JP3507407B2 - Method for detecting denatured lipoprotein in blood and diagnostic kit for arteriosclerosis - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] Oxidatively modified lipoprotein exists in the form of a complex with an acute phase reactive substance, a blood coagulation / fibrinolytic system-related protein, and a bactericidal substance produced by inflammatory cells. It was discovered that in the complex of lipoprotein and various proteins, there are a complex in which 4-hydroxy-2-nonenal is produced (strong oxidation) and a complex in which it is not produced (weak oxidation), This is a method for specifically detecting denatured lipoprotein having a strong degree of oxidation using an anti-4-hydroxy-2-nonenal antibody.

[0002]

The low-density lipoprotein that has undergone oxidative modification is taken up by macrophages through the scavenger receptor of macrophages. It is known that macrophages that have taken up a large amount of low-density lipoprotein cholesterol foam and play an important role in the formation of atherosclerotic lesions. That is, oxidized low-density lipoprotein (abbreviated as oxidized LDL) is considered to be a substance directly related to the development of atherosclerosis. Recently, the presence of oxidized HDL was confirmed, and (N
akajima, T et al. Ann Clin Biochem, 37: 179-186, 200.
0) Like oxidized LDL, it is considered to be a substance directly related to the development of arteriosclerosis.

Therefore, if information on oxidatively modified lipoproteins is obtained in the circulating blood, it can be expected to be useful for elucidating the mechanism of arteriosclerosis, evaluating the efficacy of therapeutic agents, and applying it to clinical tests. However, the actual condition of oxidatively modified lipoprotein in blood is almost unknown, and the present inventors have filed Japanese Patent Application No. 8-317162.
No. 11-109001, Japanese Patent Application No. 11-207913, Japanese Patent Application 2000-0
The reality is that the method disclosed in 12210 is becoming clear.

However, each of the above-mentioned inventions has a wide variety of objects to be measured, and it is complicated to measure each of them separately. Here, it is extremely effective if the properties common to these various types of complexes can be found. The present invention has been made in view of such circumstances, and an object thereof is to provide a more practical and effective method for diagnosing arteriosclerosis and a diagnostic kit.

[0005]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have established a complex of oxidatively modified LDL and various proteins in LDL isolated and purified from blood and an oxidatively modified HDL in HDL.
As a result of the analysis of the physical properties of the complex of the protein with various proteins, the oxidation-modified lipoprotein has a different degree of oxidation, and the weak oxidation-modified lipoprotein (α2 macroglobulin or Serum amyloid A (SAA) and the oxidation-modified lipoprotein are Body etc. and strength oxidative denatured lipoprotein (α1 antitrypsin, fibrinogen, CRP,
Lactoferrin, a complex of myeloperoxidase and oxidative denatured lipoprotein) exists, and in particular, strong oxidative denatured lipoprotein is formed on the inner wall of blood vessel, and in the strong oxidative denatured lipoprotein, 4-hydroxy-2-nonenal It was found that the formation is characteristic.

[0006] After that, as a result of further research and examination, it was found that the denatured lipoprotein in which only phospholipids present in the surface layer of lipoprotein in the circulating blood are in an oxidized state is a weakly oxidized denatured lipoprotein, Blood levels are also relatively high (1-2% of total lipoproteins), and this denatured lipoprotein is considered to be a risk factor for arteriosclerosis. Furthermore, ester type cholesterol in the core of the lipoprotein is present. The strong fatty acid oxidized lipoprotein (the blood concentration is very low, which is several hundredth of that of weakly oxidized denatured lipoprotein) in which 4-hydroxy-2-nonenal was also formed by oxidation of fatty acid of The present inventors have found the fact that a protein can be a marker for arteriosclerosis, and arrived at the present invention.

That is, Japanese Patent Application No. 8-317162 and Japanese Patent Application 2000
-012210 was developed to establish a method for detecting denatured lipoprotein in blood, thus completing the present invention.
Α2-Macroglobulin / LDL complex and Serum amyloid A (SAA) / LDL complex (mildly oxidized lipoprotein: arteriosclerosis risk factor) of Japanese Patent Application No. 2000-012210 and 4-hydroxy-2-nonenal of the present invention By differentially measuring the denatured lipoprotein (containing strong oxidative denatured lipoprotein: a marker of arteriosclerosis) containing erythrocyte, early diagnosis of arteriosclerosis and evaluation of drug efficacy during administration of therapeutic agents for arteriosclerosis can be performed more appropriately.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. 1. Relationship between oxidized LDL by various lipid oxidizing agents and 4-hydroxy-2-nonenal formation during α1 antitrypsin complex formation Using LDL and α1-antitrypsin as an example, during oxidation by various oxidizing agents The relationship between the formation state of oxidized LDL / α1 antitrypsin complex and the amount of 4-hydroxy-2-nonenal formed in the formed oxidized LDL was examined. As shown in Fig. 1, the oxidized LDL / α1 antitrypsin complex is formed by any oxidant oxidation, but 4-hydroxy-2-nonenal is produced only when a water-soluble oxidant is used. I found that it was hard to be done. It is considered that only the phospholipids in the surface layer are in the oxidized state.

2. Various protein / LDL complexes and 4-hydroxy-2
-Relationship with nonenal / LDL Using various clinical samples (serum), various proteins / LDL complexes (α1 antitrypsin / LDL complex, SAA / LDL complex, CRP /) in the LDL fraction of each sample were used. 4-hydroxy-in the same LDL fraction as LDL complex, α2 macroglobulin / LDL complex, fibrinogen / LDL complex, myeloperoxidase / LDL complex, albumin / LDL complex were used as examples)
The amount of 2-nonenal is measured (anti-hydroxy-2-nonenal antibody is used as a solid phase antibody, and anti-human ap is used as a labeled antibody).
(ELISA using oB antibody) and its relationship were examined. As shown in Fig. 2, complex of α1-antitrypsin, CRP, fibrinogen, myeloperoxidase protein and oxidized LDL and 4-hydroxy-2-nonenal were observed. There was a strong relationship between the concentrations, and SAA / LDL, α2 macroglobulin / LDL, and albumin / LDL complexes did not show a relationship with the 4-hydroxy-2-nonenal concentration.

3. Identification of various proteins contained in 4-hydroxy-2-nonenal-containing lipoproteins (LDL, HDL) in LDL and HDL fractions of clinical specimens of clinical specimens (serum) exhibiting hyperlipidemia The LDL and HDL fractions were separated by ultracentrifugation and treated with an anti-4-hydroxy-2-nonenal antibody affinity column to isolate and purify the LDL and HDL fractions containing 4-hydroxy-2-nonenal. This 4-hydroxy-2-nonenal containing LDL and HDL
After SDS-PAGE of various trace proteins contained in the protein, immunostaining of various proteins by chemiluminescence method was performed.
-The presence of α1 antitrypsin, fibrinogen, lactoferrin, myeloperoxidase, and CRP was confirmed in nonenal-containing LDL and HDL.

4. Detection Example of LDL Containing 4-Hydroxy-2-nonenal 1) Anti-α1 antitrypsin modified 4-hydroxy-2-nonenal Monoclonal antibody was added to 0.05M Tris-HCl (0.15M NaC
pH 8.0) buffer solution containing 10 μg / ml, and dispensed to a microplate at 100 μl / well. 2) After physically adsorbing at 4 ° C overnight, wash with deionized water 3 times before use, and use 0.1% sucrose and bovine serum albumin,
Dispense 0.05 M Tris-HCl buffer solution (pH 7.5) containing 0.05% sodium azide at 10 μl / well, leave at room temperature for 30 minutes or longer, discard the solution, and dry at 4 ° C. The dried microplate is washed 3 times with 250 μl / well of deionized water.

3) 55 mg / ml Mouse Gam on a microplate
Dispense 1% bovine albumin solution containing ma Globulin and Goat Gamma Globulin in 100 µl / well, and add 50 µl of sample (serum diluted 5 times with PBS) or standard solution. 4) Incubate at 37 ℃ for 1.5 hours. 5) Wash with 250 μl / well of 0.005% Tween20 solution 5 times. 6) Dispense 100 μl / well of biotin-labeled Fab′-conjugated IgG-apo B / 427 monoclonal antibody made up to 1.6 μg / ml with 1% BSA solution. 7) Incubate at 37 ℃ for 1.5 hours. 8) Wash 5 times with 250 μl / well of 0.005% Tween20 solution as in 5). 9) 1 HRP labeled avidin D (Vector laboratories)
Dilute 15,000 times with 1% casein solution and dispense 100 μl / well.

10) Incubate at 37 ° C. for 30 minutes. 11) Wash 5 times with 250 μl / well of 0.005% Tween20 solution. 12) Add 100 parts of coloring reagent consisting of hydrogen peroxide solution and TMBZ solution.
Dispense μl / well and incubate at room temperature for 30 minutes. 13) Dispense 100 μl / well of 1M aqueous phosphoric acid solution to stop the reaction. 14) Measure the light at the main wavelength of 450 nm and the sub wavelength of 630 nm. 15) Calculate the concentration of 4-hydroxy-2-nonenal-containing modified LDL (oxidized LDL) in the sample from the calibration curve obtained by artificially adjusting modified LDL (oxidized LDL).

5. Detection Example of HDL Containing 4-Hydroxy-2-nonenal 1) Anti-α1 antitrypsin modified 4-hydroxy-2-nonenal Monoclonal antibody was added to 0.05M Tris-HCl (0.15M NaC
pH 8.0) buffer solution containing 10 μg / ml, and dispensed to a microplate at 100 μl / well. 2) After physically adsorbing at 4 ° C overnight, wash with deionized water 3 times before use, and use 0.1% sucrose and bovine serum albumin,
Dispense 0.05 M Tris-HCl buffer (pH 7.5) containing 0.05% sodium azide at 10 μl / well, leave at room temperature for 30 minutes or longer, discard the solution, and dry at 4 ° C. The dried microplate is washed 3 times with 250 μl / well of deionized water. 3) 55 mg / ml Mouse Gamma Globulin on a microplate
And Goat Gamma Globulin containing 1% bovine albumin solution
Dispense 100 μl / well and add 50 μl of sample (serum diluted 5 times with PBS) or standard solution.

4) Incubate at 37 ° C. for 1.5 hours. 5) Wash with 250 μl / well of 0.005% Tween20 solution 5 times. 6) Biotin-labeled Fab'-conjugated IgG-apoA1 / polyclonal antibody (DAKO) made up to 1.6 μg / ml with 1% BSA solution was 100 μl /
Dispense well. 7) Incubate at 37 ℃ for 1.5 hours. 8) Wash 5 times with 250 μl / well of 0.005% Tween 20 solution as in 5). 9) 1 HRP labeled avidin D (Vector laboratories)
Dilute 15,000 times with 1% casein solution and dispense 100 μl / well.

10) Incubate at 37 ° C. for 30 minutes. 11) Wash 5 times with 250 μl / well of 0.005% Tween20 solution. 12) Add 100 parts of coloring reagent consisting of hydrogen peroxide solution and TMBZ solution.
Dispense μl / well and incubate at room temperature for 30 minutes. 13) Dispense 100 μl / well of 1M aqueous phosphoric acid solution to stop the reaction. 14) Measure the light at the main wavelength of 450 nm and the sub wavelength of 630 nm. 15) Calculate the concentration of modified HDL (oxidized HDL) containing 4-hydroxy-2-nonenal in the sample from the calibration curve obtained by artificially adjusted modified HDL (oxidized HDL).

6. Blood 4-hydroxy-2-nonenal-containing oxidatively modified lipoprotein concentration and serum lipid (cholesterol,
Relationship with HDL cholesterol concentration Figure 3A and B show oxidized LD containing 4-hydroxy-2-nonenal in blood.
The relationship between L and oxidized HDL concentration and serum lipid concentration was shown. It was confirmed that 4-hydroxy-2-nonenal-containing oxidatively-denatured lipoprotein was frequently present in hyperlipidemia in any of the oxidatively-denatured lipoproteins.

7. Modes of presence of two types of oxidatively modified LDL present in blood and their clinical significance (hypothesis) By taking oxidatively modified LDL as an example, the mode of existence of denatured lipoproteins having different degrees of oxidation in blood Its clinical significance is shown in FIG.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the formation of a complex of oxidized LDL by various oxidizing agents and the amount of 4-hydroxy-2-nonenal produced.

[Fig. 2] Various protein / LDL complexes and HNE / LDL (advanced peroxide LD
It shows the relationship with L).

FIG. 3: Oxidized LDL containing 4-hydroxy-2-nonenal in blood,
It is a diagram illustrating the relationship between the concentration of oxidized HDL and the concentration of serum lipid.

FIG. 4 shows the mode of existence of denatured lipoproteins having different degrees of oxidation existing in blood and their clinical significance.

Continuation of the front page (56) Reference JP-A-8-178921 (JP, A) JP-A-9-104699 (JP, A) JP-A-10-19890 (JP, A) JP-A-10-142226 (JP , A) Biochem. J. , 1992, vol. 288, p. 249-254 Biochemistry, October 18, 1994, vol. 33, no. 41, p. 12487-12494 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/53

Claims (8)

(57) [Claims] 1. A modified lipoprotein (modified lipoprotein: including oxidized lipoprotein) obtained by oxidative modification of lipoprotein, an acute phase reactive substance, a blood coagulation / fibrinolysis system-related protein, or an inflammatory cell is produced. A method for detecting a complex with a bactericidal substance using an anti-4-hydroxy-2-nonenal (HNE) antibody. 2. α1-antitrypsin, fibrinogen, C-reactive protein (CRP), fibronectin,
The method for detecting denatured lipoprotein according to claim 1, wherein a complex of a denatured lipoprotein and an acute phase reactive substance selected from α1-antichymotrypsin, α1-acid glycoprotein or a complement component is used as a measurement target. 3. A coagulant selected from tissue factor, plasminogen, prothrombin, thrombin, antithrombin 3 or plasmin activator inhibitor 1.
The method for detecting denatured lipoprotein according to claim 1, wherein a complex of a fibrinolytic system-related protein and denatured lipoprotein is used as a measurement target. 4. The method for detecting denatured lipoprotein according to claim 1, wherein a complex of a bactericidal substance produced by inflammatory cells selected from myeloperoxidase, lactoferrin, lysozyme or basic protein and denatured lipoprotein is used as a measurement target. . 5. The method for detecting denatured lipoprotein according to claim 1, which uses an immunoassay selected from enzyme immunoassay, latex agglutination, immunoluminescence assay or immunochromatography. 6. The immunoreaction detection reagent selected from anti-hydroxy-2-nonenal (HNE) antibody and anti-human ApoB antibody or anti-human ApoA1 antibody labeled with a labeling substance, according to claim 2 to 4. Method for detecting denatured lipoprotein. 7. A mouse myeloma cell line and 4-hydroxy-2-
A monoclonal antibody produced by a hybridoma obtained by fusing nonenal (HNE) with α1 antitrypsin, and spleen cells of a mammal immunized with 4-hydroxy-2-nonenal modified α1 antitrypsin, 4-hydroxy-2-, which is contained in denatured lipoprotein, does not react with native α1 antitrypsin or plasma protein
The method for detecting denatured lipoprotein according to claim 2, wherein a monoclonal antibody that specifically recognizes nonenal is used. 8. An arteriosclerosis comprising an antibody that specifically binds to 4-hydroxy-2-nonenal in the denatured lipoprotein according to any one of claims 2 to 4 and an immune reaction detection reagent. Diagnostic kit.