There were two cases of patient 7 and patient 8 that were considered to be somewhat effective considering the above conditions comprehensively. (Table 6, Cases 7 and 8)

These 17 patients were divided into 2 groups according to the presence or absence of leukocyte type HLA-DR15. Tables 4 and 5 show the patient calendars before taking the therapeutic agent containing whey protein antibody.
When comparing the two groups before the start of treatment, there is no significant difference in patient age, disease duration, and rheumatoid factor. However, in the severity of rheumatoid arthritis (Ochi classification), the HLA15-negative group is multi-joint or mucilaneous. There were many serious medical conditions. The value of DAS28, which is an index of disease activity, is significantly higher in patients with negative HLA-DR15 than in patients with positive leukocyte HLA-DR15 for both DAS28 (CRP) and DAS28 (ESR). It was bad. mHAQ is not statistically significant in the assessment of mHAQ (physical function) in patients' daily lives (DR15 negative group 12.2 ± 7.2 vs. DR15 positive group 6.4 ± 6.6, p = 0 117) was large in the patients in the DR15 negative group, and 2+ was also observed in the DR15 negative group in terms of the degree of bone destruction, indicating that the medical condition was bad.

Next, the effects after 3 months of administration of whey protein antibody in these 17 patients were divided into 2 groups according to the presence or absence of HLA-DR15, and the evaluation results are shown in Table 6.
The evaluation of the therapeutic effects of rheumatoid arthritis pharmacotherapeutic agents is usually conducted by examining the percentage change {(previous value−post value / previous value) × 100} based on the measured values before and after drug use for each test item. It is usually practiced that when an improvement of 20% or more is observed, the inspection has been improved. Table 6 shows the case where an improvement of 20% or more is obtained in bold.

From the results of Table 6, it can be seen at a glance that the rate of change (indicated in bold) showing an improvement of 20% or more is higher in the DR15 negative group. That is, in the HLA-DR15 negative group, the effect of whey protein antibody was 44 (= 44/90) in the 90 evaluation points of 9 patients and 10 test items, whereas the change rate was 44% (= 44/90). -DR15 positive group was 11/80, significant at p <0.01. In addition, the sum of the eight examination items that resulted in successful changes, that is, TJC, SJC, CRP, ESR, MMP-3, mHAQ, VAS-2, and VAS-3, is shown in the evaluation score column of Table 6. The average ± score score of the DR15 negative group was 4.0 ± 2.87, whereas that of the DR15 positive group was 1.1 ± 1.7, and the results of the statistical test are shown at the bottom. Indeed, it was a significant difference with p = 0.025. Furthermore, according to the final evaluation in consideration of systemic symptoms shown in the column of representative improvement points in Table 6, the DR15 positive group was effective in 1/8 cases, whereas the DR15 negative group was 8/9 cases. It was effective in.

The above results show that when using whey protein antibodies in patients with rheumatoid arthritis, first diagnose the patients with rheumatoid arthritis based on the determination of HLA-type DR15, and select patients with negative DR15. Thus, it was found that whey protein antibody works effectively and can treat rheumatoid arthritis patients more effectively.

Preparation of in vitro diagnostic agent for HLA-DR15 differentiation Based on the method disclosed in Patent Document 8 (Japanese Patent Laid-Open No. 5-192198), as the DR15 gene probe, the sequences of DRB7011 and DRB8603J in Table 1 and the common probe (Common) For the TGCAGACACAACTAGGG sequence, a single-stranded DNA containing a sequence in which each sequence was repeated about 50 times was prepared. The obtained single-stranded DNA was immobilized on a microtiter plate as follows. First, in a 0.75M sodium chloride, 0.15M tris-hydrochloric acid, and 0.15M magnesium chloride (pH 8.0) solution, the single-stranded DNA derived from DRB7011J and DRB8603J has a concentration of 4 μg / mL. A mixed solution of these two probes was used. The single-stranded DNA derived from the common probe was a 4 μg / mL solution. These single-stranded DNA solutions were added to a microtiter plate at 100 μL per well. The plate was covered and allowed to stand at 37 ° C. for 16 hours. Then, the liquid in the well was removed, and the plate was left at 37 ° C. for 30 minutes to dry. The drying plate was irradiated with 5 × 10 ⁵ μJ of ultraviolet rays using a UV irradiation apparatus (Stratalinker 2400 (Stratagene, La Jolla, Calif.)), And then washed with 0.2 mL of washing buffer (1M sodium chloride, 0.1M Tris- It was washed three times with hydrochloric acid, 2 mM magnesium chloride, 0.1% Tween 20 (pH 9.3), and left at a temperature of 4 ° C. until use.
Thus, an in-vitro diagnostic agent for an autoimmune disease patient effective for the antibody treatment and / or endotoxin absorbent treatment of the present invention in which the DR15 gene probe and the common probe were immobilized on the stationary phase in the well was obtained.

Next, DNA extracted from human leukocytes was used as a sample, and 30 cycles of growth were performed by PCR using the sequences of DRBAM-C and DRBAM-B described in paragraph [0046] as biotin-labeled primers. The obtained amplification product was heated at 96 ° C. for 10 minutes, heat-denatured by rapid ice cooling, and 5 μL thereof was added to a hybridization solution (pH 7.0 solution by adding 0.75 M sodium chloride, 0.075 M sodium citrate and hydrochloric acid). Of the above-mentioned single-stranded DNA, which is an in vitro diagnostic agent of the present invention, was allowed to stand at 58 ° C. for 1 hour for hybridizing. After discarding the well solution, the well was washed three times with 300 μL of a TMAC solution (3 M tetramethylammonium chloride, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5) heated to 65 ° C. using a microplate washer. Further, the wells were washed three times with 300 μL of enzyme dilution solution (0.1 M sodium chloride, 0.1 M Tris-HCl, ₂ mM MgCl ₂ , 0.05% Tween 20, pH 7.5 solution). Next, 100 μL of peroxidase-labeled avidin diluted 5000-fold with an enzyme diluent was added and left at room temperature for 15 minutes. After removing the solution, the plate was washed 3 times with 300 μL of enzyme diluent. Chromogenic substrate (ABTS) solution (1.5 mM 2,2′-azino-bis- (3-ethylbenzothiazoline-6-sulphonic acid) diammonium, 0.015% hydrogen peroxide solution, 0.2 M tartrate buffer After adding pH 4.4) and reacting at room temperature for 15 minutes, the absorbance at 415 nm was measured.

The 17 patients shown in Table 4 were diagnosed with the in vitro diagnostic kit according to the present invention obtained here, and DR15 negative (-) and positive (+) was diagnosed. The results are as shown in Table 7, and each patient was determined to be negative / positive for DR15. This is the same as the result based on the DR type diagnosis according to Example 1, and antibody treatment or endotoxin absorber treatment was performed. A successful autoimmune disease patient could be diagnosed.

Example of preparation of endotoxin absorbent (1) Epoxy acrylic resin polymyxin B conjugate RPMB
3 million units of polymyxin sulfate (manufactured by Pfizer Inc.) was dissolved in 200 mL of 0.1 M NaCl solution, sodium hydroxide was added to adjust the pH to 8, and the mixture was placed in a beaker. 4 g of polyacrylic resin having an epoxy active group (Amberzyme; manufactured by Rohm and Haas, USA) was added thereto, and the mixture was stirred for 72 hours with a stirring propeller. The resin was washed with 1000 mL of purified water on a 5 μm membrane filter, placed in a beaker containing 50 mL of 1 M glycine solution adjusted to pH 8.0 by adding sodium hydroxide in advance, left overnight, filtered, and then poured 2 liters of purified water. And washed. The water remaining in the resin was placed in a desiccator and dried to obtain 3.9 g of a polymyxin binding resin (hereinafter referred to as a spherical RPMB) which is an epoxy acrylic resin polymyxin B conjugate.

(2) Epoxy acrylic resin polymyxin B conjugate RPMB-1
4 g of a polyacrylic resin having an epoxy active group (trade name: Amberzyme, manufactured by Rohm and Haas, USA) was placed in a mortar. 3 million units of polymyxin B sulfate was dissolved in 200 mL of 0.1 M NaCl solution, and the pH was adjusted to 8 by adding sodium hydroxide. The prepared polymyxin solution was added little by little, and the resin was crushed using a pestle. All polymyxin solutions were added and stirred with a magnetic stirrer for 72 hours. The resin was washed by pouring 100 mL of purified water on a 5 μm membrane filter, and this was placed in a beaker containing 50 mL of 1 M glycine solution adjusted to pH 8.0 by adding sodium hydroxide in advance, left overnight, filtered, and purified. Washed with 2 liters of water. The water remaining in the resin was placed in a desiccator and dried to obtain 3.2 g of a polymyxin binding resin (RPMB-1) which is an epoxy acrylic resin polymyxin B conjugate.

Patients with autoimmune diseases such as rheumatoid arthritis patients are diagnosed with the in vitro diagnostic agent of the present invention to determine whether the patient's HLA type DR15 is negative or positive in advance, and the present invention is applied to patients with negative DR15. By administering the antibody therapeutic agent or endotoxin absorber, it is possible to more effectively treat patients with autoimmune diseases, which is useful for effective treatment of patients with autoimmune diseases in this field.

Claims (14)

1. A method for diagnosing a patient with an autoimmune disease effective for antibody treatment and / or endotoxin absorber treatment, wherein a patient who is negative for DR15 of an HLA-type DR gene expressed in a patient's immune system cells is selected.
2. 2. The antibody treatment and / or endotoxin absorbent treatment according to claim 1, wherein DR15 negative patients are selected using a labeled primer of an HLA-type DR gene and a stationary phase probe complementary to the DR15 gene. For effective diagnosis of autoimmune disease patients.
3. The diagnostic method according to claim 1 or 2, wherein the autoimmune disease is rheumatoid arthritis.
4. In vitro diagnosis of an autoimmune disease patient effective for antibody therapy and / or endotoxin absorbent therapy, comprising an HLA-type DR gene labeled primer and a stationary phase probe complementary to the DR15 gene as essential elements medicine.
5. It comprises, as essential elements, a labeled primer for an HLA-type DR gene, a stationary phase probe complementary to the DR15 gene, and a stationary phase probe complementary to a gene sequence commonly included in the DR gene. An in vitro diagnostic agent for autoimmune disease patients effective for antibody therapy and / or endotoxin absorber therapy.
6. The in vitro diagnostic agent according to claim 4 or 5, wherein the autoimmune disease is rheumatoid arthritis.
7. A therapeutic agent for autoimmune disease patients, which contains an immunoglobulin and / or an endotoxin absorber and is negative for DR15 of the HLA-type DR gene expressed in immune system cells.
8. The therapeutic agent for an autoimmune disease according to claim 7, wherein the autoimmune disease is rheumatoid arthritis.
9. The therapeutic agent for autoimmune diseases according to claim 7 or 8, wherein the immunoglobulin is derived from an antibacterial toxin antibody.
10. 10. The antibacterial toxin antibody according to any one of claims 7 to 9, wherein the antibacterial toxin antibody contains any one or more of an anti-endotoxin antibody, an anti-lipid A antibody and an antibody against a bacterial toxin having superantigenicity. A therapeutic agent for autoimmune diseases.
11. The therapeutic agent for autoimmune diseases according to any one of claims 7 to 10, wherein the antibacterial toxin antibody is an antibody derived from milk.
12. Autoimmunity characterized by orally administering a therapeutic agent containing an immunoglobulin and / or an endotoxin absorber to a patient with an autoimmune disease in which DR15 of the HLA-type DR gene expressed in immune system cells is negative How to treat the disease.
13. The method for treating an autoimmune disease according to claim 12, wherein the autoimmune disease is rheumatoid arthritis.
14. The treatment of autoimmune disease according to claim 12 or 13, wherein the dose of the therapeutic agent is 10 mg to 10 g as human immunoglobulin per person per day, 1 g to 100 g as whey protein, or 10 mg to 10 g as endotoxin absorber. Method.