JPH0933525A - Method and kit for measuring oxidized hdl - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure an oxidized high density lipoprotein(HDL) with high sensitivity using two kinds of antibodies, i.e., an antibody for oxidized lipid and an antibody for apolipoprotein. SOLUTION: An antibody for oxidized lipid is an antibody which can recognize the oxidizing part of a lipid composing an HDL and can be bonded therewith and includes an antibody which can recognize the oxidizing part of phospholipid composing HDL, e.g. lysophosphatidylcholine and can be bonded therewith. An antibody for apolipoprotein is an antibody which can recognize an apolipoprotein composing an HDL and can be bonded therewith and includes an antibody which can recognize an apolipoprotein A1 composing HDL, for example, and can be bonded therewith. Oxidized HDL in a sample is measured by sandwich method using such antibodies.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically measuring oxidized high density lipoprotein (HDL) and a kit used therefor.

[0002]

2. Description of the Related Art Plasma lipoprotein plays an important role in transporting lipid, which is hydrophobic, to the periphery of tissue by forming a hydrophilic complex by binding apolipoprotein to lipid. Lipoproteins are chylomicrons (0.950 g / ml or less) and ultra-low density lipoproteins (VLDL) (0.950-1.006 g / ml) depending on the difference in hydration density and molecular weight by ultracentrifugation or electrophoresis. ),
Low density lipoprotein (LDL) (1.006 to 1.0
63 g / ml), high density lipoprotein (HDL)
(1.063 to 1.210 g / ml).

Dietary lipids are absorbed from the small intestine to become chylomicron, and are taken up by the chylomicron remnant into the liver. However, they are once released from the liver as VLDL into the blood and then passed through intermediate density lipoprotein (IDL). It becomes LDL. LDL has a structure in which apolipoprotein B100 is mainly bound to a central nucleus composed of phospholipids, cholesterol and neutral fat, and is mainly responsible for transporting blood cholesterol to the periphery. The major cholesterol that accumulates is considered to be derived from LDL, so it is also called bad cholesterol. On the other hand, in HDL, apolipoprotein A1 takes up free cholesterol in the liver, small intestine, etc.
It becomes DL and LCAT rides on the bloodstream and circulates to the periphery.
Cholesterol is further taken up by the enzyme and mature H
It becomes DL. Therefore, it plays a role of transporting cholesterol from the periphery and is also called good cholesterol.

Further, in recent studies, it has been reported that LDL, which has been denatured in vivo to become an oxidant, is a cause of arteriosclerosis. That is, LDL is normally reabsorbed through the LDL receptor mainly in the liver and processed, but in the early stage of arteriosclerosis, macrophages migrating into the intima of the blood vessel oxidize LDL via the scavenger receptor. It has been reported that it is specifically taken up and foamed, resulting in the formation of atherosclerotic plaques in the intima (Proc. Natl. Acad. Sci. USA, 78,6).
499-6503 (1981), N. Eng. J. Med., 320, 915-924 (198
9), Am. J. Pathol., 135, 815-825 (1989)). On the other hand, HD
Oxidation phenomenon of L was also confirmed, and HDL was oxidized before LDL oxidation.
Oxidation progresses, and HDL itself acts to stop the oxidation of free radicals that are harmful to the living body at the HDL stage. When HDL oxidation progresses above a certain level, LDL oxidation starts, and this oxidized LDL is arteriosclerosis. It is inferred that it may be the cause (Proc. Natl. Acad. Sci. USA, 88, 6457-6461 (1991)).

[0005]

Thus, it is considered that there is a close relationship between the mechanism of arteriosclerosis and oxidized lipoprotein, and it is possible to specifically measure oxidized lipoprotein, particularly oxidized HDL. If possible, it would be useful for analysis of the mechanism of arteriosclerosis or clinical diagnosis. Conventionally, as an immunological assay for oxidized lipoprotein, a method of assaying oxidized lipoprotein as a complex with β 2 -glycoprotein I (PCT / JP94 / 01594
No.), a measurement method using an antibody against oxidized LDL (Clinic
Chimica. Acta., 218, 97-103 (1993)) and the like have been reported.

[0006]

[Means for Solving the Problems] As a result of intensive studies to develop a simpler and more sensitive method for measuring oxidized HDL, the present inventors have focused on the constituent components of HDL, The present inventors have completed the present invention by discovering that oxidized HDL in a sample can be measured with high sensitivity by using two types of antibodies against apolipoprotein.
That is, the present invention is directed to the presence of oxidized HD present in the sample.
The present invention relates to a method for measuring L by a sandwich method, and relates to a method for measuring oxidized HDL, which comprises using two kinds of antibodies as an antibody, an antibody against oxidized lipid and an antibody against apolipoprotein.
The present invention also provides a kit for measuring oxidized HDL present in a sample by a sandwich method,
The present invention relates to a kit for measuring oxidized HDL, which comprises using two kinds of antibodies, an antibody against oxidized lipid and an antibody against apolipoprotein.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The sample used in the present invention is oxidized H 2
There is no particular limitation as long as it contains DL, and specifically, blood itself or a blood fraction such as serum or plasma can be used as a sample. The assay method of the present invention is characterized by using two kinds of antibodies, an antibody against oxidized lipid and an antibody against apolipoprotein. Here, the antibody against the oxidized lipid is HDL.
It means an antibody capable of recognizing the oxidation site of the lipid that constitutes and is capable of binding to it. As a specific example of such an antibody, an antibody that recognizes and binds to the oxidation site of phospholipids constituting HDL such as lysophosphatidylcholine as used in Examples described later is exemplified. be able to. In addition, an antibody against apolipoprotein is HD
It means an antibody capable of recognizing and binding to apolipoprotein constituting L. As a specific example of such an antibody, an antibody that recognizes and binds to apolipoprotein constituting HDL such as apolipoprotein A1 as used in Examples described later can be exemplified.

Any such antibody (polyclonal antibody or monoclonal antibody) can be prepared by a conventional method. For example, an antibody against an oxidized lipid uses an oxidized lipoprotein such as oxidized HDL or oxidized LDL as an antigen, prepares an antibody by a conventional method using the antigen, and then selects the lipoprotein or apolipoprotein from the obtained antibodies. An antibody that does not bind to the protein but specifically binds only to the oxidized lipoprotein may be selected. Further, an antibody against apolipoprotein can also be prepared by a conventional method using an antigen isolated and purified from serum or an antigen prepared by a recombinant DNA technique. Furthermore, there are already commercially available antibodies, and in this case, this commercially available product can also be used. The type of each of the above-mentioned antibodies used is a polyclonal antibody, a monoclonal antibody, or an active fragment thereof [F (ab ′) ₂ , Fab ′, etc.].
There is no particular limitation.

The present invention is intended to measure oxidized HDL in a sample by the sandwich method using the above-mentioned antibody. The sandwich method itself does not change at all in conventional methods and procedures. That is, it is general to use a solid phase reagent in which one of the above antibodies is bound to a carrier and a labeling reagent in which the other antibody is labeled with a labeling agent.

As the carrier used for preparing the solid phase reagent, those which are usually used, for example, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon are used. ,
Polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, synthetic organic polymer compounds such as polymethylene methacrylate, dextran derivatives (Sephadex, etc.), agarose gel (Sepharose, biogel, etc.), cellulose (paper disc, filter paper, etc.) Examples thereof include inorganic polymer compounds such as saccharides, glass, silica gel, and silicone, and these may have functional groups such as amino groups, carboxyl groups, carbonyl groups, hydroxyl groups, and sulfhydryl groups introduced therein.

The shape of the carrier is flat (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, film-like, fine particle (latex particle, etc.), capsule-like, It may have any shape such as an endoplasmic reticulum, and a carrier having a suitable shape may be appropriately selected according to the measuring method. The carrier and the antibody are bound by a known method such as a physical adsorption method, an ionic binding method, a covalent binding method, or an encapsulation method [eg, "immobilized enzyme" (edited by Ichiro Chibata, March 20, 1975, Co., Ltd.). (Published by Kodansha Co., Ltd.)], and the physical adsorption method is particularly preferable because it is simple. Further, the above-mentioned binding may be carried out directly, or may be carried out between the two substances via another substance. The solid phase reagent thus obtained may be subjected to a blocking treatment using a usual blocking agent such as gelatin or BSA in order to suppress non-specific binding.

On the other hand, as the labeling agent, radioactive isotopes (eg ³² P, ³ H, ¹⁴ C, ¹²⁵ I etc.), enzymes (eg β-galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, catalase) are used. , Glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, etc., coenzyme / prosthetic group (eg, FAD,
FMN, ATP, biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein inovulvamil, etc.), rhodamine derivatives (eg, tetramethylrhodamine B isothiocyanate, etc.), umbelliferone and 1-anilino-8-. Fluorescent dyes such as naphthalene sulfonic acid, luminol derivatives [for example, luminol, isoluminol,
N- (6-aminohexyl) -N-ethylisoluminol and the like] and the like. The binding between the antibody and the labeling agent is described in a publication [eg, "Seikagaku Kagaku Kenkyu Kozai 5 Immunobiochemical Research Method", Tokyo Kagaku Dojin Co., Ltd., (1986), pages 102-112). It may be performed by appropriately selecting from such known methods.

As a measurement procedure using such a solid phase reagent and an antibody reagent, an ordinary sandwich method procedure may be adopted as it is. That is, the solid phase reagent and the test sample are reacted, and if necessary, after BF separation, the antibody reagent is further reacted (two-step method), or the solid phase reagent, the test sample, and the antibody reagent are simultaneously reacted (one-step method). ), And the oxidized HDL in the sample by a method known per se thereafter.
Can be detected or quantified.

Regarding the details of the sandwich method,
For example, refer to the following documents. Hiroshi Irie "Radioimmunoassay" (Kodansha Co., Ltd., published May 1, 1979) Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Co., Ltd.)
Medical Institute, December 15, 1982) Clinical pathology Extra edition special issue No. 53 "immunoassay for clinical examination-technology and application-" (Clinical Pathology Press,
Published in 1983) "Biotechnology Encyclopedia" (CMC, Inc.)
(Published October 9, 1986)

"Methods in ENZYMOLOGY Vol.70" (Im
munochemical techniques (Part A)) "Methods in ENZYMOLOGY Vol.73" (Immunochemical
techniques (Part B)) "Methods in ENZYMOLOGY Vol.74" (Immunochemical
techniques (Part C)) "Methods in ENZYMOLOGY Vol.84" (Immunochemical
techniques (Part D: Selected Immunoassay)) "Methods in ENZYMOLOGY Vol.92" (Immunochemical
techniques (Part E: Monoclonal Antibodies and Gene
ral Immunoassay Methods)) [is published by Academic Press Co.]

The kit of the present invention is for carrying out the above-mentioned assay method, and comprises, for example, the following reagents. Solid-phased antioxidant lipid antibody Labeled anti-apolipoprotein antibody In this kit, the solid-phased antibody and the labeled antibody may be replaced with each other. Further, a labeled anti-immunoglobulin antibody may be used instead of the labeled antibody, and in that case, it is composed of the following reagents. Solid-phase anti-oxidizing lipid antibody Anti-apolipoprotein antibody Labeled anti-immunoglobulin antibody In addition to the above reagents, if necessary, measure from reagents such as color reagent, reaction stopping reagent, standard antigen reagent, sample pretreatment reagent, etc. A suitable reagent according to the method may be appropriately selected and attached to the kit of the present invention.

[0017]

EFFECT OF THE INVENTION Oxidized HDL which is simple and practical according to the present invention
For the first time, it has become possible to provide the assay method and kit.

[0018]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited thereto. Examples <Method> (1) Preparation of oxidized LDL Human LDL (Organon Teknika Corp., Durham, NC) 60
0 μg was added to 2 ml of a phosphate buffer containing 5 μM of copper sulfate and reacted at 37 ° C. for 24 hours to obtain oxidized LDL.

(2) Preparation of Antioxidant Lipoprotein Antibody Lipoproteins (LDL, HD were prepared from hybridomas obtained by cell fusion of splenocytes of BALB / C mice immunized with oxidized LDL and myeloma cells P3U1 according to a conventional method.
L) and apolipoprotein (Apo A1, Apo B100)
Monoclonal antibodies were obtained by selecting clones that produce antibodies that do not bind to, but that bind only to oxidized lipoprotein. Of these antibodies, the monoclonal antibody 9F5-3a was used this time.

(3) Immobilization and blocking of antibody Monoclonal antioxidative lipoprotein antibody solution (9F5-
3a 1.3 μg / ml: 0.05 M NaHCO ₃ −
100 μl of Na ₂ CO ₃ buffer solution (pH 9.5) was added to a 96-well microplate (Nunc Immunoplate I) every minute, and 4
The reaction was carried out overnight at 0 ° C. Remove antibody solution by suction, 0.05%
After washing with a phosphate buffer containing Triton X-100, 200 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) was dispensed into the wells, reacted at 37 ° C. for 1.5 hours to perform blocking, and the blocking solution was removed by suction after the reaction. And then 0.05
It was washed with a phosphate buffer containing Triton X-100 and then dried.

(4) ELISA method 100 μl of a sample or standard concentration solution was added to each well of the 96-well microplate and reacted at 37 ° C. for 1.5 hours. After washing with a phosphate buffer containing 0.05% Triton X-100, anti-a was added as a labeled secondary antibody.
100 μl of a horseradish peroxidase labeled product of poA1 antibody (manufactured by Cosmo Bio Inc.) was added and reacted at 37 ° C. for 1.5 hours. After reaction 0.05% Triton X-10
Was washed with a phosphate buffer containing 0, and a coloring solution (1.5% AB
Citrate buffer containing TS and 30% H ₂ O ₂ , pH
4.0) was poured every 100 μl to develop a color, and the absorbance was measured at 415 nm. The serum sample used was diluted 10-fold with phosphate buffer. Further, as the standard solution, the prepared oxidized HDL is used as a protein amount of 0 to
It was adjusted to 1000 ng / ml and used. [ABTS: 2,
2'-azino-bis (3-ethylbenzthiazorine-6-sulfonic aci
d) diammonium salt]

<Results> (1) Standard curve The above ELISA prepared using a standard solution of oxidized HDL.
The standard curve of the method is shown in FIG. As shown in FIG. 1 and FIG. 2, linearity was exhibited from 15.625 ng / ml to 1000 ng / ml, and good reactivity that converges to 0 point was observed. (2) Crossability LDL, oxidized LDL, HDL for the present ELISA method,
The reactivity of each oxidized HDL is shown in FIG. as a result,
It was revealed that this ELISA method can specifically measure oxidized HDL.

(3) Analysis of specificity of the present ELISA method The influence of malondialdehyde (MDA) treatment and lysophosphatidylcholine (LPC) for the purpose of analyzing the reason why the present ELISA method can specifically measure oxidized HDL. I checked. As shown in Table 1, MDA-ized HDL was used in this ELIS.
When measured as the amount of oxidized HDL by A, the measured value increased slightly more than 3-fold, whereas in the case of MDA-oxidized HDL, it slightly decreased, and MDA-modified LDL and MDA-oxidized LDL were slightly decreased.
Was reduced to about 1/2. On the other hand, when LPC was added at the same time as the MDA treatment, it was markedly decreased in all cases, and when LPC was added in advance, the amount of oxidized HDL could hardly be detected. Further, when LPC was made to coexist during HDL oxidation by MDA and the influence of LPC in the measurement of oxidized HDL was examined, as shown in Table 2, in any case, 1.0-5.0 μg of LPC could not detect oxidized HDL at all. There wasn't.

[0024]

[Table 1]

[Table 2]

Next, the effects of antioxidants and oxidizing agents were examined. As a result, as shown in Table 3, 0.1-1.0 mM of α-tocopherol (α-Tocopherol as an antioxidant was used.
pherol) coexists with MDA during HDL oxidation
It was confirmed that the amount of oxidized HDL was lower than that of the oxidation of DA alone, and the degree thereof was proportional to the dose of α-tocopherol. In addition, catalase and superoxide dismuta as oxidants
se: SOD) was allowed to coexist during HDL oxidation by MDA, and the effect was examined. As shown in Table 4, the amount of oxidized HDL was inversely proportional to the dose of catalase, but the effect of SOD was hardly observed.

[0026]

[Table 3]

[Table 4]

From these results, the reason why the present ELISA method can specifically measure oxidized HDL is α
-LP inhibited by tocopherol or catalase
It was thought that the C-derived OH radicals were closely related. Also, because it is significantly inhibited by LPC,
L on oxidized HDL that appears as a result of oxidization of HDL
It is considered that the PC is also involved.

(4) Basic examination of this ELISA assay method for clinical application 4-1: Dilution test of serum sample A dilution test was carried out on 3 kinds of serum samples of any patients showing high levels of oxidized HDL. As shown in FIG. 5 and FIG. 5, there was a plateau tendency from the original concentration to the measured value of the 1/8 diluted sample, but at 1/16 dilution or more, it decreased linearly in proportion to the dilution ratio. From this, the subsequent measurement of the serum sample was performed by diluting 1/10. 4-2: Addition recovery test Oxidized HDL was added to a 10-fold diluted serum sample and the recovery rate was observed. The results are shown in Table 5.

[0029]

[Table 5]

4-3: Clinical study In contrast to 60 healthy subjects, 17 angina pectoris, 9 myocardial infarction
For example, 9 cases with angina and myocardial infarction and 45 cases with diabetes were measured and compared for oxidized HDL by this ELISA. As shown in FIG. 6, a high average value was observed in all diseases as compared with the average in healthy cases, but the highest tendency was observed in angina cases. When these cases were compared with the TBARS measurement value, which is a lipid peroxide measurement method, as shown in FIG. 7, the correlation coefficient γ =
Although 0.37041 was observed, the correlation was less than this in all other diseases. Therefore, it was suggested that oxidized HDL could be a completely different clinical parameter of cardiovascular disease caused by arteriosclerosis.

[0031]

[Brief description of drawings]

FIG. 1 shows a standard curve in the ELISA method.

FIG. 2 shows a standard curve in the ELISA method.

FIG. 3 shows crossability in the ELISA method.

FIG. 4 shows the dilutability of serum samples.

FIG. 5 shows the dilutability of serum samples.

FIG. 6 shows the results of measurement of oxidized HDL in serum samples.

FIG. 7 shows the correlation between the measured HDL value and TBARS.

Claims (6)

[Claims] 1. A method for measuring oxidized high density lipoprotein (HDL) present in a sample by a sandwich method, comprising two types of antibodies, an antibody against oxidized lipid and an antibody against apolipoprotein. A method for measuring oxidized HDL, which comprises using an antibody. 2. The antibody against the oxidized lipid is HDL
The assay method according to claim 1, which recognizes an oxidation site on the phospholipid. 3. The antibody against apolipoprotein is one which recognizes apolipoprotein A1.
The described measurement method. 4. A kit for measuring oxidized high density lipoprotein (HDL) present in a sample by a sandwich method, which comprises two types of antibodies, an antibody against oxidized lipid and an antibody against apolipoprotein. A kit for measuring oxidized HDL, which comprises using an antibody. 5. The antibody against the oxidized lipid is HDL
The kit according to claim 4, which recognizes an oxidation site on the phospholipid of the above. 6. The antibody against apolipoprotein is one which recognizes apolipoprotein A1.
Kit described.