JP2003279571A - Inflammatory disease detection method, detection medicine, and preventive treatment-searching method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for detecting an inflammatory disease detection method, an inflammatory disease detection medicine, and a method for searching for the inflammatory disease detection medicine. <P>SOLUTION: In the inflammatory disease detection method, an antigen that is generated by the oxidization of phospholipid is used to measure oxidation lipoprotein contained in an organism sample. In the measurement of the oxidation lipoprotein, use of an antibody for recognizing the low-density lipoprotein for a secondary antibody enables the oxidation low-density lipoprotein to be determined, which is especially useful for detecting a patient with an inflammatory disease. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for detecting an inflammatory disease, an inflammatory disease detecting agent for carrying out this method, and a method for searching for an inflammatory disease preventive / therapeutic agent.

[0002]

2. Description of the Related Art Inflammation is a type of defense reaction that biological tissues show against physical, chemical, or infectious stimuli, such as the local accumulation of body fluids, plasma proteins, and leukocytes and cells caused by the immune response. Often accompanied by proliferation. Originally, inflammation is a reaction that eliminates harmful stimuli from the living body and restores local structure and function, but when inflammatory reaction is strongly expressed, it is treated as an inflammatory disease in which normal tissues and cells are damaged. Be the target. The onset site of such an inflammatory disease may be localized, and it is called arthritis, hepatitis, dermatitis, etc. depending on the site, but it may develop in most parts of the whole body, so it may occur in multiple sites. Or may have systemic inflammation. Further, like other diseases, it is distinguished from acute, subacute, chronic, etc. according to the time course of the onset.

On the other hand, as a clinical test method for inflammatory diseases,
The method of measuring the erythrocyte sedimentation rate (erythrocyte sedimentation), the method of measuring C-reactive protein (CRP) in blood, etc. are common. Of these, erythrocyte precipitation is not necessarily an index that reflects only inflammation because it is promoted by anemia, decrease in albumin, increase in gamma globulin, increase in fibrinogen, and the like. Also, CRP is not only an inflammatory disease,
It is known to be elevated due to circulatory disorders such as thrombus and infarction, tissue necrosis and tumors such as lymphoid tumors. Therefore, at present, diagnosis is performed based on a plurality of measurements in which these clinical test values are combined with the white blood cell count, the presence or absence of fever, and the like. In order to reduce the burden on the subject and improve the accuracy of diagnosis, it is desired to develop a more specific inflammation inspection method or a new inspection method having a different measurement principle.

On the other hand, lipoproteins that have undergone oxidation are called oxidized lipoproteins. Oxidized lipoproteins have different degrees of oxidation. For example, lipids such as phospholipids and cholesterol, which have a relatively low degree of oxidation and only a small amount of oxidation, proceed to oxidize and are generated by lipid peroxidation. The above-mentioned aldehydes further react with apoprotein to form crosslinks, and the peptide chain is further cleaved. For this reason, it is general that the type of oxidized lipoprotein of interest varies depending on the measurement method.

For example, in Japanese Patent Laid-Open No. 7-238098, in view of the fact that oxidized lipoprotein is associated with the development of human atherosclerotic lesion, a monoclonal antibody that specifically recognizes a specific oxidized lipoprotein accumulated in the lesion, In particular, using an antibody that specifically recognizes the antigen FOH1 that appears in human atherosclerotic lesions, the antibody is contacted with a sample, and the reactivity of the antibody with respect to the sample is measured to determine the atherosclerotic lesion-related antigen in the sample. Detected and by said antibody the antigen of human blood vessel
Atherosclerosis is diagnosed by detecting FOH1. Further, in JP-A-8-304395, after diluting plasma to an appropriate concentration, it is contacted with an antibody that recognizes an antigen produced by the oxidation of phospholipids, and the oxidized lipoprotein bound to the antibody is further treated with the lipoprotein. Disclosed is a method for measuring oxidized lipoprotein, which comprises contacting with an antibody that recognizes. Further, in JP-A-9-288106, oxidized lipoprotein is measured by using a specific compound obtained by artificially oxidizing phospholipid as a standard substance for measurement. A calibration curve is prepared using the specific substance as a standard substance, and the results obtained by an actual assay are compared with the calibration curve to quantitatively measure the oxidized lipoprotein. However, any of the methods described in the above publications suggests the relationship between cardiovascular diseases such as arteriosclerosis and specific oxidized lipoproteins, and only applies them to diagnosis. Therefore, there is no description about the detection of inflammatory diseases or the search for preventive / therapeutic agents for inflammatory diseases using the method.

On the other hand, a low-density lipoprotein (LDL) fraction isolated from synovial fluid of a patient with rheumatoid arthritis, which is one of inflammatory diseases, contains a substance having a negative charge more than usual LDL. (L. Dai et al., Free Rad
ical Research, 32, 479, 2000, and MEJames et al., L
ipids, 33: 1115, 1998), it is presumed that a substance having a higher negative charge rate than the normal LDL is oxidized LDL.
However, this method is not suitable for LDL by centrifugation.
After fractionating the fractions, perform agarose gel electrophoresis,
It is intended to separate and detect normal LDL and a substance having a higher negative charge rate than normal LDL, and the operation is extremely complicated. Therefore, it is difficult to measure a large number of clinical specimens by the method and clarify the clinical diagnostic significance. In particular, it is necessary to measure the oxidized LDL value of a large number of samples in order to evaluate whether or not a particular drug is effective as a prophylactic drug for inflammatory diseases, and the effectiveness of the drug based on such a large number of measured values is required. It can be said that this method is extremely unsuitable as a method for searching for sex. Moreover, the findings regarding substances that are more negatively charged than LDL are only the results that were observed in a part of the investigated patients among the very small number of patients, and the relationship between oxidized lipoprotein and rheumatoid arthritis was clarified. Not what I did. In addition, in the reports of L. Dai et al. And MJ James, there is no reliable guarantee that the negatively charged substance is oxidized LDL. Even if it is oxidized LDL, it can be separated by this method because apo-B, which is a constituent of LDL, is formed by the aldehyde produced by the peroxidation of lipids constituting LDL.
It is only the oxidized LDL produced by binding to the lysine residue of the protein. Therefore, this method cannot measure oxidized LDL, which has a low degree of oxidation, in which only fatty acid or cholesterol is oxidized.

[0007] Oxidized LDL is also found in the blood of juvenile rheumatism patients and patients with cirrhosis due to excessive alcohol intake.
There is a report that the antibody against E. coli is high (G. Simonini et al, Th
e Journal of Rheumatology, 28, 198, 2001). However, the method does not directly measure the presence of oxidized lipoproteins, but rather the oxidized LDL is present in these patients.
Is only an indirect analogy of high. Also,
High levels of lipid peroxide in the blood of patients with atopic dermatitis,
It has been reported to be even higher in patients with cataracts (Y. Niwa et al., Archives of Dermatology, 130, 138).
Page 7, 1994), there is no mention of oxidized lipoproteins. Furthermore, there is a report that administration of carbon tetrachloride to mice causes an increase in lipid peroxide in blood when liver damage is caused (Y. Watanabe et al., Industrial H.
ealth, 35: 285, 1997), nothing is mentioned about oxidized lipoproteins.

That is, although these documents suggest that oxidative stress is increased by inflammation, the relationship between the increase in oxidative stress and the accumulation of oxidative lipoprotein is not clear, and further, oxidative lipoprotein The relationship with inflammatory diseases is also unknown.

[0009]

DISCLOSURE OF THE INVENTION The present invention is completely different in measurement principle from conventional methods and agents for detecting inflammatory diseases.
It is an object of the present invention to provide a simple and novel method for detecting an inflammatory disease, a detecting agent, and a method for searching for a preventive / therapeutic agent for an inflammatory disease.

[0010]

[Means for Solving the Problems] The present inventors have newly found that there is a clear relationship between inflammatory diseases and elevation of oxidized lipoprotein, and based on this new finding, A method for detecting inflammatory disease was completed by measuring oxidized lipoprotein using an antibody that recognizes an antigen expressed by oxidation. That is, the above-mentioned subject is solved by the following.

The present invention uses an antibody that recognizes an antigen produced by the oxidation of phospholipids to measure oxidized lipoprotein contained in a biological sample obtained from a patient suspected of having an inflammatory disease or an animal model of inflammatory disease. The present invention provides a method for detecting an inflammatory disease. Based on the finding that the amount of oxidized lipoprotein increases in inflammatory diseases, an antigen-antibody complex between a specific antibody and oxidized lipoprotein contained in a biological sample is formed, and the complex is used for inclusion in a biological sample. Oxidized lipoprotein can be easily and accurately quantified to detect whether or not it is the disease.

The present invention also provides a drug for detecting inflammatory diseases, which comprises as an active ingredient an antibody that recognizes an antigen produced by the oxidation of phospholipids. According to the detection drug,
Oxidized lipoprotein can be measured simply and accurately, and whether or not an inflammatory disease can be easily detected.

In addition, the present invention provides a model animal for inflammatory disease after administration of a drug whose effect is to be detected, or a biological sample collected from the model animal, or a living body sampled from a patient or animal having inflammatory disease. The drug is added to a sample, and oxidized lipoprotein contained in the biological sample is
A method for searching a prophylactic / therapeutic drug for inflammatory diseases, which comprises evaluating the prophylactic / therapeutic effect of a drug by measuring using any of the following methods (i) to (iv) Is. Whether or not a specific drug is effective as a prophylactic / therapeutic drug for inflammatory diseases is reacted with a biological sample obtained from an animal to which the drug is administered, and the oxidized lipoprotein contained therein is contacted with an antibody to By measuring, the effectiveness of the drug can be easily searched.

(I) An antigen-antibody complex is formed between an antibody that recognizes an antigen produced by the oxidation of phospholipids and an oxidized lipoprotein contained in the biological sample, and the oxidized lipoprotein contained in the complex is measured. Method.

(Ii) Using an antibody that recognizes an oxidized low-density lipoprotein together with an antibody that recognizes an antigen produced by the oxidation of phospholipids, an antigen-antibody complex with the oxidized lipoprotein contained in the biological sample is formed. A method for measuring oxidized low-density lipoprotein as oxidized lipoprotein contained in the complex, (iii) the above-mentioned (ii), wherein the antibody that recognizes oxidized low-density lipoprotein is an antibody that recognizes apo B protein Method (iv) The monoclonal antibody DL is an antibody that recognizes an antigen produced by the oxidation of phospholipids.
The method according to any one of (i) to (iii) above, which is H3.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The first aspect of the present invention includes a biological sample obtained from a patient suspected of having an inflammatory disease or an animal model of inflammatory disease using an antibody that recognizes an antigen produced by the oxidation of phospholipids. The method for detecting inflammatory diseases comprises measuring oxidized lipoprotein.

The types and amounts of oxidized lipoproteins contained in biological samples obtained from subjects with various inflammatory diseases were measured. As a result, increase in oxidized lipoproteins, particularly oxidized low density lipoproteins was observed in all inflammatory diseases. Was observed. In this method for measuring oxidized lipoprotein, an antibody against an antigen produced by the oxidation of phospholipids (hereinafter, also referred to as “primary antibody”) is brought into contact with a biological sample of a subject, and the oxidized lipoprotein contained in the sample and It is a method of forming an antigen-antibody complex with a primary antibody, measuring oxidized lipoprotein using the complex, and calculating the amount of oxidized lipoprotein contained in a biological sample, and is excellent in quantification of a target substance. When an antibody that recognizes oxidized low-density lipoprotein (hereinafter, also referred to as "secondary antibody") is used together with the primary antibody, the oxidized low-density lipoprotein in the oxidized lipoprotein can be measured, The morbidity of an inflammatory disease can be detected from the measured value. Hereinafter, the present invention will be described in detail.

The method for detecting an inflammatory disease of the present invention comprises a step of bringing an antibody against an antigen produced by the oxidation of phospholipid into contact with a biological sample obtained from a patient suspected of having an inflammatory disease or an animal model of inflammatory disease, It comprises a step of measuring oxidized lipoprotein using an antigen-antibody complex formed by a reaction between an antibody and oxidized lipoprotein contained in the sample. If a calibration curve is prepared in advance using a specific oxidized lipoprotein as a standard substance, the oxidized lipoprotein can be easily quantified.

Examples of the primary antibody that recognizes the antigen produced by the oxidation of phospholipids include an antibody that recognizes the antigen produced by the oxidation of phosphatidylcholine in the presence of a peptide, and specifically, a hybridoma. Cell line FOH1a / DLH3 (consignment number FERM B
P-7171) produced monoclonal antibody D
LH3 (JP-A-7-238098, JP-A-8-3
04395, JP-A-9-288106, H.
Itabe et al., Journal of Biological Chemistry, 269, 1527.
4 pages 1994). The antibody DLH3 is
For example, animals such as mice are immunized with a homogenate of lesion blood vessels including human atherosclerotic foci as an antigen, and antibody-producing cells are collected from splenocytes of animals with increased antibody titers, and HGPRT (hypoxanthine-guanine phosphoribosy
It is an antibody obtained from this cell line by forming a hybridoma with a tumor cell of a l transferase) -deficient strain and screening the hybridoma by its reactivity with copper sulfate-oxidized LDL. Therefore, it recognizes the common part between the substance group existing in the lesion and the heterologous antigen substance used in the screening, that is, LDL oxidized with copper sulfate, and has high specificity for the antigen produced by the oxidation of LDL in the lesion. Have. Moreover, when the oxidized lipoprotein is measured using the antibody, it becomes easy to select the standard substance used for preparing the calibration curve for calculating the oxidized lipoprotein amount from the measured value, and the calibration curve shows low concentration. Can be easily and accurately quantified from the oxidized lipoprotein of.

That is, when the oxidized lipoprotein is detected using an antibody that recognizes the oxide of the protein portion of the oxidized lipoprotein as an epitope, the epitope is bound by a covalent bond as a part of the polypeptide chain of the lipoprotein. Therefore, it is difficult to be incorporated into the lipoprotein after being synthesized, isolated and purified as such. Therefore, when an antibody that recognizes this epitope is used as an antibody against oxidized lipoprotein, the lipoprotein itself as a standard substance is oxidized, for example, in the presence of copper ions, or the lipoprotein or a specific amino acid alone is chemically modified. It is necessary to synthesize and use ones, there are many kinds of reactions, and it is difficult to obtain reproducible oxides and to purify them to generate standard substances with unity. . However, when the antibody DLH3 is used, the antibody is an antibody that recognizes an antigen produced by the oxidation of phospholipids. Therefore, the obtained data is calibrated using a compound obtained by oxidizing the phospholipid as a standard substance. It is possible to create lines and it is extremely easy to operate. From this point of view, the primary antibody used in the present invention is not particularly limited as long as it is an “antibody capable of recognizing an antigen produced by the oxidation of phospholipids”,
Can be widely used.

To measure oxidized lipoprotein using an antigen-antibody complex which is a conjugate of the primary antibody and oxidized lipoprotein contained in a biological sample, an immunological measuring method can be adopted. For example, radioimmunoassay (RIA), enzyme immunoassay (ELIS)
A), a fluorescent immunoassay (FIA), a luminescent immunoassay, an agglutination immunoassay, an immunoturbidimetric method, an immunoparasitic method and the like. Among these methods, in the present invention,
Immunological methods, especially radioimmunoassay,
It is preferable to use an enzyme immunoassay, a fluorescent immunoassay and a luminescent immunoassay. Further, the measurement method using the primary antibody may be either a competitive method or a sandwich method.

The operation and procedure of each of the above-mentioned methods can be carried out according to a conventional method. For example, as the sandwich method, a well in which the primary antibody is bound to a solid phase is incubated with a biological sample containing oxidized lipoprotein,
After that, the solid phase is washed with a washing solution, further incubated with an anti-lipoprotein antibody labeled with an enzyme as a secondary antibody, and then the solid phase is washed again, and then the antigen-antibody complex formed on the solid phase There is a method of measuring the oxidized lipoprotein by measuring the enzyme activity of.

The secondary antibody is preferably the above-mentioned primary antibody or other secondary antibody specific to lipoprotein, particularly an antibody that recognizes oxidized lipoprotein containing a specific apoprotein. The present invention is characterized by using an antibody that recognizes an antigen produced by the oxidation of phospholipids. As described above, since the epitope recognized by the antibody does not depend on apoprotein, different lipoproteins in blood are used. The oxides can be evaluated separately. Therefore, for example, when two types of combinations of the lipoprotein-specific antibody to be measured are used together with the primary antibody, it becomes possible to quantify the oxidized lipoprotein, which is a different protein portion. As such a secondary antibody specific to lipoprotein, an antibody that recognizes oxidized lipoprotein containing a specific apoprotein is used. Antibodies that specifically bind to oxidized lipoprotein include various apoproteins such as apoAI protein,
Apo AII protein, Apo AIV protein, Apo B
There are antibodies against 100 protein, apo B48 protein, apo CI protein, apo CII protein, apo CIII protein, apo D protein, apo E protein, apoprotein (a) and the like. Generally, Apo B
100 protein is an apoprotein mainly found in LDL and very low density lipoprotein (VLDL), and most of the apoproteins in LDL are apoB proteins, which can be said to be a protein that characterizes LDL. Similarly, the apoAI protein is the major apoprotein of high density lipoprotein (HDL), the apoprotein (a) is the protein characterizing lipoprotein (a), and the apoE protein is the major apoprotein of VLDL. Therefore, by using these, each lipoprotein can be efficiently identified although there is some cross-reactivity. That is, if a secondary antibody that recognizes the apo B protein is used, the oxidized LD
L can be quantified, oxidized HDL can be quantified by using one that recognizes apo AI protein, and oxidized VLDL can be quantified by using one that recognizes apo E protein as the secondary antibody, and apoprotein (a) can be quantified as the secondary antibody. Oxidized lipoprotein (a) can be quantified by using a substance that recognizes. In the present invention, it is more preferable to use, as the secondary antibody, an antibody that recognizes oxidized lipoprotein containing apo B protein (anti-apo B antibody). This is because it is possible to accurately quantify oxidized low-density lipoprotein, which has a high correlation with inflammatory diseases. The secondary antibody may be a polyclonal antibody or a monoclonal antibody.

The secondary antibody used in the present invention further comprises an enzyme,
It is preferably labeled with a labeling compound such as a radioactive substance or a fluorescent substance. For example, as an enzyme-labeled secondary antibody, there is an antibody having an affinity for lipoprotein such as anti-human apo B rabbit antibody and labeled with a labeling enzyme such as horseradish peroxidase, bovine small intestinal alkaline phosphatase, and β-galactosidase. . Other labeling methods include radioactive substances such as ¹²⁵ I, europium, fluorescein, Oregon green, BODIP.
There are fluorescent substances such as Y and CyDye, and chemiluminescent substances such as acridinium. When a secondary antibody labeled with a radioactive substance is used, the radioactivity of the antigen-antibody complex formed on the solid phase is measured, and when a secondary antibody labeled with a fluorescent substance is used, the solid phase is used. When a secondary antibody labeled with a chemiluminescent substance is used by measuring the amount of fluorescence of the antigen-antibody complex formed above, the amount of chemiluminescence of the antigen-antibody complex formed on the solid phase is measured. By doing so, the amount of antigen can be quantified. Furthermore, a method using a biotin-labeled secondary antibody and avidin labeled with an enzyme, a method using an unlabeled antibody as the secondary antibody, and a method using an antibody against the secondary antibody as the tertiary antibody are usually used. It is also possible to apply a method for improving the detection sensitivity.

Furthermore, in order to quantify the specific oxidized lipoprotein, instead of using the secondary antibody, for example, a receptor that recognizes the specific oxidized lipoprotein can be used. As a receptor that recognizes such oxidized lipoprotein, lecithin cholesterol acyltransferase that recognizes apoAI protein, and LD of liver and small intestine cells that recognize apoB100 protein
There are L receptor, lipoprotein lipase that recognizes apo CII protein, liver apo E receptor that recognizes apo E protein, and the like, and any of them can be preferably used.

As a method for separately quantifying the oxidized lipoprotein contained in the biological sample according to its type, the oxidized lipoprotein is further purified in advance into various components by centrifugation or the like, and then the primary antibody is used. It is also possible to quantify specific types of oxidized lipoproteins. Specifically, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLD)
L) or lipoprotein (a) is fractionated in advance by centrifugation or the like, and a specific oxidized lipoprotein can be quantified by allowing the primary antibody to act on the fractionated sample. In this case, the same secondary antibody can be used to measure different oxidized lipoproteins.
A specific method for separating a specific oxidized lipoprotein by centrifugation treatment is known in the art (for example, JP-A-7-238098, JP-A-8-304395, JP-A-9-288106, and Taku Yamamura, Shinsei Chemistry Laboratory, Lipid I, p. 195, 1993, Tokyo Kagaku Dojin).

The biological sample used in the present invention is not particularly limited as long as it contains oxidized lipoprotein, and blood, plasma, serum, tears, urine, amniotic fluid, synovial fluid, spinal fluid,
Biological samples such as cell extracts and tissue extracts can be mentioned.
In particular, inflammatory diseases often exert their effects on the whole body, and among them, blood, plasma and serum are preferably used. Further, as a sample collection site in the case of locally occurring dermatitis, it is preferable to collect from the epidermis of the diseased or unrashed part. As an optimal sampling site for rhinitis, sampling from nasal discharge or nasal mucosa is preferable. The measurement of oxidized lipoprotein contained in such a biological sample is preferably performed immediately after collecting the biological sample, but a stored sample may be used. When it is stored, the storage condition is not limited as long as the amount of oxidized lipoprotein does not change, but for example, a method of storing in a dark place and a vibration-free condition under a low temperature condition of 0 to 10 ° C that does not freeze. There is. Further, the sample may be frozen or freeze-dried, and the use of a cryoprotective agent is optional. However, it is preferred that a cryoprotectant be present during freezing or during the freeze-drying step, which stabilizes the oxidized lipoprotein. The term "freeze-drying" is used in the same meaning as used in the art, that is, freezing a sample, depressurizing it in the frozen state, and removing water or a sublimable substance from the sample. Excludes and means to dry.

Examples of such cryoprotective agents include:
Sugars such as sucrose, trehalose, lactose, mannitol and glucose; polymeric substances such as dextran, dextran sulfate, pullulan, polyethylene glycol and carboxymethyl cellulose; proteins such as bovine serum albumin (BSA) and human serum albumin (HSA); glycerol And water-soluble organic solvents such as dimethyl sulfoxide and the like. In addition, the above-mentioned cryoprotective agent may be used alone or in the form of a mixture of two or more kinds. The amount of these cryoprotective agents used is not limited, but in the frozen biological sample, it is 0.1 to 50% by mass, preferably 1 to 30% by mass.
%, More preferably 5 to 20% by weight, used together.

The freeze-drying conditions are not particularly limited, but are usually -80 to 20 ° C, preferably -8.
At a temperature of 0 to 15 ° C., a pressure of 0.667 to 13.33 Pa, preferably 0.667 to 1.333 Pa, and a pressure of 12 to
Lyophilize for 72 hours, preferably 24-72 hours.
According to such a freeze-drying step, the water content in the freeze-dried product containing oxidized lipoprotein is usually 10% by mass or less, preferably 1% by mass or less. The cryoprotectant is preferably added before the freeze-drying treatment, and the amount of the cryoprotectant used is usually 1 to 20% by mass, preferably 2 to
It is 5% by mass. This is because the oxidized lipoprotein can be stabilized in this range.

In the measurement, it is preferable to dilute such a biological sample to an optimum concentration. The concentration depends on the measurement conditions and cannot be specified unconditionally, but it is diluted to, for example, 0 to 500 μg / ml, more preferably 0 to 100 μg / ml. The dilution medium is not particularly limited, but physiological saline, phosphate buffer (PBS) containing EDTA, etc. may be used.

The contact between the biological sample at the optimum concentration and the primary antibody is such that the specific reaction between the oxidized phospholipid contained in the biological sample and the primary antibody sufficiently proceeds. There is no particular limitation as long as it is, for example,
It is desirable to carry out a stationary reaction at 4 to 30 ° C., more preferably at 25 ° C. for 1 to 24 hours, more preferably for 1 to 2 hours. Further, the concentration of the primary antibody at that time may be an amount sufficiently supersaturated with respect to the amount of phospholipid that is considered to be present in the biological sample, and depending on the antibody titer of this antibody and the type of measurement method. The amount of oxidized LDL present per 1 μL of the biological sample is 0 to
When the antibody DLH3 is used as the primary antibody when it is assumed to be about 1 ng, it is preferably 0.2 to 1.0 μg, and more preferably about 0.5 μg per 1 μL of the biological sample.

In measuring the oxidized lipoprotein by the above method, a compound obtained by artificially oxidizing the lipoprotein can be used as a standard substance in the present invention. The lipoprotein used for such a standard substance may be derived from any organism, for example,
Mammals such as humans, cows, horses, goats, sheep, rabbits, dogs, and guinea pigs; birds such as chickens and quails; fishes such as salmon and herring; and lipoproteins derived from microorganisms such as bacteria and fungi. Furthermore, specific examples of lipoproteins include structural lipoproteins derived from the above sources and present in cell membranes, mitochondrial membranes, biological membranes such as myelin structural membranes and bacterial cell membranes;
Soluble lipoproteins present in plasma, egg yolk, milk and the like; and by ultracentrifugation, chylomicron, VLDL, LDL, lipoprotein X, intermediate density lipoprotein (IDL), lipoprotein (a), HDL2 and Examples thereof include HDL such as HDL3, and a lipoprotein fraction fractionated into ultra-high density lipoprotein (VHDL). These lipoproteins may be used alone or in the form of a mixture of two or more kinds. Of these, human-derived lipoproteins are preferable, and human plasma and serum-derived chylomicrons, VLDL, LDL, Lp (a), HD
L2 or HDL3, or mixtures thereof are more preferred, most preferably LDL derived from human plasma and serum
Is used.

In the present invention, the method for oxidizing lipoprotein is not particularly limited, and for example, the method described in WO 00/75189 is used. In summary of the method described in the publication, the oxidation method using lipoproteins includes a method of oxidizing lipoproteins in the presence of a metal ion; and a compound obtained by artificially oxidizing phospholipids (for example, , 1-palmitoyl-2- (9-oxononanoyl) -glucero-3-phosphocholine and 1-palmitoyl-2- (5-oxovaleroyl) -glucero-3-phosphocholine) in a suitable solvent (eg DMSO) Is added to lipoprotein. In the present invention, a method of oxidizing lipoprotein in the presence of a metal ion is preferably used.

In the present invention, the standard substance is used in place of the oxidized lipoprotein contained in the biological sample to measure the amount of oxidized lipoprotein to prepare a calibration curve, and the oxidized lipoprotein amount contained in the biological sample is The target substance can be easily quantified by calculating it in comparison with the calibration curve.

The inflammatory diseases detectable by the present invention include:
Examples include arthritis, hepatitis, dermatitis, nephritis, pancreatitis, vasculitis, lymphadenitis, rhinitis, etc., and classification by time course includes acute, subacute, and chronic. However, any inflammatory disease in which the amount of oxidized lipoprotein changes can be used as a target.

According to the method for measuring oxidized lipoprotein used in the present invention, not only inflammatory diseases but also cardiovascular diseases can be detected (JP-A-7-238098, JP-A-9-9898).
No. 288106). Therefore, when the value of oxidized lipoprotein is higher than the value of oxidized lipoprotein in healthy individuals, it is determined to be an inflammatory disease or cardiovascular disease. The value of the oxidized lipoprotein of a healthy person is selected as a healthy person who has been clinically confirmed not to be an inflammatory disease or a cardiovascular disease, a biological sample is collected from the healthy person, and the oxidized lipoprotein in the biological sample is collected. The protein can be quantified by the above method. The method for clinically confirming various inflammatory diseases is not particularly limited, but examples thereof include CRP, erythrocyte precipitation,
Indicators that are relatively common to inflammatory diseases such as fever or pain in the affected area, and test methods and symptoms characteristic of various inflammatory diseases, for example, test values and jaundice indicating liver functions such as GOT and GPT for hepatitis. In the case of rheumatoid arthritis, it may be confirmed from test values such as rheumatoid factor and symptoms such as swelling, pain, stiffness and deformity of the affected joint.

As a reference value for determining whether or not an inflammatory disease is obtained from the value obtained by the above-mentioned method for measuring oxidized lipoprotein, for example, use the average value of the values of oxidized lipoprotein of many healthy persons. You can At that time, a value obtained by adding a standard error value to the average value, a value obtained by adding a value twice the standard error value to the average value, or the like can be used. Since the amount of oxidized lipoprotein and the type thereof differ depending on the type of inflammatory disease, it is possible to make a more accurate judgment by preferably setting a reference value for each disease.

The second aspect of the present invention is a drug for detecting inflammatory diseases, which comprises as an active ingredient an antibody that recognizes an antigen produced by the oxidation of phospholipids. The antibody that recognizes the antigen produced by the oxidation of phospholipids is preferably the monoclonal antibody DLH3, and the detection agent can be used in combination with an antibody that recognizes oxidized low-density lipoprotein. The antibody that recognizes low-density lipoprotein is preferably an antibody that recognizes apo B protein.
By using the monoclonal antibody DLH3, various oxidized lipoproteins can be accurately quantified from a low concentration as described in the first invention. In particular, if an antibody that recognizes oxidized low-density lipoprotein is used in combination, the oxidized low-density lipoprotein can be quantified, and if the antibody that recognizes the oxidized low-density lipoprotein is an antibody that recognizes apo B protein, its measurement The accuracy can be improved.

The inflammatory disease detecting agent can be used for carrying out the first invention of the present application and the third invention described later. The detection agent may be, if necessary, a sample diluent, an antibody-immobilized solid phase, a reaction buffer, a washing solution, a secondary antibody (enzyme-labeled secondary antibody, fluorescent-labeled secondary antibody, radioactive-labeled 2 Secondary antibody, luminescence labeled 2
A secondary antibody, which may be a labeled secondary antibody, etc.), a receptor, a detection reagent (for example, a coloring solution), a standard substance, and the like can be included.

As the sample diluent, a biological sample or a standard substance is diluted if necessary, and a solution containing a protein such as bovine serum albumin (BSA) or casein as a surfactant or a buffer is used. be able to.

As the antibody-immobilized solid phase, materials such as polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, and other polymeric materials, glass, ceramics are used. And metals. The shape may be any of tubes, beads, plates, fine particles such as latex, sticks, etc., and as a method for immobilizing an antibody, a known method such as a physical method, a chemical method, or a combination thereof is also adopted. it can. An example of such an antibody-immobilized solid phase is a polystyrene 96-well microtiter plate for immunoassay in which an antibody or the like is hydrophobically immobilized.

The reaction buffer provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the sample, and the sample diluent can be used in the same manner. Further, a solution containing a surfactant, a buffer solution, a protein such as bovine serum albumin or casein, an antiseptic agent, a stabilizer, a reaction accelerator and the like can be used.

As the washing liquid, the above-mentioned sample diluting liquid or reaction buffer can be used.

As the secondary antibody, labeled secondary antibody and receptor, those described in the first invention can be used. In the present invention, an antibody that recognizes an oxidized lipoprotein containing apo B (anti-apo B antibody) is used as a secondary antibody in that it can accurately quantify oxidized low-density lipoprotein highly correlated with inflammatory diseases. Is more preferable. The 2
The secondary antibody may be further mixed with a buffer, a protein such as bovine serum albumin or casein, an antiseptic and the like.

The reagent for detecting the labeled substance can be appropriately selected according to the labeling enzyme of the labeled secondary antibody. For example, if the labeling compound is horseradish peroxidase, tetramethylbenzidine, orthophenylenediamine, etc. If the substrate for measuring absorbance is a fluorescent substrate such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid, a luminescent substrate such as luminol can be used. When the labeling enzyme is alkaline phosphatase, a substrate for absorbance measurement such as 4-nitrophenyl phosphate, a fluorescent substrate such as 4-methylumbelliferyl phosphate, or the like is targeted.

As the standard substance, the compound obtained by artificially oxidizing the lipoprotein described in the first invention can be used. The substance may be a liquid or a frozen frozen product. In order to prepare a dried frozen product, for example, the cryoprotectant described in the first invention is added to the oxidized lipoprotein prepared in a liquid, and freeze-dried. The freeze-drying conditions are not particularly limited, and may be the same as the freeze-drying conditions of the biological sample. By such a freeze-drying step, the water content in the freeze-dried product containing oxidized lipoprotein is usually 10% by mass or less, preferably 1% by mass or less. The amount of cryoprotectant used is usually 1
-20 mass%, preferably 2-5 mass%.

The detection agent may be a reagent kit containing the above-mentioned constitution.

As a method for quantifying oxidized lipoprotein contained in a biological sample using the reagent or the reagent kit, an antigen-antibody complex is formed by contacting the primary antibody with the biological sample, and a labeled secondary antibody is further bound. Then, by measuring the labeled compound and comparing it with a calibration curve prepared by similarly measuring with a standard substance, the oxidized lipoprotein can be easily quantified. At this time, by using a different secondary antibody,
Different oxidized lipoproteins can be measured.

Further, as a method for fractionating and quantifying oxidized lipoprotein in a biological sample by another method, the oxidized lipoprotein is purified in advance by centrifugation or the like, and then a specific type of oxidized lipoprotein is prepared using the primary antibody. Quantify the protein. As described in the first invention, HDL, LDL, VLDL
Alternatively, the lipoprotein (a) is fractionated in advance by centrifugation or the like, and the primary antibody is allowed to act on the fractionated sample to quantify the specific oxidized lipoprotein. In this case, the same secondary antibody can be used to measure different oxidized lipoproteins.

In the third aspect of the present invention, a biological sample is collected from a model animal after administration of a drug whose effect is to be detected to a model animal of inflammatory disease, or a patient or animal with an inflammatory disease. The drug is added to a biological sample, and the amount of oxidized lipoprotein contained in the biological sample is measured by the method for measuring oxidized lipoprotein according to the first aspect of the present invention to evaluate the preventive / therapeutic effect of the drug on inflammatory diseases. A method for searching for a preventive / therapeutic drug for inflammatory diseases, which is characterized in that A drug for which an effect determination is required is administered to an inflammatory disease model animal, and a biological sample is collected from the animal, or a drug for which an effect determination is added is added to a biological sample obtained from an inflammatory disease model animal or a subject with an inflammatory disease. By the way,
The method comprises the step of measuring the oxidized lipoprotein contained in the biological sample and comparing the quantitative value with the quantitative value obtained from an appropriate control experiment to determine the preventive / therapeutic effect of the drug on inflammatory diseases.

The animals used in the present invention include rats,
Animal species usually used as experimental animals such as mouse, guinea pig, hamster, rabbit, sheep, dog and monkey can be used. An animal model obtained by adding a treatment for developing an inflammatory disease to these experimental animals, an animal model genetically prone to develop an inflammatory disease, or the like is preferably used. As a treatment for developing an inflammatory disease, a known method can be used and can be appropriately selected depending on an animal species, a site causing inflammation, and the like. For example, carrageenin, mustard, dextran, ovalbumin, formalin,
There are medications such as lipopolysaccharide, oxazolone, TPA, carbon tetrachloride, and inflammatory agents such as adjuvants. Further, a suitable antigen mixed with an adjuvant or antigen alone, or yeast, Candida, tubercle bacillus, staphylococcus, streptococcus, meningococcus, mouse hepatitis virus, orally the infection source such as Coxsackie virus, Alternatively, it may be a single or frequent injection in an appropriate site such as intragastric, intradermal, subcutaneous, intraarticular, intramuscular, intraperitoneal, intravenous and intrapulmonary. As the antigen, bovine serum albumin,
Human gamma globulin, a homogenate or a homogenate of a tissue collected from an animal of the same species or a syngeneic animal, or an antiserum obtained by immunizing a homogenate of a homogenate or a homogenate of a tissue homogenate collected from an animal of the same species or a similar strain. To be As an animal model that genetically develops inflammatory diseases,
For example, NZB mouse, F ₁ mouse obtained by multiplying NZB mouse and NZW mouse and the like can be mentioned. More specifically, a rat adjuvant arthritis model developed by subcutaneously administering an adjuvant to the right hind leg of a Lewiss rat was treated with rat carbon tetrachloride by repeated oral administration of carbon tetrachloride to Wistar rats for a long period of time. As a liver injury model, a mouse oxazolone dermatitis model was prepared by repeatedly applying an acetone solution of oxazolone to the auricles of BALB / c mice for a long period of time, and a rat sepsis model developed by intravenous administration of lipopolysaccharide. Can be mentioned.

The administration method of the drug whose effect is to be detected is not particularly limited, and can be appropriately selected depending on the physical properties of the drug, the solubility in a solvent such as water, the bioavailability, and the like. For example, oral administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intramuscular administration, epidermal application, intragastric administration, intraduodenal administration, intratracheal administration, nasal administration Internal administration and the like can be mentioned. Therefore, the dosage may be appropriately selected according to the type of drug, the method of administration, the type of inflammatory disease model animal, and the like.

The type of biological sample to be collected is not particularly limited as long as it contains oxidized lipoprotein,
Examples thereof include biological samples such as blood, plasma, serum, tears, urine, amniotic fluid, synovial fluid, spinal fluid, cell extract and tissue extract. Among them, blood, plasma, serum, or an extract of inflamed tissue is preferably used. In addition, the timing of collecting the biological sample after administration, the dose, the type of the drug, the administration method,
It may be appropriately selected depending on the type of inflammatory disease model animal.

The present invention is not limited to the case where the drug whose effect is to be detected is administered to the model animal as described above, and the drug is added to a biological sample previously collected from an animal or a person and incubated for an appropriate time. The sample thus obtained can also be used as a sample for determining the effect of a drug. The biological sample in this case is not particularly limited as long as it contains oxidized lipoprotein, and blood, plasma, serum, tears, urine, amniotic fluid, synovial fluid, spinal fluid, cell extract, tissue extract, etc. Biological samples. Among them, blood, plasma, serum, or an extract of inflamed tissue is preferably used.

As a method for measuring oxidized lipoprotein in a biological sample, the method described in the first invention can be used.

On the other hand, as a method for judging the effect of the drug,
Inflammatory disease animal model and patients with inflammatory disease (control experiment of affected group) and oxidized lipoprotein of healthy animals or healthy subjects (control experiment of healthy group) were measured, and the measurement values obtained by administration of the drug were measured. When compared with these control experiments, it is judged that the drug is effective when it is lower than the value of the affected group. As an appropriate control experiment of a healthy group, for example, when a drug is administered to an animal, oxidized lipoprotein obtained from an animal before drug administration or without drug administration is used, and the drug is added to a biological sample isolated in advance. In that case, oxidized lipoprotein obtained from a biological sample before or without drug addition can be used as a control experiment. More specifically, the control experiment of the diseased group and the control experiment group of the healthy group are each measured in a plurality of cases, preferably 3 or more cases, and the difference between the respective average values is more than a predetermined judgment reference value. In some cases, it may be determined that the drug is effective. As a method of determining the judgment reference value,
For example, the standard error of the control experimental group or twice the standard error can be set.

[0057]

EXAMPLES Next, more specific contents will be shown by Examples, but it goes without saying that the present invention is not limited to those shown here.

(Example 1: Oxidized LDL measurement by sandwich ELISA) (i) Preparation of antibody DLH3 Intraperitoneally in male Balb / c mice aged 8 weeks or older,
0.5 mL / animal of pristane (2,6,10,14-tetramethylpentadecane) was injected and the animals were raised for 2 weeks.
Next, the hybridoma cell line FOH1a, which is a cell producing the desired monoclonal antibody, was added to this mouse.
/ DLH3 (Accession number: FERM BP-7171; J.
Biol. Chem. 1994. 269: 15274-15279;
No. 238,098) was intraperitoneally inoculated at 1 × 10 ⁶ / animal. After 7 to 14 days, when the ascites was sufficiently accumulated in the abdominal cavity of the mouse, the ascites was collected from the abdominal cavity using an 18G injection needle, and centrifuged at 3000 rpm for 10 minutes to collect the supernatant. An equal volume of PBS (pH 7.4) was added to this supernatant.
After adding the mixture, the same amount of saturated ammonium sulfate solution as this mixed solution was added dropwise over 1 hour with sufficient stirring, and the mixture was further stirred for 1 hour and then centrifuged at 3000 rpm for 10 minutes to obtain a supernatant. Discard and collect the precipitate. Further, this precipitate was added to PB containing 0.5 mol / L NaCl.
S (pH 7.4) was added and dissolved, and this solution was added to 0.5
Sephacryl S-300 column (2.5 cm × 100 c) equilibrated with PBS (pH 7.4) containing mol / L NaCl.
m) (manufactured by Pharmacia) and the IgM fraction was collected and used as the antibody DLH3. The antibody DLH3
The concentration was measured by measuring the absorbance at 280 nm with an optical path length of 1 cm, and the obtained absorbance was divided by 1.3, and this value was defined as the antibody concentration (mg / mL).

(Ii) Preparation of rat apo B protein Rat plasma was obtained using EDTA as an anticoagulant and the plasma was subjected to ultracentrifugation to obtain LDL having a specific gravity of 1.006 to 1.063.
Fractions were collected. This LDL fraction was dialyzed three times with phosphate buffered saline (PB), concentrated by ultrafiltration, and further V.I. G. Shore et al. (Biochemistry, 8: 4510)
1969). In addition, Sephacryl S-200HR
Apo B protein was purified by gel filtration chromatography using a column.

(Iii) Preparation of anti-rat apo B antibody A rabbit was immunized with the rat apo B protein obtained in (ii) above to prepare an anti-rat apo B polyclonal antibody.
That is, 100 μg of apo B protein was mixed with an adjuvant only for the first time, and subcutaneously was added to a Japanese white rabbit 1 weekly.
Injection (total 6 times) was performed, and serum was collected 3 to 4 days after the final immunization. Immunoglobulin was purified from the obtained serum by the capryl sulfate precipitation method. That is, 60 mmol / L sodium acetate buffer (pH 4.
0) was added to adjust the pH to 4.8, and 7 mL was added to 1 mL of serum.
5 μL of capryl sulfuric acid was added, and the mixture was stirred at room temperature for 30 minutes. After centrifugation (15,000 xg, 10 minutes), the supernatant was mixed with P
It was dialyzed against BS at 4 ° C. overnight. Next, an equal amount of saturated ammonium sulfate solution was added, and the mixture was stirred overnight at 4 ° C. After centrifugation (15000 xg, 30 minutes), the precipitate was collected, dissolved in PBS in the same volume as serum, and then 4 ° C against PBS.
Dialyzed overnight.

(Iv) Europium labeling of anti-rat apo B antibody The above anti-rat apo B polyclonal purified antibody was labeled with europium using a commercially available kit (DELFIA Eu-N1 ITC type labeling reagent: WALLAC). That is, 500 μg of the purified antibody dissolved in a labeling buffer (100 mmol / L Na ₂ CO ₃ pH 9.3) was mixed with the Eu-N1 ITC type labeling reagent and reacted at room temperature for 16 hours. Then, unreacted europium and the labeled antibody were separated by gel filtration chromatography using a Sepharose 6B column.

(V) Preparation of standard substance (rat oxidized LDL) Rat plasma was obtained using heparin as an anticoagulant, and the plasma was ultracentrifuged to collect an LDL fraction having a specific gravity of 1.030 to 1.050. . This LDL fraction is 0.25 mmol / L
Fully dialyzed against PBS containing EDTA, Superose
It was purified by gel filtration chromatography using a 6HR column. The purity of LDL was confirmed by agarose gel electrophoresis to give a single band. After protein quantification, the cells were stored at 4 ° C until the following operations.

Purified LDL was prepared with a PBS solution so that the protein concentration was 1 mg / mL. 5 μmol / copper sulfate to a purified LDL solution with a protein concentration of 1 mg / mL
L was added thereto and incubated at 37 ° C. for 3 hours, and EDTA was added thereto so that the concentration was 0.34 mmol / L to stop the oxidation reaction. The obtained oxidized LDL solution was thoroughly dialyzed against PBS containing 0.25 mmol / L EDTA to quantify the protein, and then prepared so that the protein concentration would be 1 mg / mL, and used as the oxidized LDL standard solution. Stored at 4 ° C until ready.

(Vi) Measurement by sandwich method In order to immobilize the primary antibody (antibody DLH3), 20 μm
50 mM Tris-H containing g / mL antibody DLH3
Cl solution (pH 8.0) made of plastic EIA / RI
50 μL was added to each 96-well plate for A (Nunc) and incubated overnight at 4 ° C., and then the solution was removed. Next, 150 μL of 1% bovine serum albumin dissolved in PBS was added to each well, incubated at room temperature for 1 hour, and then washed with a PBS solution containing 1% Tween-20.

1% plasma obtained from rats by heparin blood sampling
Bovine serum albumin and 4% Polyethelene Glycol # 6
50 mmol / L Tris-HCl containing 000 (pH
It was diluted 2000-4000 times with the 8.0 solution and 50 μL was added to the plate on which the above-mentioned primary antibody was immobilized. After incubating the plate at 37 ° C for 2 hours, each well of the plate was added to PBS containing 1% Tween-20.
Wash with solution. Commercially available DE was added to each well except PBS.
50 mL of the above-mentioned labeled secondary antibody (europium-labeled anti-rat apo B antibody) diluted 100 times with Assay Buffer for LFIA (WALLAC) was added to each plate and incubated at 37 ° C. for 1 hour. Then, each well of the plate was washed with PBS containing 1% Tween-20. Commercially available Enhancement Solution for DELFIA (W
ALLAC) was added, and the amount of europium contained in each well was measured by excitation wavelength 340 nm, fluorescence wavelength 615 nm, and time-resolved fluorescence from 400 μsec to 400 μsec after excitation.

Standard substance stored at 4 ° C .: oxidized LD
L standard solution as protein concentration 0-100 ng
/ ML, 1% bovine serum albumin and 4% Polyethe
50 mmol / L Tris- containing leneGlycol # 6000
A serial dilution was performed using a HCl (pH 8.0) solution, and measurement was performed in the same manner as in the above sample to prepare a calibration curve of standard oxidized LDL based on the amount of standard oxidized LDL and time-resolved fluorescence value.
Oxidation LD using the calibration curve from the amount of europium in each sample
The amount of L was calculated. In FIG. 1 showing an example of the calibration curve in FIG. 1, the amount of oxidized LDL contained in each sample is the standard oxidized LDL.
The protein amount of 1 ng was expressed as 1 U.

(Example 2: Measurement of oxidized LDL value in hepatitis model rat) Wistar rat (7 weeks old, weight 150)
~ 160 g) was fed with a commercial feed (CE-2 Nippon Clare Co., Ltd.), and water containing 35 mg / mL phenobarbital sodium (for Wako Pure Chemical Industries, Ltd. biochemistry) was freely ingested as drinking water for breeding. . After 2 weeks of breeding, carbon tetrachloride (Wako Pure Chemical Industries, Ltd. reagent special grade) was added at 10% (v /
v) The contained olive oil (Wako Pure Chemical Industries, Ltd.) was orally administered once a week using a Teflon (registered trademark) sonde 12 times in total. The dose of carbon tetrachloride was 0.04 mL / animal as carbon tetrachloride at the time of the first administration, and the dose was adjusted from the second administration depending on the change in body weight for one week from the previous administration. In other words, if weight gain is observed compared to the previous administration, the carbon tetrachloride dose is gradually increased to 1.5 times, 2 times, 3 times, 4 times the initial dose, and if no weight gain is observed, tetrachloride is added. The carbon dose was the same as the previous dose. 1
On the 6th day after the two administrations were completed, blood was collected from the tail vein,
Heparin plasma was obtained and the oxidized LDL in plasma was measured by the method described in Example 1. As a control experiment, olive oil containing no carbon tetrachloride was administered.

As shown in FIG. 2, the oxidized LDL concentration in plasma of the rat to which carbon tetrachloride was administered was higher than that of the control group.

Example 3 Measurement of Oxidized LDL Value in Dermatitis An acetone solution containing 0.5% oxazolone was applied to the right auricle of Balb / c mouse in an amount of 10 μL. Acetone solution containing 0.5% oxazolone every 2 to 3 days after 1 week was applied to the right auricle of Balb / c mouse at 10 μm each time.
L was applied for 3 weeks (10 times in total). Six hours after the last application, edema of the lesion on the right auricle was measured with a dial thickness gauge (model G, Ozaki Seisakusho, Tokyo).
Collect the lesions and apply PBS to each ear
Each μL was added, and the tissue was disrupted with a homogenizer. The crushed solution was centrifuged at 15,000 × g, and the amount of oxidized LDL was measured using the resulting supernatant as a measurement sample. As a control experiment, 10 μL of each of the above acetone solutions in which 0.5% oxazolone was dissolved was applied to the right auricle instead of acetone containing no oxazolone.

As shown in FIG. 3, the amount of oxidized LDL in the auricle by the application of oxazolone was higher than the amount of oxidized LDL in the auricle of the animal to which the solvent containing no oxazolone was applied. In particular, all individuals treated with oxazolone showed higher values than all individuals not treated. From this, it was suggested that the oxidative LDL value in the tissue causing dermatitis was measured by the measurement method of Example 1 and the inflammatory disease could be distinguished from normal.

Example 4 Adjuvant Arthritis Effect of Prophylactic / Therapeutic Agent for Inflammatory Diseases Using Rats Adjuvant arthritis is described in Tamura et al., European Journal of Pharmacology, 41.
The disease was developed according to the method described in Vol. 9, p. 269, 2001.
That is, the killed bacteria (DiDi) of mycobacterium butyricum suspended in liquid paraffin were placed in the footpads of the right hindlimbs of female Lewis rats.
0.6 mg / mL (hereinafter referred to as an adjuvant) was subcutaneously injected by fco Laboratories. Diclofenac sodium [3 mg / kg;
Sigma] was orally administered once a day for 21 days at a rate of 10 mL per 1 kg of body weight. On the day after the final administration of the drug, swelling of the right and left hind limbs was measured by a rat hind footpad edema volume measuring device, and then whole blood was collected from the thigh (D).
Administration group). As a control experiment in which arthritis was not developed, rats to which neither an adjuvant nor drug was administered (normal group) were used as a control experiment in which no drug was administered, and rats were orally administered with a solvent instead of the drug (arthritis group). ) Was used.

As shown in FIG. 4, the amount of oxidized LDL in plasma of rats (arthritis group) which developed arthritis by administration of an adjuvant was determined by the amount of plasma obtained from animals (normal group) to which no adjuvant was administered. The value was higher than the amount of oxidized LDL. Furthermore, the amount of oxidized LDL in plasma obtained from the animal to which diclofenac sodium was administered (D-administered group) was lower than that obtained from the animal to which it was not administered (arthritis group). Therefore, by using this experimental system, the effect of diclofenac sodium used as a therapeutic agent for arthritis could be detected. At this time, a positive correlation was observed between the degree of swelling of the left and right hind limbs measured in the same individual and the amount of oxidized LDL in plasma determined by the measurement method of Example 1 (FIG. 5).

[0073]

INDUSTRIAL APPLICABILITY According to the present invention, various oxidized lipoproteins can be quantified using an antibody that recognizes an antigen produced by the oxidation of phospholipids, and inflammatory diseases can be affected based on the measured values. It is possible to easily detect whether or not there is. If an antibody that recognizes low-density lipoprotein is used in addition to the antibody, the quantification of oxidized low-density lipoprotein is particularly excellent.

Since the inflammatory disease-detecting drug of the present invention contains various drugs necessary for the quantification of the oxidized lipoprotein,
Easy to detect inflammatory diseases.

Furthermore, according to the present invention, the effect of a drug whose efficacy is desired to be evaluated as a prophylactic drug for inflammatory diseases can be easily evaluated by quantifying oxidized lipoprotein. Use of the above-mentioned inflammatory disease detecting agent is effective for this method for searching for a preventive / therapeutic agent for inflammatory disease.

[Brief description of drawings]

FIG. 1 shows the results of serial dilution of standard oxidized LDL and measurement.

FIG. 2 shows plasma oxidized LDL concentrations in a normal control group and a group to which carbon tetrachloride was administered (hepatitis group). Each point represents the measured value of each individual.

FIG. 3 shows the amount of oxidized LDL in the auricle in a normal control group and an oxazolone-administered group (dermatitis group). Each point represents the measured value of each individual.

FIG. 4 shows oxidized LDL concentrations in plasma in a normal control group (normal group), an adjuvant-administered group, a drug-unadministered group (arthritis group), and an adjuvant-administered diclofenac-administered group (D-administered group). Each point represents the measured value of each individual.

FIG. 5 Plasma oxidized LDL for all individuals in FIG.
The correlation between the concentration and swelling of the right hind limb (upper figure) and the left hind limb (lower figure) is shown. The correlation with swelling of the right hind limb and the left hind limb was both 0.50.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 29/00 A61P 29/00 C12P 21/08 C12P 21/08 (72) Inventor Kaori Hamaguchi Sunto-gun, Shizuoka Prefecture 1188 Shimochikari, Nagaizumi-cho Kyowa Fermentation Industry Co., Ltd., Pharmaceutical Research Institute (72) Inventor Satoshi Nakanishi Satoshi Nakashi, Shizuoka-ken, Nagaizumi-cho 1188 Kyowa-Fermentation Industry Co., Ltd. Pharmaceutical Research Laboratory (72) Inventor, Kazuhide Hasegawa, Mayor, Sunto-gun, Shizuoka Prefecture 1188 Shimotochi, Izumi-cho, Pharmaceutical Research Institute, Kyowa Fermentation Industry Co., Ltd. (72) Inventor, Tadashi Tamura, 1188, Shimochi-cho, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture 1188, Pharmaceutical Research Laboratory, Kyowa Fermentation Co., Ltd. (72) Miho Kizaki Miyukigaoka, Tsukuba, Ibaraki Prefecture No. 2 Kyowa Fermentation Industry Co., Ltd. Tsukuba Research Institute (72) Inventor Katsuya Kobayashi Nagaizumi, Sunto-gun, Shizuoka Prefecture 1188 Machishita Dokari In the Kyowa Hakko Kogyo Co., Ltd. Pharmaceutical Research Laboratory (72) Inventor Daisuke Harada 1188 Shimochikari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture 1188 F-Term (Reference) 2G045 AA25 AA40 DA61 DA62 DA77 FB03 4B064 AG27 CA10 CA20 CC24 DA13 4C084 AA17 NA14 ZB112

Claims (9)

[Claims] 1. By measuring an oxidized lipoprotein contained in a biological sample obtained from a patient suspected of having an inflammatory disease or an animal model of an inflammatory disease, using an antibody that recognizes an antigen produced by the oxidation of phospholipids. A method for detecting an inflammatory disease. 2. An oxidized low-density lipoprotein is measured as the oxidized lipoprotein by using an antibody that recognizes the oxidized low-density lipoprotein together with the antibody that recognizes an antigen produced by the oxidation of phospholipids. The method according to item 1. 3. The antibody that recognizes oxidized low density lipoprotein is an antibody that recognizes apo B protein.
The method described. 4. The method according to claim 1, wherein the antibody that recognizes an antigen produced by the oxidation of phospholipid is the monoclonal antibody DLH3. 5. A drug for detecting an inflammatory disease, which comprises, as an active ingredient, an antibody that recognizes an antigen produced by the oxidation of phospholipids. 6. The detection agent according to claim 5, which further comprises, as an active ingredient, an antibody that recognizes oxidized low-density lipoprotein. 7. The antibody recognizing oxidized low density lipoprotein is an antibody recognizing apo B protein.
The detection drug described. 8. The detection agent according to claim 5, wherein the antibody that recognizes an antigen produced by the oxidation of phospholipid is the monoclonal antibody DLH3. 9. An inflammatory disease model animal is administered with a drug whose effect is to be detected, and then a biological sample is collected from the model animal, or the drug is added to a biological sample collected from a patient or animal with inflammatory disease. To evaluate the preventive / therapeutic effect of the drug by measuring the oxidized lipoprotein contained in the biological sample using any of the following methods (i) to (iv). A method for searching a preventive / therapeutic drug for inflammatory diseases, which comprises: (i) A method of forming an antigen-antibody complex between an antibody that recognizes an antigen produced by the oxidation of phospholipids and an oxidized lipoprotein contained in the biological sample, and measuring the oxidized lipoprotein contained in the complex, ii) An antigen-antibody complex with an oxidized lipoprotein contained in the biological sample is formed using an antibody that recognizes an oxidized low-density lipoprotein together with an antibody that recognizes an antigen produced by the oxidation of phospholipids, and the complex A method for measuring oxidized low density lipoprotein as oxidized lipoprotein contained in (iii) the method according to (ii) above, wherein the antibody that recognizes oxidized low density lipoprotein is an antibody that recognizes apo B protein; ) The above-mentioned (i) to (iii), wherein the antibody that recognizes the antigen produced by the oxidation of phospholipid is the monoclonal antibody DLH3
The method described in any one of.