JP5593502B2 - Method for measuring chemerin concentration - Google Patents

Description

  The present invention relates to a new diagnostic method for diabetes.

  Diabetes is a disease of abnormal glucose metabolism, and the number of patients is expected to reach 200 million worldwide. Diabetes is roughly classified into type 1 and type 2. Type 1 diabetes, also known as insulin-dependent diabetes, is a disease in which sugar in the blood increases abnormally because insulin secretion declines because the β-cells that secrete insulin in the pancreatic islets of Langerhans die. . Treatment of type 1 diabetes is by insulin supplementation.

  On the other hand, type 2 diabetes is insulin-independent and is caused by a decrease in insulin secretion and a decrease in insulin sensitivity. It is said that obesity, overeating, lack of exercise, stress, etc. are greatly involved in the onset of type 2 diabetes, and basic diet therapy and exercise therapy are fundamental. However, the cause of the onset of type 2 diabetes has not yet been fully elucidated, so early detection and early treatment are important, but the current situation is that early detection is not possible.

  Under such circumstances, diagnosis of diabetes, particularly type 2 diabetes is important, and development of new diagnostic means is desired.

On the other hand, Chemerin was reported as a gene induced by retinoic acid in fibroblasts as TIG2 (Tazarotene-induced gene2) in 1997, but the details of its function and structure were unknown. . Thereafter, it was reported that ChemR23, which had been cloned as an Orphan G protein coupled receptor, had TIG2 as a ligand. TIG2 has activity at 21 to 157 amino acid residues, and this active part was named chemarin (Non-patent Documents 1 to 3).
However, there is no report about the relationship between this chemerin and diabetes.
J. et al. Invest. Dermatol. 1997; 109 (1): 91-95. J. et al. Exp. Med. 2003; 198 (7): 977-985 FEBS Lett. 2003; 555 (3): 495-499.

  An object of the present invention is to provide a new diagnostic means for diabetes, particularly a diagnostic means for type 2 diabetes.

  Therefore, the present inventors measured the blood concentration of chemerin whose function is not clear, and examined the relationship with various diseases. As a result, the chemerin concentration and type 2 diabetes have a significant correlation, It was found that diabetes can be diagnosed by measuring the chemerin concentration, and the present invention was completed.

That is, the present invention provides a method for diagnosing diabetes characterized by measuring a chemerin concentration in a sample.
The present invention also provides a diagnostic agent for diabetes containing a chemerin concentration measuring reagent.

  According to the present invention, it is possible to diagnose or determine diabetes, particularly type 2 diabetes. Accordingly, early detection of type 2 diabetes enables early treatment.

The expression of chemerin (Northern blotting) in 3T3L1 cells is shown. AC is chemerin. The northern blotting result in a mouse | mouth tissue is shown. AC is chemerin. The immunoblotting result by the anti-chemarine antibody in 3T3L1 adipocyte culture supernatant is shown. In FIG. 3, mAC1 is mouse chemerin, and rhAC1 is recombinant human chemerin. The result of having measured serum in the ELISA system which used D0101 as a solid-phase antibody and PoAb and D0102 as a labeled antibody, respectively is shown. The results of measuring plasma in an ELISA system using D0101 as a solid phase antibody and PoAb and D0102 as labeled antibodies are shown. It is a figure which shows the ELISA result (Superdex200 (PHI) 1.0cm * 60cm) in the serum which carried out gel filtration purification.

BEST MODE FOR CARRYING OUT THE INVENTION

  The present inventors have identified chemerin as a gene encoding a secreted protein whose expression increases with adipogenesis using adipocytes. Chemamarin was found to be strongly expressed with adipose differentiation by Northern blotting. In addition, chemerin is strongly expressed in the liver and adipose tissue, and is secreted into the culture supernatant of adipocytes, so it has been clarified that it is one of the adipokines secreted from adipocytes.

  In the present invention, the measurement includes quantitative or non-quantitative measurement. For example, as non-quantitative measurement, it is simply measured whether or not chemerin is present, whether or not chemerin is present in a certain amount or more. Measurement of comparing the amount of chemerin with other samples (for example, control samples, etc.). Examples of quantitative measurement include measurement of the concentration of chemerin, measurement of the amount of chemerin, and the like.

  The sample is not particularly limited as long as it may contain chemerin, but a sample collected from the body of an organism such as a mammal is preferable, and a sample collected from a human is more preferable. Specific examples of the sample include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, intestinal tissue and the like. Preferred are blood, serum and plasma.

  A sample is collected from a patient, the chemerin concentration of the sample is measured, and diabetes can be diagnosed if the value is higher than the reference value obtained from the distribution of the chemerin concentration of a healthy person.

  In the method of the present invention, the chemerin concentration is preferably measured by an immunological assay using an anti-chemalin antibody. Hereinafter, the measurement method using an anti-chemalin antibody will be described in detail.

  The anti-chemalin antibody used in the present invention may specifically bind to chemerin. The origin, type (monoclonal, polyclonal) and shape of the antibody are not limited. Specifically, known antibodies such as mouse antibodies, rat antibodies, human antibodies, chimeric antibodies, humanized antibodies can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.

  Further, in the immunoassay, the anti-chemalin antibody immobilized on the support and the anti-chemalin antibody labeled with the labeling substance may recognize the same or different epitopes of the chemerin molecule.

  The anti-chemarine antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-chemamarin antibody used in the present invention, a mammal-derived monoclonal antibody is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.

  A monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, chemerin is used as a sensitizing antigen and immunized according to a normal immunization method. The obtained immune cells are fused with a known parent cell by a normal cell fusion method, and monoclonal antibodies are obtained by a normal screening method. It can be produced by screening antibody-producing cells.

Specifically, the monoclonal antibody can be produced as follows.
First, chemerin used as a sensitizing antigen for antibody acquisition is obtained by purifying from available cell culture supernatants.
Next, this purified chemerin is used as a sensitizing antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, others, rabbits, monkeys and the like are used.

  Immunization of animals with a sensitizing antigen can be performed according to a known method. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is dosed several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize by binding to a carrier protein such as albumin or keyhole limpet hemocyanin.

  Thus, after immunizing a mammal and confirming that the desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Cell.

  Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeloma cell is known in various known cell lines such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Travebridge, I.S. J. Exp. Med. (1978) 148, 313-323), R210. (Galfre G.et al., Nature (1979) 277,131-133) and the like are preferably used.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3- 46) and the like.

  More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency if desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used. Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.

  For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a polyethylene glycol (PEG) (for example, an average molecular weight of about 1000 to 6000) solution that is preliminarily heated to about 37 ° C. is usually 30. The target fusion cell (hybridoma) is formed by adding at a concentration of ˜60% (w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution such as a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. Subsequently, the usual limiting dilution method is performed, and the hybridoma producing the target antibody is screened and single-cloned.

  Screening and single cloning of the target antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, an antigen is bound to a carrier such as beads made of polystyrene or a commercially available 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, washed with the carrier, and then reacted with an enzyme-labeled secondary antibody or the like. Thus, it can be determined whether or not the target antibody reacting with the sensitizing antigen is contained in the culture supernatant. A hybridoma producing the target antibody can be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used.

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen. Examples of the hybridoma include D0101 and D0102. On December 9, 2008, these hybridomas were transferred to the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) in Japan (Corporate No. 1-1, Higashi 1-chome, Tsukuba City, Ibaraki 305-8586, Japan). As D11069, D0102 was deposited as FERM ABP-11070, respectively.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites is obtained. The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

  Further, these antibodies may be low molecular weight antibodies such as antibody fragments (fragments), modified antibodies, etc., as long as they do not lose the property of recognizing the full length or part of the protein encoded by the chemerin gene . Specific examples of the antibody fragment include Fab, Fab ', F (ab') 2, Fv, Diabody, and the like. Such antibody fragments can be obtained by digesting the Fc part of IgG with pepsin or papain, constructing genes encoding these antibody fragments, introducing them into expression vectors, and expressing them in appropriate host cells. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663, Rouseseaux, J. et al. , Method . Enzymol (1986) 121,663-669; Bird, R.E.and Walker, B.W., see Trends Biotechnol (1991) 9,132-137)..

  The antibody produced as described above can be isolated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Manufactured by GE Healthcare). In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining a chromatography column other than the affinity column, filter, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane) , Cold Spring Harbor Laboratory, 1988).

  The method for detecting chemerin contained in the sample is not particularly limited, but it is preferably detected by an immunological method using an anti-chemalin antibody. Examples of the immunological method include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, etc. An immunoassay, particularly preferred is an enzyme-linked immunosorbent assay (ELISA) (eg a sandwich ELISA). The above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.

  In these immunological measurements, it is preferable to use monoclonal antibodies for both or either of the immobilized antibody and the labeled antibody. When using monoclonal antibodies for both, it is desirable to use a combination of monoclonal antibodies that recognize different sites. Monoclonal antibodies can be used on one side and polyclonal antibodies on the other side. As the monoclonal antibody used in such a measurement system, the monoclonal antibody produced by the hybridoma D0101 or D0102 is particularly preferable. Furthermore, a measurement system using D0101 or a polyclonal antibody as a solid-phased antibody and D0102 as a labeled antibody is particularly preferable because of high detection sensitivity.

  As a general detection method using an anti-chemalin antibody, for example, an anti-chemalin antibody is immobilized on a support, a sample is added thereto, incubation is performed, the anti-chemalin antibody and the chemerin are bound, washed, A method of detecting chemerin in a sample by detecting chemerin bound to a support via a chemerin antibody can be mentioned.

  Examples of the support used in the present invention include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resins, polystyrene resins, polyacrylamide resins, nylon resins, and polycarbonate resins, and insoluble supports such as glass. Can be mentioned. These supports can be used in the form of beads or plates. In the case of beads, a column packed with these can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used. The anti-chemalin antibody and the support can be bound by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be commercially available.

  The binding between the anti-chemalin antibody and chemerin is usually performed in a buffer solution. As the buffer solution, for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like are used. Incubation is performed under conditions that are already often used, for example, incubation at 4 ° C. to room temperature for 1 to 24 hours. Washing after incubation is not particularly limited as long as it does not interfere with the binding between the chemerin and the anti-chemalin antibody. For example, a buffer containing a surfactant such as Tween 20 is used.

  In the chemerin measurement method of the present invention, a control sample may be installed in addition to the sample for which chemerin is to be detected. Examples of the control sample include a negative control sample not containing chemerin and a positive control sample containing chemerin. In this case, it is possible to detect the chemerin in the sample by comparing the result obtained with the negative control sample not containing chemerin and the result obtained with the positive control sample containing chemerin. In addition, a series of control samples with varying concentrations are prepared, detection results for each control sample are obtained as numerical values, a standard curve is created, and samples are included based on the standard curve from the sample values. It is also possible to detect chemerin quantitatively.

As a preferred embodiment of the measurement of the chemerin bound to the support through the anti-chemalin antibody, there can be mentioned a method using an anti-chemalin antibody labeled with a labeling substance.
For example, the sample is brought into contact with an anti-chemalin antibody immobilized on a support, and after washing, detection is performed using a labeled antibody that specifically recognizes chemerin.

The anti-chemalin antibody can be labeled by a generally known method. As the labeling substance, labeling substances known to those skilled in the art such as fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, radioactive substances and the like can be used. As specific examples, radioisotopes ( ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide Examples include oxidase, microperoxidase, and biotin. When biotin is used as the labeling substance, it is preferable to further add avidin to which an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody. A known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method, and periodic acid method can be used for the binding of the labeling substance and the anti-chemarine antibody.

  Examples of enzyme labeling methods for antibodies include, but are not limited to, the hinge method and the non-hinge method. In the hinge method, Fab ′ and an enzyme molecule are separated from each other by using a thiol group generated by reducing a disulfide bond in a portion called a hinge portion between an F (ab ′) 2 portion having an antigen-binding ability of antibody IgG. It is a way to combine. On the other hand, the non-hinge method does not specify which reactive group of the antibody is used, but in many cases, it is a method of binding the antibody molecule and the enzyme molecule using the amino group of the antibody.

  Specifically, a solution containing an anti-chemalin antibody is added to a support such as a plate, and the anti-chemalin antibody is fixed to the support. After washing the plate, it is blocked with, for example, BSA, gelatin, albumin or the like in order to prevent nonspecific binding of proteins. Wash again and add sample to plate. After incubation, wash and add labeled anti-chemalin antibody. After moderate incubation, the plate is washed and the labeled anti-chemalin antibody remaining on the plate is detected. Detection can be performed by methods known to those skilled in the art. For example, in the case of labeling with a radioactive substance, it can be detected by liquid scintillation or RIA. In the case of labeling with an enzyme, a substrate is added, and an enzymatic change of the substrate, for example, color development, can be detected with an absorptiometer. Specific examples of the substrate include 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine), 3,3 ′ , 5,5′-tetramethylbenzidine (TMB) and the like. In the case of a fluorescent substance, it can be detected by a fluorometer.

  As a particularly preferred embodiment of the chemerin measurement method of the present invention, there is a method using an antibody labeled with a method described in a general enzyme labeling method after removing an Fc portion unrelated to the antigen-binding ability of antibody IgG. it can.

  Specifically, a solution containing an anti-chemalin antibody is added to a support such as a plate, and the anti-chemalin antibody is immobilized. After washing the plate, it is blocked with, for example, BSA in order to prevent non-specific protein binding. Wash again and add sample to plate. After incubation, wash and add peroxidase directly labeled anti-chemalin antibody. After moderate incubation, the plate is washed, a substrate corresponding to the enzyme is added, and chemerin is detected using an enzymatic change of the substrate as an indicator.

  As another embodiment of the chemerin measurement method of the present invention, there can be mentioned a method using one or more primary antibodies specifically recognizing chemerin and one or more secondary antibodies specifically recognizing the primary antibody. .

  The measurement method of the present invention can be automated using various automatic inspection apparatuses, and a large number of samples can be inspected at a time.

  The diagnostic agent of the present invention contains a chemerin measuring reagent. The chemerin measuring reagent includes components necessary for the chemerin measuring method, for example, an anti-chemalin antibody. Here, the diagnostic agent includes a kit. When the diagnostic agent is based on the ELISA method, it may contain a carrier for immobilizing the antibody, and the antibody may be bound to the carrier in advance. When the diagnostic agent is based on an agglutination method using a carrier such as latex, it may contain a carrier to which an antibody is adsorbed. In addition, the diagnostic agent may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

Reference example 1
A 3T3L1 adipocyte was used as a gene encoding a secreted protein whose expression increases with adipogenesis, and a chemerin was identified by searching using a differential display method.

  At the start of differentiation induction, total RNA was extracted from 3T3-L1 adipocytes 2 days, 6 days, and 9 days after induction. When 15 μg of this RNA was electrophoresed and Northern blotting was performed using a mouse chemerin-specific probe, strong expression enhancement was observed with adipose differentiation (FIG. 1).

  When 15 μg of Total RNA extracted from mouse tissue was electrophoresed and analyzed by Northern blotting using a mouse chemerin specific probe, strong expression was observed in adipose tissue in addition to liver (FIG. 2).

  SDS-PAGE was performed using 10 μL of 3T3-L1 culture supernatant and 15 ng of recombinant human chemerin, and immunoblotting was performed using a human chemerin-specific polyclonal antibody. Chemamarin is secreted into the 3T3L1 adipocyte culture supernatant (FIG. 3), and was found to be one of the adipokines secreted from adipocytes.

Example 1
(1) Recombinant human chemerin preparation method The human chemerin gene was amplified by PCR from human adipose tissue cDNA (Clontech) and cloned into the expression vector pET14b vector (Novagen). The pET14b vector into which the human chemerin gene was cloned was transformed into Escherichia coli BL21 (DE3) pLysS strain (manufactured by Takara Bio Inc.) to obtain Escherichia coli expressing recombinant human chemerin. This Escherichia coli was cultured, and the extract was purified using a HisTrap HP column (manufactured by GE Healthcare Life Sciences) to obtain a recombinant human chemerin.

(2) Method for preparing mouse anti-recombinant human chemerin monoclonal antibody This recombinant human chemerin was mixed with TiterMax Pro (TiterMax), emulsified, and then subcutaneously administered to mice every 14 days.
The spleen was removed from the mouse and immune cells in the spleen and mouse myeloma cells P3x63Ag8U. 1 and cell fusion.
Cell fusion is performed by mixing 50 ml of polyethylene glycol (PEG) with an average molecular weight of 1500, which is premixed in RPMI1640 culture medium so that the amount of the immune cells is 5 times the amount of myeloma cells, and heated to 37 ° C. The target fusion cell (hybridoma) was formed by adding at a concentration of w / v) and mixing.
The cells were cultured in an RPMI 1640 medium containing HAT (hypoxanthine, aminopterin and thymidine) for 1 week. Subsequently, a normal limiting dilution method was performed, and screening and single cloning of hybridomas producing the target antibody were performed.
Screening and single cloning of the target antibody were carried out by the methods shown below. Recombinant human chemerin used as an immunizing antigen was bound to a 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, washed with the carrier, and then reacted with horseradish peroxidase-labeled anti-mouse IgG antibody. It was determined whether any antibodies that react with human chemarin were included. The hybridoma producing the target antibody was cloned by the limiting dilution method.
The hybridoma was cultured using a Hybridoma-SFM medium (manufactured by Invitrogen) at 37 ° C. under 5% carbon dioxide, and the culture supernatant was collected. The culture supernatant was concentrated by salting out with ammonium sulfate. After exchanging the concentrated solution with phosphate buffered saline, the mouse was used using a protein A column (GE Healthcare Bioscience) and a Sephacryl S-300 gel filtration column (GE Healthcare Bioscience). Anti-human chemerin monoclonal antibody was purified.
The hybridoma obtained here is designated as D0101 or D0102, and on December 9, 2008, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1, Higashi 1-chome, Tsukuba, Ibaraki 305-8586, Japan) In the middle 6), D0101 was deposited as FERM ABP-11069 and D0102 was deposited as FERM ABP-11070.

(3) Rabbit anti-human chemerin polyclonal antibody production method Recombinant human chemerin was mixed with Freund's complete adjuvant, emulsified, and then subcutaneously administered to rabbits 5 times every 14 days. Recombinant human chemerin used as an immunizing antigen was bound to a 96-well microtiter plate, reacted with antiserum collected from the immunized rabbit, washed with the carrier, and then reacted with an enzyme-labeled anti-rabbit IgG antibody. It was confirmed that an antibody that reacts with human chemerin was contained in the antiserum. After confirming that the level of anti-human chemerin antibody increases in the serum as described above, rabbit serum was collected, and a protein A column (manufactured by GE Healthcare Biosciences) and a Sephacryl S-300 gel filtration column (GE Healthcare Biosciences). Rabbit anti-human chemerin polyclonal antibody (PoAb) was purified.

(4) Measurement of chemerin concentration Serum collected from a patient or the like was diluted 100 times with a buffer solution to obtain a diluted specimen solution. A standard solution containing this diluted specimen solution and a recombinant human chemerin having a predetermined concentration was added to a 96-well microtiter plate on which a mouse anti-human chemerin monoclonal antibody D0102 was solid-phased, and then allowed to stand for 2 hours. Was bound to the mouse anti-human chemerin monoclonal antibody on the wells. After washing the well, a biotin-labeled anti-human chemerin polyclonal antibody solution was added, and then allowed to stand for 1 hour. After washing the wells, a horseradish peroxidase-labeled streptavidin solution was added and allowed to stand for 1 hour. A substrate solution containing 3,3 ′, 5,5′-tetramethylbenzidine and hydrogen peroxide was added to develop color for 10 minutes, the absorbance was measured at 450 nm, and the human chemerin in the sample was determined from the absorbance of the standard solution measured at the same time. Concentration was calculated.

  Table 1 shows the measurement results of blood chemerin concentration for 172 healthy subjects and 170 patients with type 2 diabetes.

  As shown in Table 1, the blood chemerin concentration of diabetic patients was statistically significantly lower than that of healthy subjects. Therefore, measuring the blood chemerin concentration is useful as an index for diabetes diagnosis.

Example 2
A sandwich ELISA measurement system was constructed by combining two types of antibodies from three types of rabbit anti-human chemerin polyclonal antibody (PoAb) and mouse anti-human chemerin monoclonal antibody (D0101 or D0102). The detection sensitivity was compared.
That is, as a primary reaction, recombinant human chemerin was diluted 2-fold from 5000 pg / mL, and allowed to react on a plate on which an antibody had been solid-phased at 25 ° C. for 2 hours. Subsequently, it prepared so that it might become 1 ug / mL of each antibody labeled with biotin as secondary reaction, and it was made to react by leaving still at 25 degreeC for 2 hours. Next, as a tertiary reaction, HRP-labeled streptavidin was prepared so that the absorbance in the blank was 0.05 or less, and allowed to react at 25 ° C. for 1 hour.

  As a result, as shown in Table 2, it was possible to measure chemerin in any combination. Among these, the measurement system using D0102 as the labeled antibody was about 100 times more sensitive than the case where the polyclonal antibody was used as the labeled antibody.

Example 3
Twenty cases of human plasma or serum were measured in an ELISA measurement system using D0101 as a solid phase antibody and PoAb and D0102 as labeled antibodies, respectively. As a result, a good correlation of R ≧ 0.6 was shown (FIGS. 4 and 5).
The slope of the regression line of the monoclonal antibody measurement system / evaluation kit was 1.77 to 2.04, and there was a tendency for the monoclonal antibody measurement system to be high.

  As a result of measuring the fraction obtained by gel filtration of human Pool serum in an ELISA measurement system using D0101 as a solid phase antibody and PoAb and D0102 as labeled antibodies, both measurement systems recognized almost the same fraction. (Fig. 6).

Claims (5)

A method of measuring the concentration of chemarin in blood, serum or plasma for the purpose of diagnosing diabetes . Measurement of chemerin concentration measuring method according to claim 1, wherein the measurement by immunological methods. Measurement method according to claim 1 or 2, wherein diabetes is type 2 diabetes. The measurement method according to any one of claims 1 to 3, wherein the diagnosis of diabetes is a diagnosis of diabetes itself and / or a diagnosis of the course of diabetes treatment. A diagnostic agent for diabetes containing a reagent for measuring the concentration of chemerin in blood, serum or plasma .

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