JP3491743B2 - Method for detecting denatured lipoprotein in blood - Google Patents

Description

Detailed Description of the Invention

[0001]

This invention relates to LDs existing in blood.
Denatured (especially oxidatively denatured) lipoproteins such as L, HDL, VLDL, IDL, Lp (a), Small, denseLDL. In addition to finding out that it exists in the form of a complex with, it relates to a novel method for detecting denatured lipoprotein focusing on this point. To contribute to.

[0002]

The problem of arteriosclerosis is the aorta,
It frequently occurs in coronary arteries, cerebral arteries and carotid arteries, and is a major cause of myocardial infarction and cerebral infarction. Recently, it has been found that Alzheimer's disease is also a disease closely related to arteriosclerosis. Conventionally, there is no measurement target that directly reflects the in vivo arteriosclerosis state in blood, and serum or plasma LDL, Lp (a), remnant lipoprotein, Small, dense LDL, oxidized LDL As such, it has been measured as a lipoprotein involved in arteriosclerotic lesions, which is closely related to lipid accumulation mainly in LDL. Among other things, the association between oxidized LDL and the development of atherosclerotic lesions is Steinberg, D. et al. Engl. Med. 32.
0: 915, 1989), while the injury response hypothesis proposed by Ross et al. (Ross, R. Nature. 362: 801, 1993) has been pointed out, the involvement of oxidized LDL in the progression of arteriosclerosis has been noted. It was

However, in recent studies, not only oxidized LDL but also oxidized HDL (Nakajima, T et al. Biochem Biophys Biopy
s Res Conmmun. 217: 407, 1995) is localized in the affected area of arteriosclerosis, and Omura et al. (Hiroshi Omura et al. Atherosclerosis. 2
5: No4, 126, 1997) In coronary arteriosclerosis, the amount of lipid peroxide in serum HDL was increased, and it was reported that oxidative degeneration of HDL is also involved in the formation of arteriosclerosis. . Similarly, Kusunoki et al. (Kusumi Yoshiaki, et al. Atherosclerosis. 24:
327, 1996) Denatured Lp (a) due to oxidation etc. was found to have been deposited on the intima of the arteries, and denatured Lp (a) was considered to be the mechanism of arteriosclerosis development
This suggests a possible involvement of p (a). A recent epidemiological study has pointed out that the background of the onset of Alzheimer's disease is arteriosclerosis (Kalaria, RN.Pharmacol &
Ther.72: 193, 1996), oxidative degeneration of lipoproteins and apoE phenotype are associated with the onset of arteriosclerosis and Alzheimer's disease (Prem Kumar, DRD. Et al. Am J Patho).
l.148: 2083, 1996).

That is, it is expected that VLDL is oxidatively modified by active oxygen and loses heparin-binding ability to form a sparingly soluble precipitate, whereby lipid peroxide accumulates in blood vessels and nerve tissues and damages cells. (Kazuyuki Hiramura, et al. Jpn J
Electroph. 42:27, 1998). Until recently, there has been a great deal of progress in biochemical and molecular biological research on LDL with regard to the development of arteriosclerosis, and it is often overemphasized that oxidized LDL is a major player in atherogenicity. However, as a result of recent research, not only LDL but also various oxidatively modified lipoproteins such as HDL, Lp (a), and VLDL are included as substances involved in the onset and progression of arteriosclerosis and Alzheimer's disease as described above. It became clear that

Furthermore, there is a large difference in the oxidizability of each lipoprotein, and HDL, Lp (a), Small and dense LDL are easily oxidized, and LDL and VLDL having a large particle size are also difficult to be oxidized. I understand. Also, sma classified as LDL
It was also clarified that ll and dense LDL are LDL that frequently appear in diabetic patients and cannot reflect all arteriosclerosis. Therefore, not only LDL but also HDL, which is particularly oxidizable,
It has become necessary to pay attention to the oxidative modification of Lp (a).

However, at present, there is no method for easily measuring the modified forms of these various lipoproteins in blood. Therefore, it is an object of the present study to provide a novel method for detecting various denatured lipoproteins, which is closely related to the onset and progress of arteriosclerosis and Alzheimer's disease.

[0007]

[Measures to solve the problem] Proteins, lipids, sugars, and nucleic acids are listed as the main constituents of the living body, but the most oxidizable is the lipid, which undergoes an oxygenation reaction,
So-called lipid peroxide is produced. Lipids are easily oxidized because many lipids are esters of highly unsaturated fatty acids such as linoleic acid and arachidonic acid. Lipoproteins are composed of lipids and proteins, and when lipoproteins are oxidized, both lipids and proteins undergo oxidative modification. Active oxygen is considered as a trigger for non-enzymatic oxidation of this biological lipid. The measurement of this lipid peroxide uses analytical chemistry methods such as normal phase HPLC method, and it has been proved that non-enzymatic oxidation of lipid occurs reliably in the body of healthy humans (Yamamoto Junhiro, et al. .Protein / nucleic acid / enzyme44: 1253, 1999).

As described above, at present, it is possible to grasp the total amount of lipid peroxide in blood, but there is no method for ascertaining the degree of oxidative denaturation of each lipoprotein. It was demonstrated for the first time by the method of Japanese Patent Application No. 8-317162 of the present inventors. Furthermore, the present inventor has discovered that the method disclosed in Japanese Patent Application Nos. 11-109001 and 11-207913 can also detect oxidatively modified LDL in blood.

[0009] Then, as a result of repeated research and examination, the present inventors discovered the fact that oxidatively modified forms of lipoproteins other than LDL exist in circulating blood, and reached the present invention. That is,
Japanese Patent Application No. 8-317162, Japanese Patent Application No. 11-109001 and Japanese Patent Application No. 1
The present invention was completed by developing the method of 1-207913 and establishing a detection method capable of individually measuring denatured products of various lipoproteins in blood.

More specifically, the present invention provides various lipoproteins (chylomicrons, VLDL, IDL, LDL, Lp (a), H
When DL) undergoes oxidative degeneration, it forms a complex with plasma proteins (especially α1-antitrypsin, fibrinogen or fibrin (including their respective degradation products), IgA, fibronectin, etc.), and any of these complex forms arteriosclerosis. It was completed by finding points that are highly related to illness.

[0011] Typically, the present invention provides an antibody that specifically recognizes a complex of various oxidized denatured lipoproteins and α1-antitrypsin, a specific antibody that recognizes a complex of various oxidized denatured lipoproteins and IgA, Specific antibodies that recognize the complex of various oxidatively modified lipoproteins and fibronectin, or anti-human fibrinogen antibody is used as a solid-phase antibody, and after reacting with the oxidatively modified products of each lipoprotein, labeled products such as enzymes This is a detection method in which an antibody against an apoprotein that constitutes each labeled lipoprotein is reacted to differentially measure oxidatively modified lipoprotein in blood.

In this case, it is possible to use a sample obtained by fractionating lipoprotein in blood by an ultracentrifugation method or a precipitation method using a chemical substance, or to measure serum or plasma as it is as a sample. When the lipoprotein is fractionated and used as a sample, anti-human IgA, anti-human α1-antitrypsin, or anti-human fibronectin antibody can be used as the solid phase antibody. According to this detection method, the situation in which the free radical chain oxidation reaction is progressing in the living body is taken as the oxidation state of each lipoprotein (HDL, Lp (a), Small, dense LDL are particularly oxidizable). It is possible to grasp. In addition, clinical applications of this detection are suitable for early diagnosis of arteriosclerosis and Alzheimer's disease, and evaluation of drug efficacy during administration of therapeutic agents for arteriosclerosis.

[0013]

The present invention will be described in detail below. [Measurement of modified LDL / α1-antitrypsin complex in serum or plasma] 1. IgG-O.AT-4 monoclonal antibody was added to 0.05M Tris-HCl,
Dissolve it in 0.15M NaCl pH8.0 buffer at 20 μg / ml, and dispense at 100 μl / well into a microplate. After physically adsorbing at 2.4 ° C overnight, wash 3 times with distilled water, 0.1%
Sucrose, bovine serum albumin, 0.05 M containing 0.05% sodium azide, Tris-HCl buffer adjusted to pH 7.5 100 μl /
Dispense well and leave at room temperature for 30 minutes or longer, then discard the liquid 4
Dry at ℃. Dry the microplate with distilled water 25
Wash 3 times with 0 μl / well. 3. 55 mg / ml Mouse Gamma Globulin on the microplate
And Rabbit Gamma Globulin combined 1% BSA solution 100 μl / wel
Dispense l and add 50 μl of serum or standard solution. Four. Incubate overnight at room temperature. 5. Wash with 0.005% Tween20 solution 250 μl / well 5 times. 6. The biotin-labeled Fab'-conjugated IgG-apoB-427 monoclonal antibody is adjusted to 1.6 µg / ml with 1% BSA solution, and 100 µl / well is injected. 7. Incubate at 37 ℃ for 1.5 hours. 8.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 9. HRP-labeled avidin D (Vector Laboratories) 1
Dilute 15000 times with 100% casein solution and dispense 100 μl / well. 10. Incubate at 37 ℃ for 30 minutes. 11.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 12. Dispense 100 μl / well of coloring reagent and react at room temperature for 30 minutes. Dispense 100 μl / well of 13.1M phosphoric acid aqueous solution to stop the reaction. 14. It measures light at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15. Modified LDL / α in the sample from the calibration curve obtained by artificially modified denatured LDL / α1-antitrypsin complex
Calculate the 1-antitrypsin complex concentration.

[Denatured Lp (a) / in serum or plasma
Measurement of α1-antitrypsin complex] 1. IgG-O.AT-4 monoclonal antibody was added to 0.05M Tris-HCl,
Dissolve it in 0.15M NaCl pH8.0 buffer at 20 μg / ml, and dispense at 100 μl / well into a microplate. After physically adsorbing at 2.4 ° C overnight, wash 3 times with distilled water, 0.1%
Sucrose, bovine serum albumin, 0.05 M containing 0.05% sodium azide, Tris-HCl buffer adjusted to pH 7.5 100 μl /
Dispense well and leave at room temperature for 30 minutes or longer, then discard the liquid and
Dry at ℃. Dry the microplate with distilled water 25
Wash 3 times with 0 μl / well. 3. 55 mg / ml Mouse Gamma Globulin on a microplate
And 1% BSA solution containing Rabbit Gamma Globulin 100μ1 / wel
Dispense l and add 50 μl of serum or standard solution. Four. Incubate overnight at room temperature. 5. Wash with 0.005% Tween20 solution 250 μl / well 5 times. 6. Biotin-labeled Fab'-conjugated IgG-Lp (a) polyclonal antibody is adjusted to 1.6 µg / ml with 1% BSA solution, and 100 µl / well is dispensed. 7. Incubate at 37 ℃ for 1.5 hours. 8.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 9. HRP-labeled avidin D (Vector Laboratories) 1
Dilute 15000 times with 100% casein solution and dispense 100 μl / well. 10. Incubate at 37 ℃ for 30 minutes. 11.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 12. Dispense 100 μl / well of coloring reagent and react at room temperature for 30 minutes. Dispense 100 μl / well of 13.1M phosphoric acid aqueous solution to stop the reaction. 14. It measures light at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15. Denatured Lp in the sample from the calibration curve determined by artificially adjusted denatured Lp (a) / α1-antitrypsin complex
(A) / α1-antitrypsin complex concentration is calculated.

[Denatured HDL / α1 in serum or plasma
-Measurement of antitrypsin complex] 1. IgG-O.AT-4 monoclonal antibody was added to 0.05M Tris-HCl,
Dissolve it in 0.15M NaCl pH8.0 buffer at 20 μg / ml, and dispense at 100 μl / well into a microplate. After physically adsorbing at 2.4 ° C overnight, wash 3 times with distilled water, 0.1%
Sucrose, bovine serum albumin, 0.05 M containing 0.05% sodium azide, Tris-HCl buffer adjusted to pH 7.5 100 μl /
Dispense well and leave at room temperature for 30 minutes or longer, then discard the liquid and
Dry at ℃. Dry the microplate with distilled water 25
Wash 3 times with 0 μl / well. 3. 55 mg / ml Mouse Gamma Globulin on a microplate
And 1% BSA solution containing Rabbit Gamma Globulin 100μ1 / wel
Dispense l and add 50 μl of serum or standard solution. Four. Incubate overnight at room temperature. 5. Wash with 0.005% Tween20 solution 250 μl / well 5 times. 6. The biotin-labeled Fab'-conjugated IgG-apoAl polyclonal antibody was adjusted to 1.6 μg / ml with 1% BSA solution, and 100 μl / well was dispensed. 7. Incubate at 37 ℃ for 1.5 hours. 8.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 9. HRP-labeled avidin D (Vector Laboratories) 1
Dilute 15000 times with 100% casein solution and dispense 100 μl / well. 10. Incubate at 37 ℃ for 30 minutes. 11.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 12. Dispense 100 μl / well of coloring reagent and react at room temperature for 30 minutes. Dispense 100 μl / well of 13.1M phosphoric acid aqueous solution to stop the reaction. 14. It measures light at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15. Denatured HDL / α in the sample from the calibration curve obtained by artificially prepared denatured HDL / α1-antitrypsin complex
Calculate the 1-antitrypsin complex concentration.

[Denatured VLDL / α1 in serum or plasma
-Measurement of antitrypsin complex] 1. IgG-O.AT-4 monoclonal antibody was added to 0.05M Tris-HCl, 0.
Dissolve at 20 μg / ml in 15M NaCl pH8.0 buffer and dispense at 100 μl / well into a microplate. After physically adsorbing at 2.4 ° C overnight, wash 3 times with distilled water, 0.1%
Sucrose, bovine serum albumin, 0.05 M containing 0.05% sodium azide, Tris-HCl buffer adjusted to pH 7.5 100 μl /
Dispense well and leave at room temperature for 30 minutes or longer, then discard the liquid and
Dry at ℃. Dry the microplate with distilled water 25
Wash 3 times with 0 μl / well. 3. 55 mg / ml Mouse Gamma Globuli on a microplate
n and Rabbit Gamma Globulin combined 1% BSA solution 100μ1 / we
and add 50 μl of serum or standard solution. Four. Incubate overnight at room temperature. 5. Wash with 0.005% Tween20 solution 250 μl / well 5 times. 6. Biotin-labeled Fab'-conjugated IgG-apoE polyclonal antibody is adjusted to 1.6 µg / ml with 1% BSA solution, and 100 µl / well is dispensed. 7. Incubate at 37 ℃ for 1.5 hours. 8.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 9. HRP-labeled avidin D (Vector Laboratories) 1
Dilute 15000 times with 100% casein solution and dispense 100 μl / well. 10. Incubate at 37 ℃ for 30 minutes. 11.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 12. Dispense 100 μl / well of coloring reagent and react at room temperature for 30 minutes. Dispense 100 μl / well of 13.1M phosphoric acid aqueous solution to stop the reaction. 14. It measures light at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15. Denatured VLDL / α in the sample from the calibration curve obtained using the artificially prepared denatured VLDL / α1-antitrypsin complex
Calculate the 1-antitrypsin complex concentration.

[Measurement of denatured LDL / fibrinogen or fibrin (including each degradation product) complex in serum] 1. IgG-Fibrinogen polyclonal antibody 0.05MT
Dissolve at 20 μg / ml in ris-HCl, 0.15M NaCl pH8.0 buffer, and dispense at 100 μl / well on a microplate. After physically adsorbing at 2.4 ° C overnight, wash 3 times with distilled water, 0.1%
Sucrose, bovine serum albumin, 0.05 M containing 0.05% sodium azide, Tris-HCl buffer adjusted to pH 7.5 100 μl /
Dispense well and leave at room temperature for 30 minutes or longer, then discard the liquid and
Dry at ℃. Dry the microplate with distilled water 25
Wash 3 times with 0 μl / well. 3. 55 mg / ml Mouse Gamma Globulin on a microplate
And Rabbit Gamma Globulin combined 1% BSA solution 100μ1 / wel
Dispense l and add 50 μl of serum or standard solution. Four. Incubate overnight at room temperature. 5. Wash with 0.005% Tween20 solution 250 μl / well 5 times. 6. Biotin-labeled Fab'-conjugated IgG-apoB-427 monoclonal antibody is adjusted to 1.6 µg / ml with 1% BSA solution, and 100 µl / well is dispensed. 7. Incubate at 37 ℃ for 1.5 hours. 8.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 9. HRP-labeled avidin D (Vector Laboratories) 1
Dilute 15000 times with 100% casein solution and dispense 100 μl / well. 10. Incubate at 37 ℃ for 30 minutes. 11.3. Similarly, wash with 0.005% Tween20 solution 250 μl / well 5 times. 12. Dispense 100 μl / well of coloring reagent and react at room temperature for 30 minutes. Dispense 100 μl / well of 13.1M phosphoric acid aqueous solution to stop the reaction. 14. It measures light at a main wavelength of 450 nm and a sub wavelength of 620 nm. 15. The concentration of the modified LDL / fibrinogen or fibrin (including each degradation product) complex in the sample is calculated from the calibration curve obtained by the artificially modified denatured LDL / fibrinogen complex.

[Measurement of denatured Lp (a), denatured HDL / fibrinogen or fibrin complex in serum] Lp (a) antibody and apoAl antibody may be used as the biotin-labeled Fab'-conjugated antibody as described above.

[Blood degeneration in coronary artery disease (LDL, Lp
(A), HDL concentration)] 5 in 1 segment by coronary angiography
Those with 0% or more significant stenotic lesions are defined as coronary atherosclerosis, and those diagnosed with coronary atherosclerosis based on this, CAD
(+) Group (n = 26), CAD (-) group below standard value (n = 21)
Deterioration (LDL and Lp (a) / α1-antitrypsin complex, denatured HDL / α1-antitrypsin complex were measured and comparatively examined. Showed a significantly high price (Fig.
1).

[Brief description of drawings]

1] Denatured lipoprotein (LD in blood in coronary atherosclerosis)
L, Lp (a), HDL) concentrations are illustrated.

─────────────────────────────────────────────────── ─── Continuation of the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/53

Claims (2)

(57) [Claims] 1. A high-density lipoprotein (HDL) is not denatured.
Complex of denatured lipoprotein with α1-antitrypsin
It is a method for detecting denatured lipoprotein in blood to be measured.
Te, specifically recognize α1- antitrypsin in said composite
The solid-state antibody is used as an antibody, and the enzyme and other labels are used.
Using anti-human apoA1 antibody labeled with a substance as a labeled antibody
A method for detecting denatured lipoprotein in blood. 2. A modified lipoprotein obtained by modifying Lp (a).
Blood whose complex is α1-antitrypsin
A method for detecting denatured lipoprotein in a liquid, which specifically recognizes α1-antitrypsin in the complex.
The solid-state antibody is used as an antibody, and the enzyme and other labels are used.
Anti-human Lp (a) antibody labeled with a substance is used as a labeled antibody
A method for detecting denatured lipoprotein in blood.