JP2005075803A - Antibody against human apolipoprotein j, and method for detecting human apolipoprotein j using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a new living body indicator that is useful as a new factor, when the arteriosclerosis and lifestyle-related illness of a patient are examined and provide new detection means for arteriosclerosis or the like by use of the indicator. <P>SOLUTION: Among factors relating to fat metabolism, the human apolipoprotein J can be used as an indicator relating to the conditions of arteries, particularly very good relating to the flexibility of ductus arteriosus. In other words, a new evaluation on the arteriosclerosis can be attained by using an antibody against human apolipoprotein J, in addition, by the detection method for human apolipoprotein using the antibody. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antibody for a specific biological material, and a biological material detection method capable of determining the health state of a subject by detecting the biological material using the antibody.

  High density lipoprotein (HDL) is known to act prophylactically against coronary artery disease by many epidemiological studies. However, the preventive action of HDL on coronary artery disease or the mechanism of anti-arteriosclerosis is still unclear. Anti-arteriosclerosis of HDL is determined by lipoprotein-constituting proteins such as apolipoprotein (for example, apolipoprotein AI (Apo AI)) constituting HDL and proteins bound to HDL in blood [for example, apolipoprotein E (Apo AI). E), apolipoprotein F (Apo F), apolipoprotein J (Apo J), human paraoxonase (PON), platelet activating factor hydrolase (PAF-AH) etc.], or HDL binding or action, Proteins that change the particle size, structure, and function of HDL [eg, lecithin: cholesterol acyltransferase (LCAT), lipoprotein lipase (LPL), hepatic lipase (HTGL), cholesteryl ester transfer protein (CETP), phospholipids It is thought to be due to the action of transfer protein (PLTP). In other words, the former HDL-binding protein has an antioxidant effect on lipids, or has the effect of extracting excess cholesterol accumulated in peripheral cells from the cells, while the latter protein that acts on HDL has the effect of returning the extracted cholesterol to the liver. It is thought that it works to promote the (reverse cholesterol transfer system) or to produce an acceptor having an action of extracting cholesterol from the cell. The synergistic action of these HDL-binding proteins and HDL-acting proteins seems to act in an anti-arteriosclerotic manner by acting on the reverse cholesterol transfer system in vivo.

Some of these factors related to lipid metabolism are used as indicators for detecting the progression of arteriosclerosis and the like, which are hardly recognized as subjective symptoms. In particular, serum lipid tests have been performed more generally than before, but attempts have been made to use various elements as indicators for detecting arteriosclerosis etc. in order to obtain more sensitive knowledge about arteriosclerosis etc. [For example, Unexamined-Japanese-Patent No. 11-346782 (patent document 1), Unexamined-Japanese-Patent No. 2000-333694 (patent document 2), Unexamined-Japanese-Patent No. 2001-663314 (patent document 3), etc.].
JP-A-11-346782 Japanese Patent Laid-Open No. 2000-333694 JP 2001-66314 A

  People with various lifestyle-related diseases are often accompanied by arteriosclerosis, and if arteriosclerosis can be detected easily, predict the possibility of lifestyle-related diseases and their exacerbations and take appropriate measures. It becomes possible to apply. It is also known that arteriosclerosis is deeply related to lipid metabolism, and serum LDL cholesterol level, HDL cholesterol level, etc. are used as related indices of arteriosclerosis also in general health examinations and the like. It has been broken. However, arteriosclerosis is actually complicated by various factors. For example, as arteriosclerotic phenomenon, arterial wall thickening (morphological aspect) and arterial blood vessel flexibility decrease (functional aspect) It is considered desirable to grasp the condition of the subject by examining the state of the artery from at least two sides of ().

  Therefore, the problem to be solved by the present invention is to find a biometric index that can be a new element in examining arteriosclerosis and lifestyle-related diseases of a subject, and to provide means for detecting arteriosclerosis and the like using this index. .

  As a result of intensive studies, the present inventor has surprisingly found that human apolipoprotein J, among elements related to lipid metabolism, is surprisingly used as an index that is very well linked to arterial conditions, particularly arterial vascular flexibility. The present invention was completed by finding out what can be done.

  That is, the present inventor provides an antibody against human apolipoprotein J, and further provides a method for detecting human apolipoprotein J using this antibody, thereby evaluating the state of the artery from the suppleness of arterial blood vessels. I found that it was possible.

  Thus, the present invention provides an antibody against human apolipoprotein J (hereinafter, apolipoprotein J is also referred to as Apo J), and human apolipoprotein J in a specimen is obtained using the antibody. The present invention provides a method for detecting human apolipoprotein J (hereinafter also referred to as the present detection method) that diagnoses arteriosclerosis in a subject by quantitative detection.

  As described above, this antibody is an antibody against human Apo J, that is, a monoclonal antibody or a polyclonal antibody against human Apo J.

  As a precondition for producing such an antibody, human Apo J used as an immunizing antigen is necessary.

  Apo J, also called clusterin, is one of the apolipoproteins that make up HDL in the blood and exists as a heterodimeric protein of about 80 kDa due to disulfide bonds of α and β chains of molecular weight of about 40 kDa, respectively. . In particular, Apo J is highly expressed in the brain, and is also observed in testis, ovary, and liver. HDL containing Apo J has a low fat content and is thought to function to extract cholesterol from cells.

  Human Apo J can be prepared as a natural product separated from human plasma. That is, a generally known purification method such as salting out, gel filtration chromatography, ion exchange chromatography, etc. is applied to the crude Apo J-containing fraction obtained from human plasma by conventional methods such as ultracentrifugation or salting out. To produce purified human Apo J (eg Choi et al., J Immu Methods. 131, 1990, 159-163).

  The purified human Apo J thus obtained can be used as an immunizing antigen for producing this antibody.

  In addition, a recombinant protein of human Apo J can be obtained using genetic engineering techniques, and this can be used as an immunizing antigen for producing this antibody.

  The recombinant protein is prepared by preparing a primer for gene amplification with reference to a known human Apo J gene base sequence (for example, accession number J02908 (cDNA)), and performing RT-PCR using this primer. By purifying the human Apo J recombinant protein produced from the clone obtained by cloning the human Apo J gene as a gene amplification product, and purifying the antibody by the above-mentioned commonly known purification method, It can be a human Apo J recombinant protein that can be used as an immunizing antigen for production. In addition, a modified recombinant protein encoded by a partially modified gene of human Apo J, in which the nucleotide sequence of the above human Apo J gene is partially modified, is possible as long as it does not lose its properties as an immunizing antigen against human Apo J.

  It should be noted that human Apo J as an immunizing antigen used here does not necessarily need to be the whole of human Apo J, and may be a partial peptide fragment thereof. In particular, human Apo J is composed of an α chain and a β chain, and the α chain or β chain can be used as an immune antigen.

  Some fragment peptides of human Apo J include human Apo J fragment encoded by a partial fragment of human Apo J gene, protease treated product of human Apo J, phosphite-triester method (Ikehara, M., et al , Proc. Natl. Acad. Sci. USA, 81, 5956 (1984)) and the like can be used as an immunogen as a chemically synthesized peptide by a solid phase method or a liquid phase method.

  In the human Apo J obtained as described above, in particular, when the human Apo J used as an immunizing antigen is a partial peptide of a small molecule, a hapten is bound to improve the immunoreactivity of the antibody. Can be made.

  As the hapten, a substance that can be usually used as a hapten can be arbitrarily selected. For example, umbrella hemocyanin (KLH), chicken egg albumin (OVA), bovine serum albumin (BSA) and the like are used as haptens. You can choose.

  A cross-linking agent can be used to bind the peptide and the hapten. Such a crosslinking agent can be appropriately selected according to the type of hapten to be selected. For example, when umbrella hemocyanin is selected as the hapten, m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS) or the like can be used. In addition, according to the kind of crosslinking agent to select, a required process can be given to said peptide. For example, when MBS described above is used as a cross-linking agent, it is necessary to add a cysteine residue having a sulfide bond necessary for the binding reaction to either end of the peptide. In addition, the cysteine residue in the selected peptide is preferably protected by substituting it with another amino acid or attaching a protecting group.

  In order to produce this antibody, the animal immunized with the immunizing antigen obtained as described above is not particularly limited, and mice, rats, etc. can be widely used, and bone marrow used for cell fusion. It is desirable to select in consideration of compatibility with tumor cells.

  Regardless of whether this antibody is a polyclonal antibody or a monoclonal antibody, immunization may be carried out by a general method, for example, intravenously, intradermally, subcutaneously, intraperitoneally, etc. Can be performed.

  More specifically, immunization is carried out by administering the immunizing antigen to an animal to be immunized several times according to the above technique every about two weeks, optionally in combination with a normal adjuvant.

  Polyclonal antibodies can thus be obtained as antisera from animals immunized with the immunizing antigen.

  The monoclonal antibody can be obtained from the immunized animal, for example, a hybridoma that produces this antibody as a monoclonal antibody obtained by cell fusion of the spleen cells as immune cells and cell fusion between the immune cells and myeloma cells.

  As said myeloma cell, already well-known thing, for example, SP2 / 0-Ag14, P3-NS1-1-Ag4-1, MPC11-45,6. TG 1.7 (from the mouse); 210. RCY. Ag1.2.3 (rat origin); SKO-007, GM 15006TG-A12 (above, human origin), etc. can be used.

Cell fusion between the above immunocytes and myeloma cells can be performed by generally known methods such as the method of Kohler and Milstein (Kohler, G. and
Milstein, C., Nature , 256 , 495 (1975)).

  More specifically, this cell fusion is carried out by using an auxiliary agent such as dimethyl sulfoxide in order to improve the fusion efficiency in the presence of a commonly known fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ). Hybridomas are prepared by carrying out in a normal culture medium added as necessary.

  Separation of a desired hybridoma can be performed by culturing in a normal selection medium such as HAT (hypoxanthine, aminopterin and thymidine) medium. That is, the hybridoma can be separated by culturing in this selection medium for a time sufficient for cells other than the target hybridoma to die. The hybridoma obtained in this way can be subjected to the search for a monoclonal antibody of interest and single cloning by the usual limiting dilution method.

  The target monoclonal antibody-producing strain can be searched according to a general search method such as ELISA method, plaque method, spot method, agglutination method, octalony method, RIA method.

  The hybridoma of the present invention that produces this antibody, which is a monoclonal antibody recognizing human Apo J, obtained in this way can be subcultured in a normal medium and further stored in liquid nitrogen for a long time. You can also

  The monoclonal antibody of the present invention is collected from this hybridoma by culturing the hybridoma according to a conventional method and obtaining it as a culture supernatant, or by administering it to an animal that is confirmed to be compatible with the hybridoma to be used and obtaining it as ascites A method or the like can be used.

It should be noted that immune cells are cultured in vitro in the presence of human Apo J or a part thereof, and after a certain period of time, a hybridoma of this immune cell and myeloma cell is prepared using the cell fusion means, and an antibody-producing hybridoma is obtained. The desired monoclonal antibody can also be obtained by screening (Reading, CL, J. Immunol. Meth. , 53, 261 (1982); Pardue, RL, et al., J. Cell Biol., 96 , 1149). (1983)).

  Further, the monoclonal antibody obtained as described above can be further purified by usual means such as salting out, gel filtration, affinity chromatography and the like.

 The present antibody (monoclonal antibody or polyclonal antibody) obtained as described above is an antibody having specific reactivity with human Apo J. The phrase “having specific reactivity with human Apo J” means that this antibody exhibits cross-reactivity with at least human Apo J.

  As will be described later, this antibody having such properties can be used clinically, and is also extremely useful as a reagent for the separation and purification of human Apo J itself.

  More specific contents of this antibody will be described later in Examples. The antibody thus obtained can be labeled with a labeling substance as necessary, and used in the measurement method according to the present invention described later.

Such a labeling substance is a labeling substance that provides a detectable signal by reacting the labeling substance alone or with another substance. Specifically, for example, horseradish peroxidase, alkaline phosphatase, β -Enzymes such as D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or fluorescent substances such as tetramethylrhodamine isothiocyanate, ¹²⁵ I, ¹⁴ C, ³ radioisotopes such as H, biotin Chemicals such avidin or Jigokeshigenin, or chemiluminescent substances, and the like.

  As the labeling method of the present antibody with these labeling substances, known labeling methods can be appropriately used according to the type of labeling substance to be selected.

  Moreover, it can also use for the measuring method concerning this invention etc. which are mentioned later as an immobilized antibody which fix | immobilized this antibody (including what was labeled) to the insoluble support | carrier.

  Furthermore, this antibody can be used by immobilizing it on an insoluble carrier, if necessary. As such an insoluble carrier, various insoluble carriers already used as an insoluble carrier for antibodies can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead, a ball, a filter, a membrane, or a cellulose carrier, an agarose carrier, a polyacrylamide carrier, a dextran carrier, a polystyrene carrier, Examples include insoluble carriers used in affinity chromatography such as polyvinyl alcohol carriers, polyamino acid carriers, and porous silica carriers.

  As the method for immobilizing the present antibody in these insoluble carriers, the method for immobilizing antibodies already established in various insoluble carriers can be appropriately selected according to the type of insoluble carrier to be selected.

B. As described above, this detection method is a method for detecting human apolipoprotein J that diagnoses arteriosclerosis in a subject by quantitatively detecting human apolipoprotein J in a specimen using this antibody.

  Examples of this detection method include enzyme immunoassay, radioimmunoassay, analysis by fluorometry, Western blotting, etc., but in general, the method by analysis by enzyme immunoassay is selected. It is desirable to do. The enzyme immunoassay method is also called an enzyme immunoassay method, and is a detection method using an enzyme as a labeling substance among the labeled immunoassay methods. Enzyme immunoassay methods are roughly classified into “heterogeneous enzyme immunoassay” that requires so-called B / F separation and “homogeneous enzyme immunoassay” that does not require this B / F separation. In this detection method, among these, it is preferable to select the former “heterogeneous enzyme immunoassay”, which is generally said to have high measurement sensitivity, and “enzyme-linked immunosorbent assay (ELISA)” using an immunosolvent is used. More preferably, it is selected.

  As a more specific detection mode, an enzyme immunoassay method (hereinafter also referred to as a sandwich method) by a so-called sandwich method can be exemplified. Such a sandwich method is one of the particularly preferred detection modes particularly considering the convenience of operation and the convenience of economy, especially the versatility of clinical tests.

  In carrying out this sandwich method, the present antibody is immobilized with an immobilized antibody and an enzyme such as horseradish peroxidase or biotin immobilized on a microplate having many wells, as represented by a 96-well microplate. It is preferable to use the labeled antibody.

  The sandwich method is an enzyme immunoassay method including at least the following steps (a) and (b).

  (a) A step of reacting a sample such as a blood sample with an immobilized antibody in which the monoclonal antibody of the present invention is immobilized on an insoluble carrier.

  In this step (a), usually, after the reaction, the used microplate is washed, and the unreacted specimen is removed from the immobilized antibody.

  (b) A step of reacting the present antibody labeled with horseradish peroxidase or biotin with an antigen-antibody complex formed by binding of the immobilized antibody and human Apo J in the sample.

  In this step (b), usually, after the reaction, the used microplate is washed, and the unreacted labeled antibody is removed from the immobilized antibody.

  Further, in this step (b), the labeled signal can be revealed using a means for expressing a labeled signal corresponding to the type of the labeled substance in the reacted second antibody. For example, when biotin is selected as the labeling substance, the labeling signal can be revealed using avidin or streptavidin. In addition, for example, when horseradish peroxidase is selected as the labeling substance, the substrate can be added together with a coloring substance as necessary to reveal the labeling signal.

  Thus, human Apo J in a desired specimen can be detected by detecting the developed color signal using a signal specifying means corresponding to the type of color signal.

  The specimen used in the present detection method is not particularly limited as long as human Apo J is inherently present, but the present detection method is a method for determining matters related to arteriosclerosis or the present invention. In view of indications as clinical tests, blood samples such as plasma are preferable.

  The amount of human Apo J in the specimen, which is the detection target of this detection method, is a powerful indicator of the degree of progression of arteriosclerosis in the subject. That is, the present detection method is provided in which arteriosclerosis of the specimen provider is determined based on the abnormality of human Apo J in the specimen detected by the present detection method.

  In this detection method, if arteriosclerosis has not progressed, the amount of human Apo J in the sample is a normal value (about 40 μg / ml), but if arteriosclerosis has progressed, it is quantitatively high. By indicating the value, determination of arteriosclerosis is made.

  That is, according to this detection method, if the amount of human Apo J in a sample such as a blood sample is larger than a normal value, it can be determined that the progression of arteriosclerosis is recognized.

  This detection method is particularly sensitive to functional changes in the arterial wall, specifically to a decrease in the suppleness of arterial blood vessels. In other words, it is possible to detect hardening as a previous stage of thickening of the arterial wall and hardening not related to thickening of the artery as well as thickening and hardening of the arterial wall. Conventionally, the suppleness of arterial blood vessels has been evaluated based on the propagation speed of pulse waves in the arteries, etc., but this detection method can be easily evaluated only by blood tests and the like.

  With this detection method, appropriate treatment can be performed on specimen providers determined to be “arteriosclerosis positive” before specific symptoms such as angina pectoris, myocardial infarction, cerebral infarction, etc. appear. It is possible and is a very useful method in preventive medicine.

  In the present invention, there is also provided an arteriosclerosis determination kit containing the present antibody for use in the present detection method (hereinafter also referred to as the present kit).

  In this kit, an element for providing the present antibody for detection of human Apo J in a specimen, for example, the labeled present antibody or the immobilized present antibody can be included.

  This kit is preferably a kit that can easily perform determination and detection by the sandwich method or fluorometry in correspondence with this detection method.

  According to the present invention, there is provided a detection method for human Apo J capable of evaluating the degree of arteriosclerosis, particularly the flexibility of arterial blood vessels, an antibody for use in the detection method, and a detection kit comprising the same as a component. Provided.

  Hereinafter, the best mode for carrying out the present invention will be described by way of examples. However, this is not intended to limit the technical scope of the present invention.

1. Purification of human Apo J protein
The method was partially modified with reference to the method of Choi et al. (J Immu Methods. 131, 1990, 159-163).

  Polyethylene glycol (M.W. 3,500, Sigma-Aldrich) is added to human plasma to a final concentration of 12%, and after stirring, centrifuged at 8,000 rpm, 4 ° C. for 20 minutes (BECKMAN JA-21), and the supernatant is collected. Further, polyethylene glycol was added to the collected supernatant to a final concentration of 25%, and the mixture was stirred and centrifuged, and the precipitate was collected.

  The recovered precipitate is dissolved in 10 mM Tris-HCl, pH 7.4 / 1 mM EDTA buffer (Tris / EDTA buffer), and added to DEAE Sepharose (Amersham biosciences) 2.6 × 40 cm that has been equilibrated with Tris / EDTA buffer in advance. And eluted with a linear gradient from 0 to 0.5 M NaCl. As a result, human Apo J was eluted at a NaCl concentration of 0.1 to 0.3 M.

The eluted DEAE Sepharose fraction was collected and dialyzed against Tris / EDTA buffer containing 1M ammonium sulfate (4 ° C., 16 hours). After dialysis, previously added to Phenyl Sepharose (Amersham biosciences) equilibrated with 1M ammonium sulfate-containing Tris / EDTA buffer 1.6 × 40 cm, after the linear gradient of 1~0M ammonium sulfate, and eluted with dH ₂ O . Human Apo J was eluted in the dH ₂ O fraction.

  The eluted Phenyl Sepharose fraction was dialyzed (4 ° C., 16 hours) with a 10 mM phosphate buffer (pH 7.4). Hydroxyapatite (Bio Rad) 1.0 × 20 cm previously equilibrated with 10 mM phosphate buffer (pH 7.4) was added and eluted with a linear concentration gradient of 10 to 250 mM phosphate. Human Apo J was eluted at a phosphate concentration of 50-150 mM.

  The eluted Hydroxypatite fraction was dialyzed against Tris / EDTA buffer (4 ° C., 16 hours). After dialysis, it was added to Blue agarose (Sigma Aldrich) 1.0 × 3 cm that had been equilibrated in advance with Tris / EDTA buffer, and the unbound fraction was recovered. Human Apo J was eluted in the unbound fraction.

  The blue agarose non-binding fraction was added to Sephacryl S-200 2.6 × 60 cm (Amersham biosciences) previously equilibrated with Tris / EDTA buffer and separated at a flow rate of 0.5 ml per minute. Human Apo J was eluted in fractions 20 to 26 (FIG. 1: electrophoresis image in the gel filtration column elution fraction). Thus, the obtained fraction was finally used as an antigen.

2. Human Apo J cDNA cloning, recombinant protein production, and purification mRNA was purified from human liver , and the human Apo J α subunit (1-194 amino acid residues), β subunit (195- 416 amino acid residues) and full-length cDNA were amplified and cloned. The obtained cDNA was sequenced and confirmed to be no different from human Apo J cDNA (accession number J02908). Each obtained human Apo J cDNA was inserted into an E. coli expression vector pQE-30 (Qiagen), and the vectors pQE-30 / Apo J-α, pQE-30 / Apo J-β, pQE-30 / Apo J- I got the full length. Each vector was introduced into E. coli strain JM109 to obtain E. coli JM109 / Apo J-α, JM109 / Apo J-β, and JM109 / Apo J-full length. Each recombinant Escherichia coli strain was cultured in TB medium containing 50 μg / ml ampicillin, and expression of recombinant human Apo J (hereinafter also referred to as rhApo J) was induced by 1 mM IPTG. The culture solution was centrifuged (5,000 rpm, 5 min, 4 ° C.) to precipitate E. coli expressing rhApo J. The precipitated cells are suspended in 2% Triton X-100 and 50 mM phosphate buffer (pH 8.0) containing 0.5 M NaCl, crushed by sonication on ice, and centrifuged (15,000 rpm, 30 min, 4 And the precipitate containing rhApo J was recovered. The precipitate was suspended in 50 mM phosphate buffer (pH 8.0) containing 7 M Urea, 10 mM 2-mercaptoethanol, 10 mM Imidazole, and 0.5 M NaCl, extracted by sonication on ice, and centrifuged (15,000 rpm, 30 min 4 ° C.) and the supernatant containing rhApo J was recovered. The supernatant was bound to Ni-NTA agarose (Qiagen) equilibrated with the same 50 mM phosphate buffer (pH 8.0).

  Elution was performed with 7M Urea, 10 mM 2-mercaptoethanol, 250 mM Imidazole, and 50 mM phosphate buffer (pH 8.0) containing 0.5 M NaCl, and the eluted fractions were purified with rhApo J-α, rhApo J-β, and rhApo J-full length.

3. Production of polyclonal antibodies against human Apo J
The rhApo J-full length was subjected to SDS-PAGE and then CBB staining, and a band of about 45 kDa corresponding to the rhApo J full length was excised. The obtained gel fragment was washed with PBS and then pulverized in PBS to obtain a rhApo J suspension. The rhApo J suspension was emulsified with an equal volume of Freund's complete adjuvant, and rhApo J-total 0.3 mg equivalent was administered subcutaneously to the rabbit. Thereafter, rhApo J-sustained rhApo J suspension emulsified with an equal amount of Freund's incomplete adjuvant was administered subcutaneously to rabbits three times every two weeks. One week after the final administration, blood was collected and the serum was centrifuged. The obtained serum was used as an anti-human Apo J polyclonal antibody.

4). Preparation of monoclonal antibody against human Apo J protein Purified human Apo J (25 μg) was mixed with an equal amount of Freund's complete adjuvant and immunized to the abdominal cavity of 7-week-old Balb / c mice (female). Two weeks later, booster immunization was similarly performed using Freund's incomplete adjuvant, and thereafter, immunization was performed four times, once every two weeks. Spleen cells of mice 3 days after the final immunization were mixed with mouse myeloma cells (SP2 / 0-Ag14), and cell fusion was performed using a PBS solution containing 50% polyethylene glycol (average molecular weight 1500). After the fusion, the cells were suspended in RPMI 1640 medium at a rate of 5 × 10 ⁵ cells per well and seeded in a 96-well plate. From the next day, HAT reagent was added and selective culture was performed for 7 days. Production of a specific monoclonal antibody against human Apo J in the culture supernatant of each hybridoma was screened by an ELISA method in which purified human Apo J or rhApo J was immobilized. That is, 50 ng of purified Apo J or rhApo J was immobilized on each well, 100 μL of the culture supernatant of each hybridoma was added, and the mixture was reacted at room temperature for 2 hours. After the reaction, each well was washed with a washing solution (PBS containing 0.1% Tween 20), a peroxidase-labeled anti-rabbit anti-mouse IgG antibody diluted 4,000 times was added, and reacted at room temperature for 1 hour. After the reaction, the wells were washed 5 times with a washing solution, and 100 μL of coloring solution (0.012% hydrogen peroxide, 0.4 mg / ml OPD (o-phenylenediamine dihydrochloride, Sigma-Aldrich) -containing citrate / phosphate buffer, pH 5.0 ) Was added and reacted at room temperature for 30 minutes, and the reaction was stopped by adding 2N sulfuric acid. After stopping the reaction, absorbance at a wavelength of 492 nm was measured with a microplate reader. A hybridoma culture supernatant exhibiting an absorbance of 1.0 or higher was regarded as positive. In addition, positive hybridoma culture supernatants were confirmed for reactivity to purified Apo J by Western blotting, and clones that showed specificity were selected by both methods.

  In Western blotting, 0.5 μg of purified human Apo J or rhApo J per lane was separated by 10% SDS polyacrylamide electrophoresis and transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with 0.1% Tween 20-containing PBS (washing solution), each monoclonal antibody diluted 10-fold to 100-fold with the washing solution is reacted at room temperature for 2 hours. After the reaction, the plate is washed 5 times with a washing solution, and 0.05 μg / ml of horseradish peroxidase-labeled anti-mouse IgG antibody is reacted at room temperature for 2 hours. After the reaction, the plate was washed five times with a washing solution, and then an X-ray film (Kodak) was exposed to light using a chemiluminescence reagent (PerkinElmer) to detect a specific band.

  Furthermore, the selected hybridomas are adjusted to 0.5 cells per well in RPMI1640 medium supplemented with HT reagent, seeded in a 96-well plate, and the same screening procedure is repeated three times to produce purified human Apo J A specific monoclonal antibody-producing hybridoma against was obtained.

  Finally, purified human Apo J or rhApo J was immobilized by ELISA using a microplate and Western blotting against human plasma, purified human Apo J or rhApo J. Positive clones were purified There were 11 clones producing monoclonal antibodies against human Apo J.

    Production and purification of antibodies from the obtained monoclonal antibody-producing hybridoma cells are as follows.

Balb / c mice (female) were injected intraperitoneally with 10 ⁶ to 10 ⁷ hybridoma cells / 0.5 ml per mouse. Ten days after the injection, the mouse was opened and ascites was collected. The obtained ascites was mixed with an equal amount of ice-cooled saturated ammonium sulfate solution added to the supernatant obtained by centrifugation, cooled on ice and allowed to stand for 2 hours. Further, after centrifugation at 10,000 × g for 10 minutes at 4 ° C., the supernatant is discarded, the precipitate is dissolved in a binding solution (3M sodium chloride, 1.5M glycine solution, pH 8.9), mixed with protein A sepharose, 4 ° C. Invert and mix overnight. Bound protein A sepharose was packed into a column, washed with 6 volumes of the binding solution, and then eluted 1 ml at a time with an elution solution (0.1 M citric acid solution, pH 4.0). The eluted fraction was neutralized with 0.1 ml of 2 M Tris solution (pH 10.0). The absorbance (280 nm) of each elution fraction was measured, and the monoclonal antibody elution fraction was collected and dialyzed against PBS (4 ° C., overnight) to obtain a purified monoclonal antibody.

  The isotype of the obtained monoclonal antibody was measured using a mouse monoclonal antibody isotype determination kit (Boehringer) according to the operation procedure attached to the kit. Monoclonal antibody-producing clones against purified human Apo J were IgG1 (6 clones), IgG2a (2 clones) and IgG2b (3 clones).

  The antibody specificity was determined by Western blotting for the antigen (purified human Apo J) (Fig. 2) and by preparing an antibody dilution curve by ELISA using a plate on which the antigen (purified human Apo J) was immobilized. (Fig. 3).

  The reactivity of each antibody to human Apo Jα and β chain was confirmed by Western blot using rhApo J forcibly expressed by E. coli. The obtained 11 clones were confirmed to react with rhApo Jα chain (3 clones) or rhApo Jβ chain (8 clones).

  The hybridoma producing a monoclonal antibody (3C11) that reacts with the rhApo Jβ chain is “mouse hybridoma J-3C11” (FERM P-19493), and the hybridoma producing the monoclonal antibody (4H8) is “ mouse hybridoma J-4H8 "(FERM P-19494) is deposited at the National Institute of Advanced Industrial Science and Technology.

5. Preparation of biotinylated monoclonal antibody 1 mg of the purified anti-Apo J human monoclonal antibody (4H8) obtained as described above was dialyzed with 0.1 M NaHCO ₃ (pH 8.3) solution at 4 ° C for 16 hours. . 60 μg NHS-biotin (Pierce) dissolved in DMSO was added and stirred, and reacted at room temperature for 4 hours. Dialysis was performed with PBS (5 L) at 4 ° C. for about 16 hours to obtain a biotinylated monoclonal antibody.

6． Construction of Apo J protein quantitative measurement system in human plasma
100 μL of each immobilized antibody solution (2.5 μg / ml) diluted in PBS containing 0.05% NaN ₃ was added to a 96-well microplate, and allowed to stand at 4 ° C. overnight to be immobilized. The solution in each well was discarded, and the solution was completely removed with a paper towel or the like. A blocking solution prepared by dissolving BSA (Sigma Aldrich) in PBS was added in an amount of 200 μL per well and allowed to stand at room temperature for 2 hours. Each well was washed twice with 300 μL of washing solution (0.1% Tween 20 / PBS). 100 μL each of a measurement sample (purified human Apo J or rhApo J, rhApo J culture supernatant, control plasma and measurement plasma specimen) diluted in advance with a washing solution was added and allowed to stand at room temperature for 2 hours. Each well was washed 5 times with a washing solution. 100 μL of biotin-labeled antibody solution (0.5 μg / ml) diluted with a washing solution was added to each well and allowed to stand at room temperature for 2 hours. Each well was washed 5 times with a washing solution, and then 100 µL of a peroxidase-labeled avidin solution (0.05 µg / ml) diluted with the washing solution was added, and the mixture was allowed to stand at room temperature for 1 hour. After the reaction, each well was washed 5 times with a washing solution, and the coloring solution prepared just before use (0.012% hydrogen peroxide / 0.4 mg / ml OPD (O-phenylenediamine dihydrochloride) / citrate / phosphate buffer pH 5.0 ) Was added to each well at 100 μL, and the color was developed for 0.5 hour at room temperature. 25 μL of a reaction stop solution (2N H ₂ SO ₄ solution) was added to each well to stop the reaction. Measurement was performed using a microplate reader at an absorbance of 492 nm.

  A standard curve using purified human Apo J is shown in FIG. In addition, reactivity to purified human Apo J (A) and human plasma (B) is shown in FIGS. 5 (A) and 5 (B). From these results, it was shown that the reactivity in this sandwich ELISA was equivalent in purified human Apo J and human plasma.

7). Relationship between human plasma Apo J protein mass and arteriosclerosis In order to investigate the relationship between human Apo J protein and arteriosclerosis , human plasma Apo J protein mass and index of arteriosclerosis are 1) blood pressure, 2 ) Maximum carotid intima-media thickness determined by ultrasound echo (max IMT), and 3) Relationship between pulse wave velocity (PWV) measured by pulse wave velocity meter investigated.

  As an index of arteriosclerosis, the intima-media complex thickness (thickness degree, thickness) of the common carotid artery blood vessel wall is measured.

  Higher systolic blood pressure or lower diastolic blood pressure tends to be recognized due to the presence of the arteriosclerotic layer and the site where the vascular lumen is raised in the carotid artery (also called arteriosclerotic layer or plaque) It is known that there is.

  On the other hand, the pulse wave velocity is an index of arterial extensibility (flexibility), and is used as an index of arterial aging and arteriosclerosis progress. That is, it is known that the pulse wave velocity increases with hardening of the arterial blood vessel.

  For 62 healthy subjects (especially those without subjective symptoms) (mean age 61.4 ± 8.4 years old), along with the human ApoJ concentration in plasma, blood pressure, carotid artery thickness, and pulse wave velocity were measured, The relationship between human Apo J concentration in these subjects and arteriosclerosis was examined. As a result, the human Apo J concentration was positively correlated with the mean blood pressure and the pulse wave velocity (FIGS. 6 (1) and (2)). This indicates that plasma human Apo J concentrations tend to increase with the progression of arteriosclerosis. Furthermore, a significant correlation was also found with systolic blood pressure, diastolic blood pressure, and pulse pressure.

  Furthermore, since the human Apo J concentration was positively correlated with age and blood pressure, it was corrected with age and mean blood pressure, and the relationship between max IMT and PWV was examined.

  In the measurement of carotid intima-media thickness (IMT), both carotid arteries were observed with a high-resolution ultrasonic measurement device (Power Vision 6000, manufactured by Toshiba) using a 7.5 MHz probe. Conventional IMT (C-IMT) is 1-3 cm from the common carotid artery bifurcation, and the thickness of the distal intima-media complex at the average site excluding plaque is measured in three directions. Value. In addition, all the bilateral common carotid arteries were observed, and the IMT of the thickest part including the plaque was measured as the maximum IMT (max IMT).

  The pulse wave velocity was measured using a blood pressure pulse wave test device (form PWV / ABI, manufactured by Nippon Chorin Co., Ltd.), as well as systolic blood pressure and diastolic blood pressure, and brachial-ankle pulse wave velocity (brachial-ankle PWV, baPWV) were automatically measured.

  When max IMT and PWV are classified into three levels and examined, the human Apo J concentration in max IMT is 45.5 ± 4.8 μg / ml in the Low max IMT group, 46.6 ± 5.3 μg / ml in the Middle max IMT group, It was 47.5 ± 7.0 μg / ml in the High max IMT group, and no significant relationship was observed in the relationship between human Apo J concentration and max IMT (FIG. 7 (1)). On the other hand, in PWV, the human Apo J concentration is 41.9 ± 2.3 μg / ml in the Low PWV group, 46.6 ± 5.2 μg / ml in the Middle PWV group, 49.4 ± 4.7 μg / ml in the High PWV group, and the human Apo J concentration is It was shown that the Middle PWV group and the High PWV group were significantly higher (P <0.01) than the low PWV group, which is a normal group (FIG. 7 (2)).

  This suggests that human Apo J does not correlate with arterial morphological changes, i.e. common carotid wall thickness, but correlates with arterial functional changes, i.e. increased pulse wave velocity . This indicates that measuring the amount of human Apo J protein in the blood can be a new index of arteriosclerosis.

  According to the present invention, it becomes easy to detect arteriosclerosis, and the present invention is particularly useful in preventive medicine.

It is the figure which showed the finally purified human Apo J with molecular weight. It is drawing which shows the result of having confirmed the specificity of this antibody (monoclonal antibody) using the western blotting method with respect to the purified human Apo J and human plasma. It is a drawing showing the results of confirming the specificity of this antibody (monoclonal antibody) by preparing an antibody dilution curve by ELISA using a plate on which an antigen (purified human Apo J) is immobilized. It is the standard curve drawn using this antibody (monoclonal antibody) with respect to purified human Apo J. It is the figure which showed the reactivity of this antibody (monoclonal antibody) with respect to purified human Apo J and human plasma. It is the figure which showed the correlation with the average blood pressure and the pulse wave velocity of the human ApoJ density | concentration in blood. It is drawing which showed the correlation with the carotid intima-media thickness and pulse wave velocity of human ApoJ concentration in blood.

Claims (9)

An antibody against human apolipoprotein J. The antibody of claim 1, wherein the antibody is a monoclonal antibody. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is a monoclonal antibody that specifically binds to human apolipoprotein Jβ chain. A hybridoma producing the monoclonal antibody according to claim 2 or 3. A method for detecting human apolipoprotein J, comprising diagnosing arteriosclerosis in a subject by quantitatively detecting human apolipoprotein J in a specimen using the antibody according to claim 1. The method for detecting human apolipoprotein J according to claim 5, wherein the diagnosis of arteriosclerosis is a diagnosis of suppleness of arterial blood vessels. The method for detecting human apolipoprotein J according to claim 5 or 6, wherein the antibody is a monoclonal antibody. A kit for detecting human apolipoprotein J, comprising at least one antibody according to any one of claims 1 to 3. The kit for detecting human apolipoprotein J according to claim 8, wherein the antibody is a monoclonal antibody.

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