Method for quantifying adiponectin and analytical reagents used therefor

Abstract

The present invention provides: an adiponectin quantification method which enables the quantification of adiponectin in a body fluid with distinguishing adiponectin having a physiological activity from adiponectin having a pathological activity; and an analysis reagent which can be used in the method.

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying adiponectin, a substance involved in metabolism in blood, and an analytical reagent used therefor. [Background technology]

[0002] Adiponectin is a secretory protein expressed in adipocytes and thought to be involved in glucose regulation and fatty acid degradation. Since blood adiponectin levels generally show an inverse correlation with body fat mass, adiponectin is known as a beneficial adipokine and has attracted attention for its ability to suppress metabolic syndrome, which has become a problem in recent years, and its associated diseases such as arteriosclerosis.

[0003] Adiponectin is known to exist in the form of monomers, trimers, hexamers, and further polymers formed by polymerizing multiple monomers. The total amount of adiponectin can be quantified using anti-adiponectin antibodies, and this has been used as a quantitative technique. However, it is believed that adiponectin formed by polymerizing multiple monomers is primarily responsible for physiological functions. Patent Document 1 discloses a technique for quantifying high-molecular-weight adiponectin multimers, which are thought to be involved in the metabolism. This technique aims to selectively measure high-molecular-weight adiponectin using a sandwich immunoassay system in which antibodies that recognize the SS-binding site or its vicinity, which contributes to intermolecular binding of adiponectin, and that specifically recognize high-molecular-weight adiponectin when used as the primary and secondary antibodies in the sandwich immunoassay system, are used as the primary and secondary antibodies. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2009 / 078151 Summary of the Invention [Problem to be solved by the invention]

[0005] However, while adiponectin is thought to contribute to the metabolism of fats and sugars, high blood adiponectin levels may actually increase the risk of diseases related to these metabolisms, such as the risk of cardiovascular disease. Therefore, the physiological activity of adiponectin and its influence on the above-mentioned diseases cannot be accurately understood by simply quantifying the total amount of adiponectin or the amount of adiponectin in each structure such as a monomer or a polymer.

[0006] For example, in the case of high-density lipoprotein (HDL), known as "good" cholesterol, patients with coronary artery disease have increased levels of pathologically active, proatherosclerotic dysfunctional HDL. The present inventors have hypothesized that, similar to HDL, the total amount of adiponectin in the blood contains both physiologically active adiponectin and pathologically active adiponectin, and that high concentrations of pathologically active adiponectin prevent it from exerting its physiological activity. The present inventors have considered the need to accurately measure the physiological activity of adiponectin while taking these factors into consideration, and have therefore carried out extensive research.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a method for quantifying adiponectin, which enables accurate understanding of the physiological activity of adiponectin and is useful for assessing the risk of cardiovascular disease, lifestyle-related disease, etc., and an analytical reagent for use in the method. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention has the following aspects. [1] A method for quantifying adiponectin in a body fluid, which distinguishes between adiponectin having physiological activity and adiponectin having pathological activity. [2] The method for quantifying adiponectin in a body fluid, wherein T-cadherin proteins are used to quantify the physiologically active adiponectin. [3] The method for quantifying adiponectin in a body fluid, wherein the T-cadherin proteins are recombinant T-cadherin proteins. [4] The method for quantifying adiponectin in the body fluid, wherein the physiologically active adiponectin is quantified by immunoassay of a complex of adiponectin, T-cadherin proteins, and an anti-adiponectin antibody. [5] The method for quantifying adiponectin in the body fluid, wherein the adiponectin having the pathological activity is quantified using a conjugate of adiponectin and denatured low density lipoprotein. [6] The method for quantifying adiponectin in a body fluid, wherein LOX-1 protein is used to quantify adiponectin having pathological activity. [7] The method for quantifying adiponectin in a body fluid, wherein the LOX-1 protein is a recombinant LOX-1 protein. [8] The method for quantifying adiponectin in the body fluid, wherein the adiponectin having pathological activity is quantified by immunoassay using the recombinant LOX-1 protein to quantify receptor binding activity. [9] The method for quantifying adiponectin in the body fluid, wherein the amount of adiponectin having pathological activity is quantified by using the recombinant LOX-1 protein and quantifying the receptor binding activity by immunoassay of a complex of an anti-adiponectin antibody, adiponectin, and denatured low-density lipoprotein.

[10] The method for quantifying adiponectin in the body fluid, wherein T-cadherin proteins are used to quantify the physiologically active adiponectin, recombinant LOX-1 protein is used to quantify the pathologically active adiponectin, and receptor binding activity is quantified by immunoassay.

[11] The method for quantifying adiponectin in the body fluid, wherein T-cadherin proteins are used to quantify the physiologically active adiponectin, and a conjugate of adiponectin with denatured low density lipoprotein and recombinant LOX-1 protein are used to quantify the pathologically active adiponectin, and the receptor binding activity is quantified by anti-adiponectin immunoassay.

[12] The method for quantifying adiponectin in a body fluid, wherein the body fluid is blood or saliva.

[13] An analytical reagent used to quantify adiponectin in body fluids for determining cardiovascular disease, diabetes, or diabetic disease, or the stage of progression thereof, the analytical reagent comprising a recombinant T-cadherin protein for quantifying physiologically active adiponectin.

[14] An analytical reagent used to quantify adiponectin in body fluids to determine cardiovascular disease, diabetes, or diabetic disease, or the stage of progression thereof, the analytical reagent containing recombinant LOX-1 protein for quantifying adiponectin having pathological activity, and quantifying receptor binding activity against adiponectin-denatured low-density lipoprotein complexes by anti-adiponectin immunoassay. [Effects of the Invention]

[0009] According to the present invention, a method for quantifying adiponectin that enables accurate understanding of the physiological activity of adiponectin and is useful for assessing the risk of cardiovascular disease, lifestyle-related disease, etc., and an analytical reagent for use in the method are provided. [Brief explanation of the drawings]

[0010] [Figure 1]FIG. 1 is a schematic diagram showing an outline of the method for quantifying adiponectin used in the Examples of the present application. [Figure 2] FIG. 1 is a graph showing the results of a binding test between physiologically active adiponectin and T-cadherin. [Figure 3] FIG. 1 is a graph showing the results of a quantitative test of the concentration of physiologically active adiponectin in human serum samples. [Figure 4] FIG. 1 is a graph showing the comparative results of a quantitative test of the concentration of physiologically active adiponectin in a human serum sample and a plasma sample containing EDTA. [Figure 5] FIG. 1 is a graph showing the results of a binding test between adiponectin having pathological activity and denatured LDL. [Figure 6] FIG. 1 is a graph showing the results of a quantitative test of the concentration of pathologically active adiponectin in human serum samples. [Figure 7] FIG. 1 is a graph showing the results of a quantitative test of the concentration of LOX-1-bound modified LDL in human serum samples. [Figure 8] FIG. 1 shows a binding test between denatured LDL and adiponectin in a reference test of the present application. [Figure 9] FIG. 1 shows a test in which AMPK and its phosphorylation (pAMPK) were detected using an anti-adiponectin antibody. DETAILED DESCRIPTION OF THE INVENTION

[0011] The method for quantifying adiponectin and the analytical reagent according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0012] (Method for quantifying adiponectin) In the method for quantifying adiponectin of this embodiment, adiponectin having physiological activity and adiponectin having pathological activity are distinguished from each other and quantified in the quantification of adiponectin in body fluids.

[0013] Here, adiponectin having physiological activity refers to adiponectin that can bind to and exert physiological effects via adiponectin receptors, such as AdipoR1, AdipoR2, and T-cadherin. Adiponectin that has physiological activity is sometimes called functional adiponectin.

[0014] Adiponectin with pathological activity refers to adiponectin that is less able to exert its inherent physiological activity, and includes adiponectin that has lost its physiological activity, adiponectin that has significantly reduced physiological activity, adiponectin that has acquired non-physiological activity, etc. Such adiponectin is, for example, adiponectin that exhibits reduced adiponectin receptor-mediated responses. Mechanisms by which physiologically active adiponectin loses its physiological activity include, for example, chemical modification or partial or complete degradation of the constituent proteins, genetic defects and mutations, abnormalities or changes in higher-order structure, or binding to molecules that cause it to lose its activity. Adiponectin with pathological activity interacts with receptors that induce adverse reactions in the body, such as LOX-1 (lectin-like oxidized LDL receptor-1), and acquires the ability to bind to LOX-1, including through other proteins. In this embodiment, adiponectin that has become less able to exert its physiological activity due to the acquisition of such binding abilities is referred to as adiponectin with pathological activity, but the activity of adiponectin with pathological activity is not limited to the above. Adiponectin with pathological activity is sometimes called dysfunctional adiponectin.

[0015] Distinguishing and quantifying physiologically active adiponectin from pathologically active adiponectin includes, for example, quantifying each using a means capable of quantifying only physiologically active adiponectin, or a means capable of quantifying only pathologically active adiponectin, or a combination of these. It also includes a means for distinguishing and separating physiologically active adiponectin from pathologically active adiponectin and quantifying each of them. In this embodiment, either physiologically active adiponectin or pathologically active adiponectin can be quantified. It is also preferable to quantify both physiologically active adiponectin and pathologically active adiponectin. Analyzing and evaluating these results together allows for more accurate assessment of disease risk, etc.

[0016] The above-mentioned quantification can be carried out using known means for protein quantification (measuring protein amount), for example, immunological techniques. Immunological quantification and immunoanalysis can be performed using enzymes (enzyme immunoassay, EIA, ELISA), fluorescent substances (fluorescent immunoassay, FIA), or radioactive substances (radioimmunoassay, RIA), etc. Among these, ELISA is preferred because it is relatively inexpensive, simple, and can analyze a large number of samples.

[0017] For example, in this embodiment, the quantification can be performed using an ELISA method. Specifically, a compound capable of binding to the target to be quantified is immobilized on an ELISA plate. A sample is added to this ELISA plate, and the target to be quantified contained in the sample is allowed to bind to a receptor. The plate is washed to remove unbound material, and detection is performed using an antibody to the target to be quantified, thereby enabling the quantification of a complex between the target to be quantified and a compound capable of binding to the target. In this embodiment, receptors with physiological effects are used as compounds capable of binding to the target to be quantified. In this embodiment, a receptor capable of binding only to adiponectin with physiological activity and a receptor capable of binding only to adiponectin with pathological activity are used. Hereinafter, the activity of each adiponectin binding to a receptor may also be referred to as receptor binding activity. In other words, the quantification of each adiponectin in this embodiment is the quantification of the receptor binding activity to each receptor. A known method can be appropriately used to immobilize (immobilize) the receptor. For example, the receptor can be bound to a known carrier (support).

[0018] In this embodiment, adiponectin contained in a body fluid is quantified. Here, the body fluid may be, for example, blood or saliva. As a blood sample, serum or plasma may be used. In this embodiment, these body fluids are used as samples for the above-mentioned quantification.

[0019] The above-mentioned body fluid sample preferably does not contain EDTA. EDTA, a calcium ion inhibitor, may be added to samples such as plasma for storage. However, it has been reported that the binding between T-cadherin and adiponectin is calcium-dependent. Therefore, the above-mentioned quantification method can be suitably used when the sample does not contain EDTA, a calcium ion inhibitor.

[0020] (Quantification of physiologically active adiponectin) In quantifying adiponectin in body fluids, it is preferable to use T-cadherin proteins to quantify physiologically active adiponectin. The T-cadherin proteins of this embodiment include known families of T-cadherin proteins and modified proteins. T-cadherin is a receptor for physiologically active adiponectin. By quantifying adiponectin that interacts with (e.g., binds to) T-cadherin, physiologically active adiponectin can be distinguished from pathologically active adiponectin and quantified.

[0021] In quantifying adiponectin in body fluids, the T-cadherin protein is preferably, for example, a recombinant T-cadherin protein. Here, the recombinant T-cadherin protein is a T-cadherin protein obtained by expression through genetic recombination. The recombinant T-cadherin protein may be fused with other structures or mutated, as necessary.

[0022] In quantifying adiponectin in body fluids, it is also preferable to quantify physiologically active adiponectin by immunoassay of a complex of adiponectin, T-cadherin proteins, and anti-adiponectin antibody.

[0023] Specifically, adiponectin-T-cadherin protein complexes can be bound to anti-adiponectin antibodies, and the complexes can be quantified by immunoassay. For example, T-cadherin proteins can be immobilized on an ELISA plate. A reagent derived from a body fluid is injected into the ELISA plate, causing physiologically active adiponectin to bind to the T-cadherin proteins on the ELISA plate. The complexes on the ELISA plate can then be quantified using anti-adiponectin antibodies. In this embodiment, the anti-adiponectin antibody may be either a monoclonal or polyclonal antibody.

[0024] (Quantification of pathologically active adiponectin) In quantifying adiponectin in body fluids, adiponectin having pathological activity is preferably quantified using a conjugate of adiponectin and denatured low density lipoprotein. Denatured low-density lipoprotein (hereinafter referred to as denatured LDL) is a type of LDL (low-density lipoprotein) that has been denatured due to some factor. LDL is commonly known as bad cholesterol and is known to be responsible for transporting cholesterol from the liver to the periphery. Examples of denatured LDL include oxidized LDL (oxLDL).

[0025] According to the present inventors, there are verification results showing that adiponectin is concentrated in the fraction of denatured low density lipoprotein, and that the action of adiponectin is inhibited by oxidized LDL, which is a denatured LDL. In our studies, as shown in Figure 8 (discussed below), adiponectin is concentrated in the fraction corresponding to denatured LDL in human blood, and an interaction between adiponectin and denatured LDL occurs in vivo. As shown in Figure 9 (discussed below), denatured LDL has the effect of inhibiting the phosphorylation of adiponectin by AMPK (AMP kinase). In the presence of denatured LDL, adiponectin is thought to bind to denatured LDL and lose its original action via AdipoR1. Therefore, we predicted that the interaction between denatured LDL and adiponectin would cause adiponectin to exhibit pathological activity. From these results, the inventors discovered that denatured low-density lipoprotein interacts with adiponectin, causing it to lose its physiological activity, and that denatured low-density lipoprotein can be used to quantify adiponectin that has pathological activity.

[0026] In quantifying adiponectin having pathological activity, a conjugate of adiponectin and denatured low-density lipoprotein can be used to detect and quantify the conjugate of adiponectin and denatured low-density lipoprotein, for example, using a protein that specifically binds to denatured low-density lipoprotein and an anti-adiponectin antibody. By quantifying adiponectin that has interacted with (eg, bound to) denatured low density lipoprotein, adiponectin having pathological activity can be distinguished from adiponectin having physiological activity and quantified.

[0027] In quantifying adiponectin in body fluids, it is also preferable to use a protein that interacts with denatured low-density lipoprotein (LDLP) to quantify adiponectin with pathological activity. A preferred example of a protein that interacts with denatured LDL is the LOX-1 protein. LOX-1 is a molecule discovered by the present inventors (Sawamura T et al., Nature 386, 73-77, 1997) and is known as a type of lectin-like oxidized LDL receptor. The detailed structure of LOX-1 has been elucidated, and it is known to be a single-pass transmembrane protein with a lectin-like domain, which is the recognition site for oxidized LDL (see, for example, Japanese Patent Publication No. 9-98787). It is also known that soluble components exist in blood, and modified high-density lipoprotein (HDL) acts as a ligand for LOX-1 (see, for example, Japanese Patent Publication No. 2012-100585 and Japanese Patent Publication No. 6231307).

[0028] In quantifying adiponectin in body fluids, the LOX-1 protein is preferably, for example, a recombinant LOX-1 protein. Here, the recombinant LOX-1 protein is a LOX-1 protein obtained by expression through genetic recombination. The recombinant LOX-1 protein may be fused with other structures or mutated, as necessary.

[0029] In quantifying adiponectin in body fluids, it is also preferable to quantify adiponectin having pathological activity by immunoassay using recombinant LOX-1 protein to quantify receptor binding activity. It is also preferable to quantify the receptor binding activity by immunoassay of a complex of anti-adiponectin antibody, adiponectin, and denatured low density lipoprotein. Specifically, a complex of adiponectin and denatured low density lipoprotein is bound to an anti-adiponectin antibody, and the complex can be quantified by immunoassay.

[0030] In the above-mentioned immunoassay, a means for quantifying the complex of recombinant LOX-1 protein, denatured low-density lipoprotein, and adiponectin can be used. This quantification can be performed using a so-called sandwich ELISA. For example, this quantification method involves immobilizing LOX-1, a receptor for low-density lipoprotein, on an ELISA plate, and then allowing denatured low-density lipoprotein to bind to the LOX-1 on the ELISA plate. By injecting a reagent derived from a body fluid into the ELISA plate, adiponectin, which has pathological activity, is allowed to bind to the complex of LOX-1 and denatured low-density lipoprotein. The complex on the ELISA plate can then be quantified using an anti-adiponectin antibody.

[0031] The quantification of adiponectin in body fluids can be carried out by combining the above-mentioned quantification of physiologically active adiponectin with the quantification of pathologically active adiponectin. For example, T-cadherin proteins can be used to quantitate physiologically active adiponectin, and a conjugate of adiponectin with denatured low-density lipoprotein and recombinant LOX-1 protein can be used to quantitate pathologically active adiponectin, and the receptor binding activity can be quantified by anti-adiponectin immunoassay.

[0032] (Analytical reagent used for quantifying adiponectin) Next, an analytical reagent used in the above-mentioned method for quantifying adiponectin in body fluids will be described. This analytical reagent can be used to quantify adiponectin in body fluids, and can be used to quantify adiponectin in body fluids to determine cardiovascular diseases, diabetes, or diabetic diseases, or the degree of progression thereof. The analytical reagent contains the recombinant T-cadherin protein described above, which allows for the quantification of physiologically active adiponectin.

[0033] The analytical reagent may also contain recombinant LOX-1 protein for the quantification of adiponectin having pathological activity. As described above, this analytical reagent allows the quantification of adiponectin with pathological activity by quantifying the receptor binding activity of the adiponectin-denatured low-density lipoprotein complex using anti-adiponectin immunoassay, since the recombinant LOX-1 protein interacts with denatured low-density lipoprotein.

[0034] (Other configurations) The analytical reagent of this embodiment may contain other components contained in reagents for immunoassays.

[0035] The analytical reagent of this embodiment may also be provided as a kit comprising a plurality of the above-described components. For example, this kit may comprise an analytical reagent containing a recombinant T-cadherin protein for quantifying physiologically active adiponectin, and an analytical reagent containing a recombinant LOX-1 protein for quantifying pathologically active adiponectin. This kit may also comprise recombinant low-density lipoprotein for quantifying pathologically active adiponectin. This kit may also comprise a carrier, an ELISA plate, a chromogenic substrate, an antibody, or the like for use in the above-described quantification procedure. It may also comprise an ELISA plate on which the above-described protein is immobilized.

[0036] (Effects of this embodiment) According to the method for quantifying adiponectin and the analytical reagent of this embodiment, the physiological activity of adiponectin can be correctly understood, and can be used to evaluate the risk of cardiovascular disease, lifestyle-related disease, and the like.

[0037] The quantitative method and analytical reagent of this embodiment are highly likely to be useful for cardiovascular disease risk stratification by distinguishing between physiologically active adiponectin and pathologically active adiponectin. Furthermore, these quantitative methods and analytical reagents are highly likely to be applicable to arteriosclerotic diseases, particularly coronary artery disease, diabetes, and diabetic diseases. Many of these diseases are so-called lifestyle-related diseases. Therefore, all people aged 40 or older, who are at increased risk of lifestyle-related diseases, are candidates for disease risk assessment, prevention, and diagnosis, making the scope of social application extremely broad.

[0038] The quantification method of this embodiment can be applied to in vitro diagnosis for predicting cardiovascular disease risk. Since the primary purpose of the quantification method of this embodiment is to assess cardiovascular disease risk, it can be used for secondary risk assessment of patients who have already experienced adverse cardiovascular events and for primary risk assessment of healthy individuals. Furthermore, the analytical reagent of this embodiment and a kit including the same can be applied to these in vitro diagnostic agents.

[0039] (Application of this embodiment) The adiponectin quantification method and analytical reagent of this embodiment can be used, for example, in a method for diagnosing the onset of cardiovascular disease and / or diabetes or diabetic diseases by measuring physiologically active adiponectin in the quantification of adiponectin in body fluids.

[0040] Furthermore, the present invention can be suitably used in a method for diagnosing the above-mentioned cardiovascular diseases, such as arteriosclerotic diseases and coronary artery diseases.

[0041] As a diagnostic method, for example, by quantifying adiponectin in the body fluids and measuring physiologically active adiponectin, it is possible to diagnose the risk of developing cardiovascular disease, diabetes, and diabetic diseases, and / or the degree of progression of these diseases from changes in the ratio of pathologically active adiponectin to physiologically active adiponectin and changes in the ratio of physiologically active adiponectin to total adiponectin, which is the sum of the amount of physiologically active adiponectin and the amount of pathologically active adiponectin. This diagnostic method also includes the steps of using a protein standard, a healthy body fluid sample, etc. as a standard to prepare a calibration curve, a comparison table, etc., and comparing the quantitative results of the specimen sample to be diagnosed with the calibration curve, comparison table, etc. for use in diagnosis. It can also be used to determine and diagnose the onset, risk of onset, and / or progression of obesity-related diseases such as hypertension and sleep apnea syndrome. [Example]

[0042] Examples are shown below, but the present invention is not limited to these examples.

[0043] (Summary of adiponectin quantification method) 1 shows an outline of the method for quantifying adiponectin used in the Examples. Figure 1(a) shows a schematic diagram of the detection of physiologically active adiponectin. Physiologically active adiponectin is bound to T-cadherin, which can interact with this adiponectin. The adiponectin site in the complex of this physiologically active adiponectin and T-cadherin is quantified by immunoassay using anti-adiponectin. Figure 1(b) shows a schematic diagram of the detection of pathologically active adiponectin. Pathologically active adiponectin is bound to denatured low-density lipoprotein (0xLDL), which can interact with this adiponectin. Furthermore, LOX-1, which can interact with this denatured low-density lipoprotein, is bound to the denatured low-density lipoprotein. The adiponectin site in the complex of this pathologically active adiponectin, denatured low-density lipoprotein, and LOX-1 protein is quantified by immunoassay using anti-adiponectin.

[0044] (Quantification of physiologically active adiponectin) First, we examined the binding of physiologically active adiponectin to T-cadherin. The human T-cadherin gene was cloned and inserted into the pSeqTag2 vector (Invitrogen) for use in a protein expression system. Protein expression was carried out using the Expi293 Expression System (Invitrogen), and the fusion protein secreted into the medium was purified with Ni Sepharose excel resin (GE). The resulting protein was dialyzed in PBS, sterilized by filtration, and then used in experiments. Purified T-cadherin protein was immobilized on an ELISA plate, and adiponectin recombinant protein was bound to it. The bound adiponectin was detected with an anti-adiponectin antibody.

[0045] The results are shown in Figure 2. For the ELISA plate on which T-cadherin was immobilized, the amount of binding increased depending on the concentration of adiponectin added. In other words, it was demonstrated that the concentration of physiologically active adiponectin that binds to T-cadherin in a sample can be quantified using the plate on which T-cadherin was immobilized.

[0046] Next, the concentration of physiologically active adiponectin in the human serum samples was quantified. Immobilization on an ELISA plate and detection were carried out under the following conditions. Immobilization: T-cadherin protein (139-692aa) 0.15μg / well Blocking: 1% casein-Na, HEPES-NaCl, 2hr, room temperature Ligand: adiponectin in HEPES-NaCl, 2 hours at room temperature, serum in HEPES-NaCl Detection: Primary antibody anti-adiponectin antibody (rabbit polyclonal; BioVendor #RD181023100) 1μg / ml in 1% casein-Na / HEPES-NaCl for 1hr at room temperature Secondary antibody anti-rabbit IgG-HRP, 1:4000, in 1% casein-Na / HEPES-NaCl, 1 hr at room temperature

[0047] The results are shown in Figure 3. The amount of binding increased depending on the serum concentration, i.e., the concentration of adiponectin in the sample, demonstrating that the concentration of physiologically active adiponectin can be quantified.

[0048] Quantification was also performed under the same conditions as above, except that plasma containing EDTA was used as the sample. Figure 4 shows a comparison of the binding amounts between serum and plasma (EDTA-Plasma). The amount of binding was reduced in plasma containing EDTA. This is because, as previously reported, the binding between T-cadherin and adiponectin is calcium-dependent (Hug C, Proceedings of the National Academy of Sciences of the United States of America, 2004). The fact that binding was inhibited in the presence of EDTA, a calcium inhibitor, indicates that the previously known binding of T-cadherin and adiponectin occurs in the serum. These results demonstrate that it is possible to detect and quantify physiologically active adiponectin in blood samples that binds to T-cadherin using ELISA.

[0049] (Quantification of pathologically active adiponectin) Next, we examined the binding of adiponectin, which has pathological activity, to denatured LDL. LDL isolated from human plasma was oxidized in a test tube in the presence of copper ions to produce oxidized LDL, a modified form of LDL. This oxidized LDL was mixed with recombinant adiponectin protein, and the detectability of the complex between the two was examined by sandwich ELISA using the modified LDL receptor LOX-1 and anti-adiponectin antibodies. Human LOX-1 was expressed in the same protein expression system as described above, and purified recombinant LOX-1 was used.

[0050] Figure 5 shows the results of quantification of a mixed solution of purified adiponectin and oxidized LDL using an anti-adiponectin antibody. The detection of purified adiponectin increased not only depending on the concentration of purified adiponectin (AdN) but also depending on the concentration of oxidized LDL (oxLDL). This demonstrates that the complex of oxidized LDL and adiponectin can be detected by ELISA.

[0051] Next, the concentration of adiponectin having pathological activity in human serum samples was quantified. Serum samples used were blood serum from healthy individuals and blood serum from patients with arteriosclerotic disease purchased from a supplier. Immobilization on the ELISA plate and detection were carried out under the following conditions. Solid phase: LOX-1 0.15μg / well Blocking: 1% casein-Na in HEPES-NaCl, 2 hours at room temperature Ligand: Oxidized LDL adiponectin complex in HEPES-NaCl-2mM EDTA, 2hr at room temperature Detection: Primary antibody anti-adiponectin antibody (rabbit polyclonal) 1μg / ml in 1% casein-Na / HEPES-NaCl for 1hr at room temperature Secondary antibody anti-rabbit IgG-HRP, 1:4000, in 1% casein-Na / HEPES-NaCl, 1 hr at room temperature

[0052] The results are shown in Figure 6. Compared with the serum of healthy subjects, the serum of arteriosclerotic disease patients showed higher levels of the LOX1-oxidized LDL-pathologically active adiponectin complex as quantified by anti-adiponectin antibody.

[0053] Figure 7 shows the results of detecting LOX-1-bound modified LDL using the HUC20 antibody as the detection antibody. As in Figure 6, the values were higher in patients with arteriosclerotic disease than in healthy sera. The patient whose serum #1 showed high values in Figures 6 and 7 is predicted to have more advanced arteriosclerotic disease than the patient whose serum #2 showed high values. These results indicate that this test can be used to determine arteriosclerotic disease or its progression.

[0054] (Reference test) Figure 8 shows the results of detecting LDL fractionated from human plasma with an anti-adiponectin antibody. Among the LDL fractions, subfraction L5 is an electronegative LDL fraction that corresponds to denatured LDL. The figure shows that adiponectin is concentrated in subfraction L5, suggesting the possibility that denatured LDL binds to adiponectin.

[0055] Figure 9(a) shows the results of detecting AMPK and its phosphorylated form (pAMPK) using an anti-adiponectin antibody, and Figure 9(b) shows the amount detected. The amount of pAMPK detected by the anti-adiponectin antibody decreased in an oxLDL concentration-dependent manner, suggesting that oxidized LDL may inhibit the phosphorylation of AMPK by adiponectin. These results suggest that oxidized LDL may act on adiponectin, inhibiting its function and converting physiologically active adiponectin into pathologically active adiponectin. [Industrial Applicability]

[0056] According to the present invention, there are provided a method for quantifying adiponectin, which enables accurate understanding of the physiological activity of adiponectin and is useful for assessing the risk of cardiovascular disease, lifestyle-related disease, etc., and an analytical reagent for use in the method. The method and analytical reagent can be used, in particular, to determine the risk or progression of cardiovascular disease, diabetes, and diabetic diseases.

Claims

1. In quantifying adiponectin in body fluids, adiponectin having physiological activity capable of binding to an adiponectin receptor; and adiponectin having a pathological activity in which the physiological activity defined by binding to the adiponectin receptor is reduced, and The method for quantifying adiponectin, wherein the body fluid is blood.

2. The method for quantifying adiponectin according to claim 1, wherein T-cadherin proteins are used to quantify adiponectin having physiological activity defined by binding to the adiponectin receptor in the quantification of adiponectin in the body fluid.

3. 3. The method for quantifying adiponectin according to claim 2, wherein the T-cadherin proteins are recombinant T-cadherin proteins.

4. The method for quantifying adiponectin according to any one of claims 1 to 3, wherein in quantifying adiponectin in the body fluid, the quantification of adiponectin having physiological activity defined by binding to the adiponectin receptor is carried out by immunoassay of a complex of adiponectin, T-cadherin proteins, and an anti-adiponectin antibody.

5. A method for quantifying adiponectin according to any one of claims 1 to 4, wherein in quantifying adiponectin in the body fluid, the adiponectin having pathological activity is quantified using a conjugate of adiponectin and denatured low density lipoprotein.

6. 6. The method for quantifying adiponectin according to claim 1, wherein LOX-1 protein is used to quantify adiponectin having pathological activity in the quantitative determination of adiponectin in a body fluid.

7. 7. The method for quantifying adiponectin according to claim 6, wherein the LOX-1 protein in the body fluid is a recombinant LOX-1 protein.

8. The method for quantifying adiponectin according to claim 7, wherein in quantifying adiponectin in the body fluid, the adiponectin having pathological activity is quantified by immunoassay using the recombinant LOX-1 protein to quantify its binding activity to an adiponectin receptor.

9. The method for quantifying adiponectin according to claim 8, wherein the amount of adiponectin having pathological activity in the body fluid is quantified by using the recombinant LOX-1 protein and quantifying its binding activity to an adiponectin receptor by immunoassay of a complex of an anti-adiponectin antibody, adiponectin, and denatured low density lipoprotein.

10. The method for quantifying adiponectin according to any one of claims 1 to 9, wherein in quantifying adiponectin in the body fluid, T-cadherin proteins are used to quantify adiponectin having physiological activity defined by binding to the adiponectin receptor, and recombinant LOX-1 protein is used to quantify adiponectin having pathological activity, and the binding activity to the adiponectin receptor is quantified by immunoassay.

11. The method for quantifying adiponectin according to any one of claims 1 to 10, wherein in quantifying adiponectin in the body fluid, T-cadherin proteins are used to quantify adiponectin having physiological activity defined by binding to the adiponectin receptor, and a conjugate of adiponectin with denatured low density lipoprotein and recombinant LOX-1 protein are used to quantify adiponectin having pathological activity, and the binding activity to the adiponectin receptor is quantified by anti-adiponectin immunoassay.

12. An analytical reagent used to quantify adiponectin in body fluids for determining cardiovascular disease, diabetes, or diabetic disease, or the stage of progression thereof, comprising: a recombinant T-cadherin protein for quantifying physiologically active adiponectin capable of binding to an adiponectin receptor; An analytical reagent, wherein the body fluid is blood.

13. An analytical reagent used to quantify adiponectin in body fluids for determining cardiovascular disease, diabetes, or diabetic disease, or the stage of progression thereof, comprising: a recombinant LOX-1 protein for quantifying adiponectin having reduced physiological activity and pathological activity, which can bind to an adiponectin receptor; The binding activity of adiponectin to the adiponectin receptor was quantified by anti-adiponectin immunoassay for the complex of adiponectin and denatured low density lipoprotein. An analytical reagent, wherein the body fluid is blood.

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