JP2002181820A - Diagnosis kit for disease resulting from arteriosclerosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an excellent diagnosis kit for a disease resulting from arteriosclerosis. SOLUTION: Taking denatured remnant lipoprotein (denatured IDL) resulting from denaturation of remnant lipoprotein (IDL) in blood and denatured low- density lipoprotein (denatured IDL: oxidized LDL is also included) resulting from denaturation of low-density lipoprotein in blood as subjects of measurement, measurement is carried out by using an antibody recognizing aldehyde formed in the denatured LDL, or an anti-4-hydroxy-2-nonanol antibody reactive with the oxidized LDL and a complex of oxidized LDL and protein, an anti- malonidialdehyde antibody, or an anti-acrolein antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a kit for diagnosing atherosclerotic disease by measuring denatured LDL (including oxidized LDL) and denatured remnant lipoprotein in blood.

[0002]

The arteriosclerosis is the aorta,
It occurs frequently in the coronary arteries, cerebral arteries, and carotid arteries, and is a major cause of myocardial infarction and cerebral infarction. Recently, Alzheimer's disease has also been found to be a disease that is highly associated with arteriosclerosis. Conventionally, in blood, there is no measurement target that directly reflects the state of arteriosclerosis in vivo, and LDL cholesterol, Lp
(a), LDL such as remnant lipoprotein, small dense LDL
It has been measured as a lipoprotein involved in arteriosclerotic lesions, which is closely related to vascular wall lipid accumulation mainly in blood vessels. Above all, the association between oxidized LDL and the development of atherosclerotic lesions has been shown by Steinberg (D. et al. Engl. Med. 320:
915, 1989), while the involvement of oxidized LDL in the progression of arteriosclerosis has attracted attention since being pointed out by the injury response hypothesis proposed by Ross et al. (Ross, R. Nature. 362: 801, 1993).

On the other hand, recent studies have revealed that remnant lipoprotein, which is an intermediate of VLDL or chylomicron metabolism, is a lipoprotein having a strong atherosclerosis-inducing property. Remnant lipoproteins are neutral fat (TG) -rich lipoproteins such as chylomicron, which is produced in the small intestine and transports dietary fat, and VLDL, which is produced in the liver and transports endogenous fat, is lipoprotein lipase in the blood. It refers to an intermediate metabolite that has been remodeled and reduced in size by the action of enzymes such as these. That is, remnant lipoprotein is a general term for chylomicron remnant and VLDL remnant, and both are TG
It is a rich lipoprotein rich in apoE and cholesterol (Tada Norio. History of Medicine, 181: 868, 1997).

[0004] A large-scale epidemiological study showing that hyperTGemia is a risk factor for coronary artery disease has shown that Prospective Cardiovascular Munster in subjects without coronary artery disease.
(PROCAM) Study (1996, Germany, subject: male 4849
Example), The Physician´s HealthyStudy (1996, USA, 574 subjects), Copenhagen Male Study (1998,
Denmark, subjects: 2906 males), The Baltimore Coronary Observation Long
-Term Study (COLTS) (1998, USA, 350 cases), Th
e Bezafibate Infarction Prevention (BIP) Study (19
In 1999, Israel, the subject was 11,532 cases).

[0005] It has been considered that the cause of the delay in the study on the mechanism of atherosclerosis progression in hyperTGemia compared to hypercholesterolemia is due to its diversity. That is, high TG
Factors related to the mechanism of atherosclerosis progression in hyperemia are hyper-remnantemia, low HDL cholesterol, and small dense
LDL, blood coagulation / fibrinolysis abnormalities, insulin resistance, etc. are very diverse. Since this remnant lipoprotein is a TG-rich lipoprotein, its increase can be regarded as an increase in serum TG, but an increase in serum TG does not necessarily mean an increase in remnant. The increase in remnant lipoprotein in blood can be estimated by the appearance of a broad-β pattern on lipoprotein electrophoresis, but Lp (a) also migrates to this site on electrophoresis, making its differentiation difficult. (Norio Tada, Atherosclerosis,
26: 98,1998). In addition, the total amount of cholesterol carried into the intima of the arterial wall by this remnant lipoprotein is LDL.
(Gianturco SH et al., J. Cl
in. Invest, 82: 1633, 1988) Furthermore, remnant lipoproteins are deeply involved in the formation of atherosclerotic lesions because they are taken up by macrophages and promote foaming like oxidized LDL without being denatured.

Under these circumstances, the present inventors have developed a method for detecting denatured LDL (oxidized LDL) present in blood (Japanese Patent Application No. 8-31).
No. 7162, Japanese Patent Application No. 11-109001, Japanese Patent Application No. 11-2027913, Japanese Patent Application 20
No. 00-012210, Japanese Patent Application No. 11-24440), together with these oxidized LDLs, identify the denatured remnant lipoproteins that are formed in atherosclerotic foci and migrate into the blood, and are expected to be present in the bloodstream. An object of the present invention is to provide a better diagnostic method for atherosclerotic disease by devising a novel method for detecting both, after clarification.

[0007]

The epidemiological and experimental studies so far have revealed that lipoprotein abnormalities are closely related to the onset and progression of arteriosclerosis. It has been shown that LDL is taken up by macrophages via scavenger receptor or LDL receptor by oxidative denaturation and promotes its foaming.

On the other hand, remnant lipoproteins are receptors that macrophages recognize remnant lipoproteins without oxidative denaturation like LDL (LDL receptor-related protein, membrane-bound protein 2).
It is known that macrophages become foam cells when taken up via the membrane binding protein 235). Furthermore, in hyperremnantemia, LDL is easily oxidized and small and has a high density (Small dense LDL), both of which result in a double hit state that acts to promote cholesterol accumulation in the blood vessel wall. I have.

Therefore, LDL (mainly Small)
It is expected that both denatured products of dense LDL) and remnant lipoproteins coexist. Furthermore, since both of these components are likely to be transferred to the blood with plaque rupture, if denatured remnant lipoprotein can be detected in the blood simultaneously with oxidized LDL, a better marker for diagnosing atherosclerotic lesions (Fig. 1).

As a result of further studies, it has been found that lipoprotein having the characteristics of remnant lipoprotein and denatured lipoprotein and acid-denatured LDL obtained by denaturing small and high-density LDL
Have found the fact that they coexist in blood and have led to the present invention. That is, the diagnostic kit for arteriosclerotic lesions according to the present invention targets a modified remnant lipoprotein (denatured IDL) obtained by denaturing remnant lipoprotein (IDL) in blood. In addition, denatured low-density lipoprotein (denatured LDL: oxidized LDL) obtained by denaturing low-density lipoprotein (LDL) in blood
And denatured remnant lipoprotein (IDL) obtained by denaturing remnant lipoprotein (IDL) in blood. Specifically, oxidative denaturation in blood
The purpose of the present invention is to diagnose arteriosclerotic diseases by simultaneously or separately measuring LDL and denatured remnant lipoprotein.

In the present invention, it is preferable to use an antibody that recognizes the aldehyde formed in the modified LDL. Also,
It is preferable to use an anti-4hydroxy 2-nonenal antibody, an anti-malondialdehyde antibody, or an anti-acrolein antibody, more preferably, oxidized LDL, and anti-4hydroxy2 having reactivity with a complex of oxidized LDL and a protein. -It is preferable to use a nonenal antibody, an anti-malondialdehyde antibody, or an anti-acrolein antibody. Here, the protein is α1 antitrypsin, lactoferrin, fibrinogen, myeloperoxidase, human albumin, casein, CRP,
Alternatively, an antibody which is at least one of SAA1 and has reactivity with a complex with all of the proteins is desirable.

It is also preferable to measure the denatured remnant lipoprotein in blood using an immunoglobulin (IgG) derived from an animal that does not specifically recognize human protein components such as denatured LDL and denatured remnant lipoprotein. .

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below. [1. LDL Particle Size in Serum with High Levels of Oxidized LDL LDL in Serum with High Levels of Oxidized LDL in the Blood Oxidized LDL Measurement Method (Japanese Patent Application No. 8-317162) previously invented by the present inventors.
When the particle size was measured, as shown in FIG. 2, small LDL particles were observed in all cases. In addition, on agarose electrophoresis, small particles LDL increased in negative charge and migrated to the positive side (FIG. 3).

[2. Particle Size of Oxidized LDL Oxidized LDL particles isolated and purified from LDL fractionated by ultracentrifugation based on the mode of existence of oxidized LDL in blood previously discovered by the present inventors (Japanese Patent Application No. 2000-012210). As a result of examining the size, all the oxidized LDLs tended to be small particles (FIG. 4).

[3. Confirmation of remnant lipoprotein coexisting in oxidized LDL Oxidized LDL in LDL fraction obtained from serum of 8 cases was isolated and purified,
When electrophoresed using polyacrylamide gel,
It was confirmed that the oxidized LDL was a small particle and that the remnant lipoprotein coexisted (FIG. 5).

[4. ] Confirmation of IgG binding lipoprotein present in oxidized LDL Monoclonal antibody Ox that specifically recognizes α1 antitrypsin of oxidized LDL (α1 antitrypsin / LDL complex)
Reaction with LDL fractionated by ultracentrifugation using Affinity carrier with AT-4 immobilized Affinity carrier and 7D4 antibody (mouse globulin) that shows no specificity for human components, then isolated by each carrier The resulting components (oxidized LDL and IgG-binding lipoprotein) were stained with an OxAT antibody and an apoB100 antibody.
As a result, as shown in FIG. 6, the presence of an apo B100 component that was reactive only with the 7D4 antibody was recognized (IgG-binding lipoprotein).

[5. ] Isolation and purification of IgG-binding lipoprotein present in LDL fraction of normolipidemic blood and its properties (1) Antibody that does not react LDL fractionated by ultracentrifugation with human components (anti-horse ferritin antibody: rabbit IgG) The IgG-binding lipoprotein was isolated by passing through a fixed Affinity column. The feature is that the fragmentation of apoB100 is almost the same as that of oxidized LDL, but the aldehyde (4-hydroxy-2-nonenal) present in oxidized LDL was not recognized (FIG. 7).

[6. IgG present in LDL fraction of hyperlipidemia
Isolation and purification of binding lipoprotein and its properties (2) After isolating IgG-binding lipoprotein from LDL of hyperlipidemic patients in the same manner as above, the fragmentation state and size of apoB100 were examined. The fragmentation of apoB100 was comparable to oxidized LDL. Further, when the particle size was examined by gel filtration analysis, it was almost the same size as IDL (remnant lipoprotein) (FIG. 8).

[7. ] Confirmation of apoE in AT / LDL complex (oxidized LDL) and IgG-binding LDL As described above, the presence of apoprotein E in the IgG-binding lipoprotein isolated and purified from the LDL fraction was confirmed. The IgG-binding lipoprotein was found to have apoE (FIG. 9).
Therefore, it was found that the IgG-binding lipoprotein was remnant lipoprotein (denatured remnant lipoprotein).

[8. [Preparation of Various Aldehyde Antibodies] [Preparation of Antigen] Various aldehyde antibodies used in the present invention are prepared as follows. Preparation of antigen is 4-hydroxy 2
-Nonenal, or malondialdehyde, or acrolein 5 mg / ml solution and α1 antitrypsin (α1AT) solution of the same concentration are mixed at room temperature, respectively, and a conjugate of 4hydroxy 2-nonenal and α1AT, or After forming a conjugate of malondialdehyde and α1AT or a conjugate of acrolen and α1AT, each conjugate was subjected to desalting column treatment to remove free aldehyde.

[Immunity to animals] These conjugates (antigens)
1 mg each in α1AT concentration with phosphate buffered saline
/ ml solution, and an equal volume of this solution and Freund's adjuvant were mixed. Emulsions were obtained by intraperitoneal injection of 500 μl of 6-week-old mice (Balb / c mice) prepared for each type of aldehyde. Was administered. This work
This was performed three times every two weeks.

[Cell fusion] Four days after the final immunization, spleen lymphocyte cells collected from the spleen of each mouse were fused with mouse myeloma cells (P3-X63-Ag8-U1). Fusion was carried out according to a conventional method using a 50% polyethylene glycol 4000 solution as a fusion accelerator, and a fusion time consisting of addition, mixing, and dilution operations of the fusion accelerator was performed for 10 minutes at 37 ° C. Then H
The fused cells after the completion of the fusion were dispersed in an AT medium, and then 200 μl of each aldehyde / α1AT conjugate was dispensed into each well of 10 96-well microplates and cultured at 37 ° C. in the presence of 5% carbon dioxide. .

[Selection and unification of an anti-4-hydroxy-2-nonenal / α1AT monoclonal antibody or an anti-malondialdehyde / α1AT monoclonal antibody or an acrolein / α1AT monoclonal antibody-producing hybridoma]
After a week, 100 μl of the HAT medium in each well is aspirated, and the HAT medium (containing hypoxanthine / thymidine / 10% fetal bovine serum)
RPMI medium) was dispensed into each well, and after 2-3 days, antibody-producing hybridomas were selected for each aldehyde.

The selection method is as follows. For antibodies against 4-hydroxy-2-nonenal-bound α1AT, 4-hydroxy-2-nonenal-bound α1AT, oxidized LDL, oxidized LDL / α1AT, oxidized LDL / lactoferrin, oxidized LDL / fibrinogen, oxidized LDL / myeloperoxidase, Oxidized LDL / human albumin, oxidized /
100 μl of the culture supernatant of the hybridoma-forming colony was dispensed into each well of a 96-well microplate on which casein, oxidized LDL / CRP, and oxidized LDL / SAA1 complex were immobilized, and reacted. Add 100 μl of mouse immunoglobulin antibody, react with antigen-antibody, operate according to the usual methods of washing, coloration and ELISA, and react with the target antibody (reactive with as many kinds of antigens and oxidized LDL and various proteins / LDL complexes as possible). , An antibody that does not react with α1AT) was produced. Hybridomas producing malondialdehyde and acrolein antibodies were also selected in the same manner as in the above-mentioned 4-hydroxy-2-nonenal.

Next, colonies showing the desired antibody production were collected and cloned by the limiting dilution method so as to obtain single hybridoma colonies. In this method, the collected colonies were diluted with a HAT medium, and spread to each well of a 96-well microplate together with feeder cells such that the number of hybridomas was 1 or less per well. Perform the above operation twice to obtain a monocloned anti-4-hydroxy
2-nonenal / α1AT antibody or anti-malondialdehyde
A plurality of hybridomas each producing an / α1AT antibody or an anti-acrolein / α1AT antibody were obtained.

[Ascites of each aldehyde / α1AT antibody] Pristane (immunosuppressant) was intraperitoneally administered to 8-week-old mice (Balb / c mice). Three to seven days later, a plurality of the antibody-producing hybridomas were intraperitoneally administered to the mice prepared for each hybridoma, and after about seven days, the ascites-purified antibody was recovered from the peritoneal cavity of the mice.

[Purification of Antibodies] Each antibody obtained by ascites was subjected to salting-out separation twice with 50% ammonium sulfate, purified by dialyzing with phosphate buffered saline, and purified for each aldehyde. Was obtained.

[9. ] Demonstration of the presence of various aldehydes in oxidized LDL Aldehydes (4-hydroxy-2-nonenal, malondialdehyde, acrolein) in various oxidized LDLs isolated and purified from hyperlipidemic LDL fractions were examined using various anti-aldehyde antibodies. As a result, the presence of aldehyde was confirmed in the oxidized LDL (FIG. 10).

[10. ] Method for measuring oxidized LDL in serum [Antibodies] 1. anti-4hydroxy 2-nonenal monoclonal antibody Anti-human apo B-427 monoclonal antibody

[Reagents used] 1. 30 mM CHAPS solution CHAPS was added to 0.1% Tris-HCl buffer containing 0.5% BSA and 0.15 M NaCl.
(pH 8.0). 2.5% skim milk solution 0.1% Tris-HCL buffer solution containing 0.65M NaCl
H8.0). (Using 5% skim milk for blocking prevents the binding of denatured remnant lipoprotein to IgG.) 3.1% casein solution Milk casein is dissolved in 0.1M Tris aqueous solution containing 0.15M NaCl, and then adjusted to pH 8.0 with hydrochloric acid. adjust.

4. Tween 20 solution of 0.05% Tween 20 is dissolved in 0.01 M phosphate saline (PBS). Five. After dissolving 40mg of TMBZ (Dojindo Chemical) with 50ml of methanol
Mix 50 ml of 0.1 M Tris aqueous solution to prepare 100 ml of TMBZ solution. Also, adjust a 0.035 M citric acid aqueous solution containing 0.0375% aqueous hydrogen peroxide (adjusted at the time of use). At the time of use, equal amounts of both reagents are mixed to give a coloring reagent. 6. 1M phosphoric acid aqueous solution 7. Biotin-labelled anti-human apoB-427 monoclonal antibody apoB-427 Biotin maleimide (Vecto
r Laboratories) is labeled with Fab′-modified anti-human apoB-427 monoclonal antibody.

[Measurement procedure] Anti-4hydroxy-2-nonenal (HNE) monoclonal antibody was added to 0.05 M Tris-HCl, 0.15 M NaCl pH 8.0 buffer at 5 μg /
Dissolve in 100 ml and dispens into the microplate at 100 μl / well. 2. After physical adsorption overnight at 4 ° C, wash 3 times with 250 µl / well of distilled water. 3. 55μg / ml Mouse Gamma Globulin and Rabbit Gamma Gl
A 5% skim milk solution containing obulin is dispensed at 100 μl / well into a HNE antibody solid phase microplate, and 50 μl of serum diluted 10-fold with a 30 mM CHAPS solution is added thereto.

4. Incubate at room temperature for 1 hour. 5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution. 6． Biotin-labeled anti-human apoB-427 Fab′-labeled monoclonal antibody is diluted 3000 times with a 1% BSA solution and dispensed at 100 μl / well. 7． Incubate at room temperature for 1 hour. 8.5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution in the same manner as described above.

9. HRP-labeled avidin D (Vector Laboratori
es) (20000 times) with 1% casein solution, 100 μl
Dispense / well. Ten. Incubate at room temperature for 30 minutes. 11.5. Wash 5 times with 250 μl / well of 0.05% Tween20 solution in the same manner as described above. 12． Dispense 100 μl / well of the coloring reagent and allow it to react at room temperature for 20 minutes. 13. Dispense 100 μl / well of 1 M phosphoric acid aqueous solution to stop the reaction. 14. The light is measured at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15． Calculate the oxidized LDL concentration in the serum from the calibration curve.

[11. ] Method for measuring denatured remnant lipoprotein in serum [Antibodies] 1. Anti-Horse Ferritin polyclonal antibody (IgG-binding lipoprotein: Use an anti-Horse Ferritin antibody that does not react with human components to measure denatured remnant lipoprotein) Anti-human apo B-427 monoclonal antibody

[Reagents used] 1. 30 mM CHAPS solution CHAPS was added to 0.5% BSA in 0.1 M Tris-HCl buffer containing 0.15 M NaCl.
(pH 8.0). 2. 1% BSA solution Dissolve BSA in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. 3. 1% casein solution Milk casein is dissolved in a 0.1M Tris aqueous solution containing 0.15M NaCl, and then adjusted to pH 8.0 with hydrochloric acid. 4. 0.05% Tween20 solution Tween20 is dissolved in 0.01 M phosphate saline (PBS).

5. After dissolving 40 mg of the coloring reagent TMBZ (Dojindo) in 50 ml of methanol
Mix 50 ml of 0.1 M Tris aqueous solution to prepare 100 ml of TMBZ solution. In addition, a 0.035M citric acid aqueous solution containing 0.0375% hydrogen peroxide solution is prepared (adjusted at the time of use). At the time of use, equal amounts of both reagents are mixed to give a coloring reagent. 6. 1M phosphoric acid aqueous solution 7. Biotin-labelled anti-human apoB-427 monoclonal antibody apoB-427 Biotin maleimide (Vecto
r Laboratories) is labeled with Fab′-modified anti-human apoB-427 monoclonal antibody.

[Measurement procedure] Anti-Horse Ferritin polyclonal antibody was added to 0.05M Tris-
Dissolve in HCl, 0.15M NaCl pH8.0 buffer at 5 μg / ml and dispense into a microplate at 100 μl / well. 2. After physical adsorption overnight at 4 ° C, wash 3 times with 250 µl / well of distilled water. 3. 55μg / ml Mouse Gamma Globulin and Rabbit Gamma Gl
A 1% BSA solution containing obulin was dispensed at 100 μl / well into an anti-Horse Ferritin antibody solid phase microplate, and 30 mM CHAP
Add 50 μl of serum diluted 10-fold with S solution. Four. Incubate at room temperature for 1 hour.

5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution. 6． Biotin-labeled anti-human apoB-427 Fab′-labeled monoclonal antibody is diluted 3000 times with a 1% BSA solution and dispensed at 100 μl / well. 7． Incubate at room temperature for 1 hour. 8.5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution in the same manner as described above. 9． 1 HRP-labeled avidin D (Vector Laboratories)
Dilute 20,000-fold with 100% casein solution and dispense 100 μl / well. Ten. Incubate at room temperature for 30 minutes.

11.5. 250 μl of 0.05% Tween20 solution
Wash 5 times with / well. 12． Dispense 100 μl / well of the coloring reagent and allow it to react at room temperature for 20 minutes. 13. Dispense 100 μl / well of 1 M phosphoric acid aqueous solution to stop the reaction. 14. Photometry is performed at 450 nm main wavelength and 620 nm sub wavelength. 15． The concentration of denatured remnant lipoprotein in the serum is calculated from the calibration curve.

[12. ] Simultaneous measurement of oxidized LDL and denatured remnant lipoprotein in serum [Antibody] Anti 4-hydroxy 2-nonenal monoclonal antibody (HN
E antibody) Anti-human apo B-427 monoclonal antibody

[Reagents used] 1. 30 mM CHAPS solution CHAPS was added to 0.5% BSA in 0.1 M Tris-HCl buffer containing 0.15 M NaCl.
(pH 8.0). 2. 1% BSA solution Dissolve BSA in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. (Ig by blocking with 1% BSA
G-linked lipoprotein: denatured remnant lipoprotein can bind to the Fc site.) 3.1% casein solution Milk casein is dissolved in 0.1M Tris aqueous solution containing 0.15M NaCl, and then adjusted to pH 8.0 with hydrochloric acid. 4. 0.05% Tween20 solution Tween20 is dissolved in 0.01 M phosphate saline (PBS).

5. After dissolving 40 mg of the coloring reagent TMBZ (Dojindo) in 50 ml of methanol
Mix 50 ml of 0.1 M Tris aqueous solution to prepare 100 ml of TMBZ solution. In addition, a 0.035M citric acid aqueous solution containing 0.0375% hydrogen peroxide solution is prepared (adjusted at the time of use). At the time of use, equal amounts of both reagents are mixed to give a coloring reagent. 6. 1M phosphoric acid aqueous solution 7. Biotin-labeled anti-human apoB-427 monoclonal antibody Fabo-biotin maleimide (Vecto
r Laboratories) is labeled with Fab′-modified anti-human apoB-427 monoclonal antibody.

[Measurement Procedure] Anti-HNE monoclonal antibody was added to 0.05M Tris-HCl, 0.15M
Dissolve in NaCl pH 8.0 buffer at 5 μg / ml and dispense into a microplate at 100 μl / well. 2. After physical adsorption overnight at 4 ° C, wash 3 times with 250 µl / well of distilled water. 3. 55μg / ml Mouse Gamma Globulin and Rabbit Gamma Gl
A 1% BSA solution containing obulin is dispensed into a HNE antibody solid-phase microplate at 100 μl / well, and 50 μl of serum diluted 10-fold with a 30 mM CHAPS solution is added thereto. Four. Incubate at room temperature for 1 hour.

5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution. 6． Biotin-labeled anti-human apoB-427 Fab′-labeled monoclonal antibody is diluted 3000 times with a 1% BSA solution and dispensed at 100 μl / well. 7． Incubate at room temperature for 1 hour. 8.5. Wash 5 times with 250 μl / well of 0.05% Tween 20 solution in the same manner as described above.

9. HRP-labeled avidin D (Vector Laboratori
es) (20000 times) with 1% casein solution, 100 μl
Dispense / well. Ten. Incubate at room temperature for 30 minutes. 11.5. Wash 5 times with 250 μl / well of 0.05% Tween20 solution in the same manner as described above. 12． Dispense 100 μl / well of the coloring reagent and allow it to react at room temperature for 20 minutes. 13. Dispense 1 M phosphoric acid aqueous solution at 100 μl / well to stop the reaction. 14. The light is measured at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15． The concentration of oxidized LDL and denatured remnant lipoprotein in the serum is calculated from the calibration curve.

[13. ] Relationship between Serum Lipids and Oxidized LDL and Denatured Remnant Lipoprotein Concentration A method for accurately measuring serum remnant lipoprotein concentration has not been established. In general, it is known that when remnant lipoprotein is increased, both hypercholesterolemia and hypertriglyceridemia are exhibited. As a result of measuring oxidized LDL and denatured remnant lipoprotein in these cases, increase in oxidized LDL and denatured remnant lipoprotein was observed in some subjects with increased remnant lipoprotein, and the progression of arteriosclerosis was observed. It was suggested that there was a case that could be indicated (FIGS. 11 and 12).

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a hypothesis that oxidized LDL and denatured remnants are present in blood.

FIG. 2 is a drawing showing LDL particle size in AT / LDL complex-high serum.

FIG. 3 is a drawing showing the relationship between LDL particle size and charge.

FIG. 4 is a drawing showing particle sizes of various protein / LDL complexes.

FIG. 5 is a drawing showing remnant lipoproteins present in oxidized LDL.

FIG. 6 is a drawing showing IgG-binding lipoproteins present in the LDL fraction.

FIG. 7 is a drawing showing characteristics of an IgG-binding lipoprotein present in a normolipidemic LDL fraction.

FIG. 8 is a drawing showing the characteristics of an IgG-binding lipoprotein present in a hyperlipidemia LDL fraction.

FIG. 9 is a drawing showing that apoE was not observed in the AT-LDL complex (oxidized LDL) but apoE was observed in the IgG-binding lipoprotein.

FIG. 10 is a drawing showing aldehydes in various protein / LDL complexes.

FIG. 11 illustrates the relationship between oxidized LDL and serum lipids.

FIG. 12 illustrates the relationship between denatured remnant lipoproteins and serum lipids.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AG27 CA10 CA20 CC24 CE04 CE06 DA13 4H045 AA30 BA55 CA42 DA76 DA86 EA50 FA72 GA06 GA26

Claims (9)

[Claims] 1. A kit for diagnosing atherosclerotic lesions, wherein a modified remnant lipoprotein (denatured IDL) obtained by denaturing remnant lipoprotein (IDL) in blood is to be measured. 2. Denatured low-density lipoprotein (LDL) obtained by denaturing low-density lipoprotein (LDL) in blood and denatured remnant lipoprotein (IDL) in blood. A kit for diagnosing atherosclerotic lesions, characterized by measuring denatured remnant lipoprotein (denatured IDL). 3. The kit for diagnosing arteriosclerotic disease according to claim 1, wherein an antibody recognizing aldehyde formed in the modified LDL is used. 4. The kit for diagnosing arteriosclerotic disease according to any one of claims 1 to 3, wherein the kit uses an anti-4hydroxy 2-nonenal antibody, an anti-malondialdehyde antibody, or an anti-acrolein antibody. 5. The method according to claim 1, wherein an anti-4hydroxy-2-nonenal antibody, an antimalondialdehyde antibody, or an antiacrolein antibody reactive with oxidized LDL and a complex of oxidized LDL and protein is used. A kit for diagnosing atherosclerotic disease according to the above. 6. The method according to claim 5, wherein the protein is at least one of α1 antitrypsin, lactoferrin, fibrinogen, myeloperoxidase, human albumin, casein, CRP, and SAA1. kit. 7. The method according to claim 1, wherein an immunoglobulin (IgG) derived from an animal that does not specifically recognize human protein components such as denatured LDL and denatured remnant lipoprotein is used to measure denatured remnant lipoprotein in blood. Item 4. A diagnostic kit for arteriosclerotic disease according to Item 2. 8. The arteriosclerotic composition according to claim 1, wherein an immunological assay such as an enzyme immunoassay, a latex agglutination reaction, an immunoluminescence assay, or an immunochromatography is used. Kit for diagnosis of disease. 9. It comprises at least an anti-4hydroxy-2-nonenal antibody, an enzyme-labeled anti-human apoB100 antibody, and a reagent for measuring an enzyme, and the denatured LDL (including oxidized LDL in blood) is determined by immunoassay. 9. The kit for diagnosing arteriosclerotic disease according to any one of claims 1 to 8, wherein the modified remnant lipoprotein is measured.