JP2010241744A - Antibody to Usia diponectin and method for measuring Usia diponectin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means and method for measuring bovine adiponectin, which must utilize endogenous glucose instead of ingested glucose due to the presence of rumen.
The present invention is obtained by immunizing a rabbit with a peptide having an amino acid sequence that may have a deletion, substitution or addition of one or several amino acids in the amino acid sequence of ussia diponectin, An anti-ussia diponectin antibody characterized by having the reactivity of
[Selection] Figure 1

Description

  The present invention relates to antibodies against ussia diponectin. The present invention also relates to a reagent for detecting siasia diponectin containing the antibody and a method for detecting siasia diponectin using the antibody.

  Adipocyte complement-related protein (adipocyte complement-related protein) is a hormone that is secreted from fat cells and involved in glucose metabolism. Adiponectin is a secreted protein consisting of 244 amino acids, has a 66 amino acid collagen-like motif, and has homology with C1q and collagens X and VII of the complement system (Non-patent Document 1). There are various effects such as increased insulin sensitivity, suppression of arteriosclerosis, anti-inflammation, suppression of myocardial hypertrophy (Non-patent Document 2). The adiponectin concentration in the blood of humans and mice can be measured using a known immunological assay or a commercially available ELISA kit (Patent Document 1), but the assay or kit is applied to other animals. It is not possible.

  Cows, on the other hand, must use endogenous glucose rather than ingested glucose because of the characteristic ruminant presence. For this reason, adiponectin, which is involved in glucose metabolism, seems to be deeply involved in cattle. However, no means or method has been found for detecting Usia diponectin. For this reason, the physiological action of bovine adiponectin has not been clarified, and a measuring instrument for detecting and quantifying bovine adiponectin simply and with high sensitivity has been desired.

WO2005 / 038458 pamphlet

K. Maeda, K. Okubo, I. Shimomura, T. Funahashi, Y. Matsuzawa and K. Matsubara, Biochem. Biophys. Res. Commun. Vol.221 pp.286-289, 1996 Y. Arita, S. Kihara, N. Ouchi, K. Maeda, H. Kuriyama, Y. Okamoto, M. Kumada, K. Hotta, M. Nishida, M. Takahashi, T. Nakamura, I. Shimomura, M. Muraguchi, Y. Ohmoto, T. Funahashi, Y. Matsuzawa, Circulation. 105, 2893-2898, 2002

  The object of the present invention is to provide means and methods for measuring Usia diponectin.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have succeeded in producing an antibody that recognizes ussia diponectin in rabbits, and by using this antibody, siasia diponectin is highly sensitive, convenient and specific. The present invention has been completed.

That is, this invention is the following (1)-(4).
(1) It is obtained by immunizing a rabbit with a peptide having an amino acid sequence which may have deletion, substitution or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, and bovine An anti-ussia diponectin antibody characterized by having reactivity with adiponectin.
The antibody may be a polyclonal antibody or a monoclonal antibody.
(2) A method for detecting ussia diponectin, comprising contacting a bovine-derived sample with the antibody, and detecting binding of ussia diponectin and the antibody in the sample.
(3) A method for purifying Usa diponectin, which comprises contacting a ussia diponectin-containing sample with the antibody, and dissociating ussia diponectin bound to the antibody.
(4) A reagent for detecting siasia diponectin, comprising the antibody.

  The present invention provides an antibody that reacts with ussia diponectin. By using such an antibody, ussia diponectin in a sample can be detected with high sensitivity, convenience and specificity. This detection result is useful for elucidation of bovine sugar metabolism. Further, by using the present invention, it is possible to purify siasia diponectin from a sample.

It is a calibration curve (signal versus concentration) generated using assay signals obtained with known standard solutions of adiponectin.

  The present invention provides an antibody that recognizes and reacts with ussia diponectin. Although a partial peptide of mouse adiponectin is used for the production of the antibody, the antibody according to the present invention is characterized by having reactivity with siadiadiponectin, unlike the conventional antibody.

1. Anti-Ussia diponectin antibody The anti-Ussia diponectin antibody according to the present invention can be produced by immunizing a rabbit using a partial peptide of mouse adiponectin as an antigen.

  As an antigen, a partial peptide of mouse adiponectin is used. The nucleotide sequence of mouse adiponectin is registered in GenBank with accession number U37222, and the amino acid sequence is registered with accession number AAA80543. Specifically, a peptide consisting of amino acids 111 to 247 (SEQ ID NO: 1) in the amino acid sequence of mouse adiponectin shown in SEQ ID NO: 3 is used. In the present invention, the peptide used as an antigen has one or several amino acids deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 1 as long as it has antigenicity for raising an antibody against ussia diponectin. It may be a peptide consisting of an amino acid sequence. For example, 1 to 5, preferably 1 to 3 amino acids of the amino acid sequence shown in SEQ ID NO: 1 may be deleted, and 1 to 5, preferably 1 to 1, are added to the amino acid sequence shown in SEQ ID NO: 1. Three amino acids may be added, or one in which 1 to 5, preferably 1 to 3 amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted with other amino acids may be used as an antigen. it can. In particular, it is preferable that one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are conservatively substituted. A “conservative substitution” is known in the art and refers to the replacement of an amino acid with an amino acid that exhibits similar properties to that amino acid. Further, for example, a peptide comprising an amino acid sequence showing at least 80% or more, preferably 90% or more, particularly preferably 95% or more of the sequence homology or identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is also used as an antigen. be able to. The homology or identity of amino acid sequences can be easily determined by methods known in the art.

  Antigenicity for raising an antibody against ussia diponectin means an ability as an antigen capable of producing an anti-ussia diponectin antibody and an ability to react with an anti-ussia diponectin antibody. Whether or not a certain peptide has the antigenicity can be confirmed by preparing an antibody against the peptide and detecting whether or not the prepared antibody reacts with a full-length (wild type) siadiponectin.

  The antigen may be chemically synthesized based on the amino acid sequence shown in SEQ ID NO: 1 or generated by transforming a host with a nucleic acid encoding it and recovering the peptide expressed in the host can do.

  In the case of chemical synthesis, according to a known peptide synthesis method, for example, a peptide for antigen can be synthesized using a commercially available peptide synthesizer or a commercially available peptide synthesis kit. When using a genetic recombination technique, a nucleic acid encoding an antigen is prepared according to a method known in the art and linked to an expression vector so that the target antigen peptide can be expressed in a host cell. An antigenic peptide can be prepared by introducing a transformant and culturing the transformant.

  In order to enhance antigenicity, a carrier protein may be bound to the antigenic peptide used. For example, carrier proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA) can be bound. These carrier proteins are known in the art and commercially available kits are also sold.

  The immunogen is prepared by dissolving the antigen peptide obtained as described above or the antigen peptide bound to the carrier protein in a buffer. If necessary, an adjuvant may be added for effective immunization. Examples of the adjuvant include commercially available Freund's adjuvant (FCA) and incomplete Freund's adjuvant (FIA). These adjuvants can be used alone or in combination.

  Immunization is performed by administering the prepared immunogen to rabbits by injection into the vein, subcutaneous, intraperitoneal, or footpad. The rabbit to be used is not particularly limited as long as it is a rabbit conventionally used in the art, and examples thereof include Japanese white species and New Zealand white species. Immunization intervals are not particularly limited, and immunization is performed 1 to 5 times at intervals of several days to several weeks. Thereafter, in the case of polyclonal antibodies, serum is collected 20-90 days after the last immunization, and immunoassays such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) ), Etc., and collect on the day when the maximum antibody titer was shown. Thereafter, the reactivity of the polyclonal antibody specific for ussia diponectin present in the serum is measured by the above-described immunoassay or the like. Antiserum can be used directly for immunological measurement, but the antibody in antiserum should be purified and used by affinity chromatography, protein A or protein G affinity chromatography, etc. using Usia diponectin protein Is preferred.

  When producing a monoclonal antibody, antibody-producing cells are collected 20 to 90 days after the final immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like. In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed according to a conventional method. Next, the target hybridoma is selected from the cells after the cell fusion treatment. For example, the cell suspension is appropriately diluted with a medium and then spread on a microtiter plate. A selective culture medium is added to each well, and thereafter the selective culture medium is appropriately exchanged to perform cell culture. Cells that grow from about 30 days can be obtained as hybridomas. Next, the culture supernatant of the hybridoma that has proliferated is screened for the presence of antibodies that react with ussia diponectin. The screening of hybridomas may be carried out in accordance with a usual method, and for example, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), or radioimmunoassay (RIA) can be employed. Cloning of the fused cells is performed by a limiting dilution method or the like to establish a hybridoma that produces the target monoclonal antibody. As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  Furthermore, by splicing a gene from an antibody molecule having reactivity to ussia diponectin prepared as described above with a gene from a human antibody molecule having an appropriate biological activity, a chimeric antibody (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454) be able to. Also, single chain antibodies (US Pat. No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Ward et al. , 1989, Nature 334: 544-546), F (ab ′) 2 fragments, Fab fragments, and the like can also be produced using techniques known in the art.

2. Detection of Usia diponectin It is possible to detect Usia diponectin in a sample using the antibody prepared as described above. This detection can be performed based on any method as long as it is a measurement method using an antibody, that is, an immunological measurement method. For example, Usia diponectin can be detected by immunohistochemical staining and immunoelectron microscopy, as well as immunoassay (enzyme immunoassay (ELISA, EIA), fluorescent immunoassay, radioimmunoassay (RIA), immunochromatography, Western blotting, etc. ) Etc. can be implemented.

  The target sample is not particularly limited. For example, a tissue or cell sample derived from bovine (adipocyte, heart, pancreas, kidney, liver, etc.), biological fluid sample (blood, serum, plasma, urine) , Cerebrospinal fluid, saliva, sweat, ascites, etc.). For example, from the viewpoint of easy collection, it is preferable to use blood, serum or plasma as a sample.

  In the detection method of the present invention, ussia diponectin is detected by binding ussia diponectin in a sample to the antibody according to the present invention and detecting the binding. In the present invention, “detection” includes not only detecting the presence or absence of ussia diponectin but also quantitatively detecting siasia diponectin.

  Immunoassays for Usia diponectin typically involve contacting a sample to be tested with an antibody according to the present invention and detecting the bound antibody using techniques known in the art. “Contact” means that the siasia diponectin present in the sample and the antibody according to the present invention can be brought into close proximity so that they can bind, for example, a liquid sample and an antibody-containing solution are mixed. Such operations as applying the antibody-containing solution to the solid sample and immersing the solid sample in the antibody-containing solution are included.

  The immunoassay may be performed in either a liquid phase system or a solid phase system. From the viewpoint of ease of detection, it is preferable to use a solid phase system. Further, the format of the immunoassay is not limited, and a direct solid phase method, a sandwich method, a competitive method, or the like may be used.

  The anti-ussia diponectin antibody according to the present invention specifically binds to usa diponectin and can detect it under both denaturing conditions (reducing conditions) and non-denaturing conditions. Therefore, the antibody according to the present invention can be used in Western blotting without pretreatment of a sample with a reducing agent or the like, or in various other types of immunoassays.

  The assay can be performed by a known method (Ausubel, F.M. et al., Short Protocols in Molecular Biology, Chapter 11 “immunology” John Wiley & Sons, Inc. 1995). Further, the complex of ussia diponectin and the antibody is separated by a known separation means (chromatography, salting-out method, alcohol precipitation method, enzyme method, solid phase method, etc.), and the signal of the label is detected. Good.

  As an example of an immunoassay, for example, when a solid phase system is used, the antibody may be immobilized on a solid support or carrier (resin plate, membrane, beads, etc.), or the sample may be immobilized. For example, the antibody is immobilized on a solid support, and the support is washed with an appropriate buffer, and then treated with the antibody of the present invention. Next, the solid support is washed a second time with a buffer to remove unbound antibody. Then, by detecting the amount of bound antibody on the solid support by a conventional means, it is possible to detect the binding between ussia diponectin and the antibody in the sample.

  The binding activity of the antibody can be measured according to a well-known method. A person skilled in the art can determine an effective and optimal measurement method for each assay according to the type and format of the immunoassay employed, the type of label used, the target of the label, and the like.

  In one embodiment of the present invention, in order to easily detect the reaction between the antibody of the present invention and ussia diponectin present in a sample, the reaction of the present invention is directly detected by labeling, Alternatively, it is detected indirectly by using a labeled secondary antibody or a biotin-avidin complex. Examples of labels that can be used in the present invention and detection methods thereof are described below.

  In the case of enzyme immunoassay, for example, peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, amylase and the like can be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. These enzymes and antibodies can be bound by a known method using a cross-linking agent such as glutaraldehyde or maleimide compound.

  In the case of a fluorescence immunoassay, for example, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) or the like can be used. These fluorescent labels can be bound to antibodies by conventional techniques.

  In the case of a radioimmunoassay, for example, tritium, iodine 125, iodine 131 and the like can be used. The radioactive label can be bound to the antibody by a known method such as the chloramine T method or the Bolton Hunter method.

  For example, when the antibody of the present invention is directly labeled with a label as described above, the sample is brought into contact with the labeled antibody of the present invention to form an adiponectin-antibody complex. Then, unbound labeled antibody is separated, and the amount of adiponectin in the sample can be measured from the amount of bound labeled antibody or the amount of unbound labeled antibody.

  For example, when a labeled secondary antibody is used, the antibody of the present invention is reacted with a sample (primary reaction), and the resulting complex is further reacted with a labeled secondary antibody (secondary reaction). The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. By the primary reaction and the secondary reaction, an adiponectin-antibody of the present invention-labeled secondary antibody complex or an antibody-adiponectin-labeled secondary antibody complex of the present invention is formed. Then, the unbound labeled secondary antibody is separated, and the amount of adiponectin in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.

  When the biotin-avidin complex system is used, a biotinylated antibody is reacted with a sample, and avidin (avidin, streptavidin, extraavidin, etc.) added with a label is reacted with the obtained complex. Since avidin can specifically bind to biotin, the binding between the antibody and adiponectin can be measured by detecting the signal of the label added to avidin. Although the label added to avidin is not particularly limited, for example, an enzyme label (peroxidase, alkaline phosphatase, etc.) is preferable.

  Detection of the labeled signal can also be performed according to methods known in the art. For example, in the case of using an enzyme label, a substrate that develops color by degradation by enzymatic action is added, and the enzyme activity is obtained by optically measuring the amount of degradation of the substrate. The amount of antibody is calculated from the comparison. The substrate varies depending on the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), diaminobenzidine (DAB), etc. When alkaline phosphatase is used as the enzyme, paranitrophenol, p-nitrophenyl phosphate, or the like can be used. The fluorescent label can be detected and quantified using, for example, a fluorescence microscope or a plate reader. When a radioactive label is used, the radiation dose emitted by the radioactive label is measured with a scintillation counter or the like.

  The antibodies according to the invention can also be used histologically for in situ detection of adiponectin, such as immunohistochemical staining (eg immunostaining) or immunoelectron microscopy. In situ detection can be performed by excising a histological sample from a test cow (such as a paraffin-embedded section of tissue) and contacting it with a labeled antibody.

  The present invention also relates to a reagent for detecting ussia diponectin comprising the antibody of the present invention. In the detection reagent of the present invention, the anti-ussia diponectin antibody may be labeled. Further, the anti-ussia diponectin antibody may be immobilized on a solid phase support (for example, a membrane, beads, etc.).

  In addition to the antibody of the present invention, the detection reagent may contain components useful for performing an immunological measurement method. Such components include, for example, competitors for use in immunoassays, buffers, sample processing reagents, labels, and the like.

By using the detection reagent of the present invention, the above-described detection of ussia diponectin can be performed easily and simply.
Further, as described above, bovine sugar metabolism and lipid metabolism can be determined by detecting and quantifying ussia diponectin in a sample. Regarding glucose metabolism, it has been reported that insulin sensitivity calculated by the insulin clamp test and adiponectin concentration correlate. Therefore, abnormalities in glucose metabolism can be examined by measuring the blood concentration of ussia diponectin in the test cow and comparing it with the blood concentration of bovine showing normal sugar metabolism or the average concentration of many cows. Regarding lipid metabolism, it is considered that the function of the energy combustion system is reduced due to a decrease in adiponectin concentration, and fat is deposited in various organs. Therefore, abnormalities in lipid metabolism can be examined by measuring the blood concentration of ussia diponectin in the test cow and comparing it with the blood concentration of bovine showing normal lipid metabolism or the average concentration of a large number of cows.

3. Other Uses Since the antibody of the present invention specifically reacts with usa diponectin as described above, it can be used for purification of usa diponectin. The antibody of the present invention can be used in any purification method known in the art as long as it is an antibody-based protein purification method. In this case, the antibody of the present invention is preferably immobilized on a solid phase such as a bead or a membrane.

  Specifically, the purification can be performed by contacting a ussia diponectin-containing sample with the anti-ussia diponectin antibody of the present invention, allowing the antibody to capture siasia diponectin, and dissociating the siasia diponectin bound to the antibody. .

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

  Antibodies against ussia diponectin were prepared as follows. As an antigen, a mouse adiponectin peptide (ATGen, catalog number ADI3001) which was expressed in Escherichia coli and was purified by a usual chromatography technique was used as a peptide consisting of amino acid sequences 111 to 247 of mouse adiponectin.

  The above antigen was immunized by subcutaneous injection against two female Japanese white rabbits. In addition, the serum of the rabbit before immunization was extract | collected for subsequent titer evaluation. During the first immunization, FCA (Freund's complete adjuvant) was used as an adjuvant, and FICA (Freund's incomplete adjuvant) was used for the second and subsequent times. Immunization was performed by boosting 4 times every 2 weeks from the initial immunization. Primary serum was collected 8 days after the 4th booster, and secondary serum was collected on the 39th day.

The reactivity of the antibody-containing serum prepared in Example 1 with respect to ussia diponectin was evaluated as follows.
For the serum before injection and on the 39th day, the titer against ussia diponectin was measured. The titer was obtained by diluting rabbit serum in 8 levels of 1/1000 to 1/128000, and determining the reactivity to the peptide at each dilution rate by ELISA using a 96-well plate.

The outline of the measuring method is as follows.
1. Plate coating:
Standard adiponectin diluted to 1 μg / ml with the coating solution was coated at 100 μl / well on the microwell surface of the test plate. The coating solution is 0.015 M sodium bicarbonate, 0.035 M sodium bicarbonate (pH 7.4).

2. Plate washing:
Remove the coating solution and add and remove wash buffer (approximately 400 μl per well). Repeat this wash cycle three times. Wash buffer is 0.05M Tris, 0.15M sodium chloride (pH 7.4, containing 0.05% Tween20).

3. Plate blocking:
Blocking buffer containing protein and detergent (5% skim milk in wash) is added to microwells (300 μl per well).

4). Reaction of antibodies with samples and standards:
Prepare a plate separately from the measurement plate, and gradually increase the antibody (75 μl / well) diluted 5000 times with Can get signal (registered trademark) (Toyobo) Solution1 and Can get signal (registered trademark) from 1000 ng / ml stepwise. Add test standard or C bovine test plasma sample (75 μl / well) diluted to 3.91 ng / ml. The reaction is allowed to proceed for 2 hours at room temperature with shaking using a microplate shaker. The test plasma sample of bovine uses EDTA as a blood coagulant and a 10-fold diluted solution by Can get signal (registered trademark).

5). Addition of antibody / antigen solution:
Following the washing, the antigen-antibody reaction solution prepared in 4 is added (100 μl / well), and the reaction is allowed to proceed for 1 hour at room temperature while shaking using a microplate shaker.

6). Addition of secondary antibody:
Following washing, biotin-labeled goat IgG specific for rabbit IgG (Zymed) diluted 10,000-fold with Can get signal® (Toyobo) Solution 2 is added to each well.

7). Addition of ExtraAvidin (SIGMA):
After washing, extra avidin diluted with a washing solution is diluted 10,000 times with a washing solution (100 μl / well), and the reaction is allowed to proceed at room temperature for 1 hour while shaking using a microplate shaker.

8). Color development with pNPP reagent:
Add the reaction buffer (0.1 M glycine, 1 mM magnesium chloride, 1 mM zinc chloride, pH 10.4) in which P-nitrophenyl phosphate (SIGMA) was dissolved to 1 mg / ml (200 μl / well) Incubate for 10 minutes.

9. Stop reaction with NaOH:
Add a stop solution (3N sodium hydroxide) (50 μl / well) and measure the absorbance. The color development by the absorptiometer is read as absorbance (405 nm) and shows the amount of ussia diponectin present in the test sample. The actual concentration is the standard made using the adiponectin of known concentration. Determined by reading against the curve.

Ten. reading:
When a sufficient assay signal is obtained, the signal is measured, for example, with a microplate spectrophotometer or a fluorimeter.

11． Data processing:
A calibration curve (signal versus concentration) is constructed using assay signals obtained with known standard solutions of adiponectin. A calibration curve is used to interpolate the concentration of ussia diponectin in the test sample.
The calibration curve obtained according to the above reaction is shown in FIG. 1, and the absorbance and concentration of the bovine plasma sample are shown in Table 1.

  From the results shown in FIG. 1 and Table 1, it was shown that ussia diponectin could be accurately quantified within the ELISA tolerance limit using the antibody prepared in Example 1.

SEQ ID NO: 1: Synthetic peptide corresponding to a part of mouse adiponectin SEQ ID NO: 2: Nucleotide sequence of mouse adiponectin SEQ ID NO: 3: Amino acid sequence of mouse adiponectin

Claims (5)

  It is obtained by immunizing a rabbit with a peptide having an amino acid sequence that may have deletion, substitution or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, and with siasia diponectin An anti-ussia diponectin antibody characterized by having reactivity.   The antibody according to claim 1, which is a polyclonal antibody or a monoclonal antibody.   A method for detecting ussia diponectin, comprising contacting a bovine-derived sample and the antibody according to claim 1 or 2 and detecting binding of ussia diponectin and the antibody in the sample.   A method for purifying Usa diponectin, which comprises contacting a ussia diponectin-containing sample with the antibody according to claim 1 or 2 and dissociating ussia diponectin bound to the antibody.   A reagent for detecting ussia diponectin, comprising the antibody according to claim 1 or 2.

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