JPH10142226A - Arteriosclerosis measuring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure an onset of arteriosclerosis with high accuracy by making a denatured low specific gravity lipoprotein (LDL).α1 anti-trypsin polymeric complex in blood serum or blood a measured object. SOLUTION: A denatured LDL.α1 anti-trypsin polymeric complex forming a complex with denatured LDL, the component of arteriosclerosis, is a measured object. An immonoassay such as an enzyme immunization, a latex agglutination method, a luminous enzyme immunization or an immunochromatography is used to detect the denatured LDL.α1 anti-tripsin polymeric complex in blood serum or blood. An immunologic tissue staining method can also be used. The denatured LDL.α1 anti-trypsin polymeric complex can be singularly detected by antihuman polymeric α1 anti-trypsin monoclonal antibody, that is, this antibody does not detect α1 anti-trypsin but singularly detects the denatured LDL.α1 anti-trypsin polymeric complex.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a kit for measuring the onset of arteriosclerosis occurring in a living body due to circulatory diseases, diabetes, etc., and more particularly to a modified LDL / α1 antitrypsin polymer complex in blood. The present invention relates to a measurement kit for measuring the state of arteriosclerosis in a living body by measuring.

[0002]

2. Description of the Related Art Conventionally, there is no measuring method for measuring the state of atherosclerosis in a living body in blood, and there is a measuring method for measuring lipoprotein (a) or remnant cholesterol in serum or plasma. These are all measured as arteriosclerosis risk factors.

[0003]

However, in recent years,
It has been clarified that the above-mentioned measurement target does not reflect arteriosclerosis actually occurring in a living body, and thus the concentration of the above-mentioned measurement target in serum or plasma is
The fact is that it does not reflect arteriosclerosis. The present invention has been made in view of the above problems, and is a measurement kit capable of measuring the onset of arteriosclerosis with high accuracy by measuring a novel component that replaces the conventional measurement object. The purpose is to provide.

[0004]

As a cause of arteriosclerosis, denatured (oxidized) low-density lipoprotein (LDL) is known. Antitrypsin becomes a polymer,
The present inventors have discovered that a complex is formed with this modified LDL and present in blood, thereby completing the present invention. That is, in the present invention, a modified LDL-α1 antitrypsin polymer complex, which forms a complex with a modified LDL that is a cause of arteriosclerosis, is measured. In addition, in order to detect denatured LDL / α1 antitrypsin polymer complex in serum or plasma, commonly used immunological methods such as enzyme immunoassay, latex agglutination, luminescent enzyme immunoassay and immunochromatography are used. A measuring method can be used. Alternatively, an immunological tissue staining method can be used.

[0005] The denatured LDL which is the cause of arteriosclerosis is LDL oxidized by active oxygen generated in a living body, or LDL to which lipid peroxide or a degradation product thereof ingested as food is bound, and α1 antitrypsin. Becomes a polymer by this modified LDL
And a complex, and exists in blood as a modified LDL-α1 antitrypsin polymer complex. Therefore, detecting a denatured LDL-α1 antitrypsin polymer complex is to detect a denatured LDL that is a cause of arteriosclerosis. The present inventors have found that the anti-human polymer α shown in the Examples
It was confirmed that the denatured LDL / α1 antitrypsin polymer complex can be specifically detected by 1 antitrypsin monoclonal antibody. That is, as shown in FIG. 1, this antibody did not detect native α1 antitrypsin, but specifically detected a denatured LDL · α1 antitrypsin polymer complex.

As shown in FIG. 2, in gel filtration analysis, α1 antitrypsin is polymerized by denatured LDL as compared with native α1 antitrypsin having a molecular weight of 52,000 in blood, and denatured LDL · α1 antitrypsin polymer complex Was formed. It was also confirmed that the modified LDL-α1 antitrypsin polymer complex was present in the serum of arteriosclerosis patients. As shown in FIG. 3, the value of the method of the present invention was high in the hemoglobin A1c high diabetes group or the high cholesterol group. As described above, denatured LDL-α in serum or plasma
By measuring 1 antitrypsin polymer complex, the state of arteriosclerosis occurring in a living body can be measured.
That is, by measuring the denatured LDL-α1 antitrypsin polymer complex in serum or plasma, the state of arteriosclerosis in a living body can be measured, and arteriosclerosis can be diagnosed.

[0007]

EXAMPLE As an example, a method for producing a monoclonal antibody will be described. 1. Preparation of anti-human high molecular weight α1 antitrypsin monoclonal antibody [Preparation of antigen] Purified α1 antitrypsin was prepared. [Immunization of animals] This antigen was treated with phosphate buffered saline for 1 hour.
mg / ml solution, and emulsion obtained by mixing equal amounts of this solution and Freund's adjuvant,
500 μl intraperitoneally to 6 week old mice (Balb / C mice)
Was administered. This operation was performed three times in total every two weeks. [Cell fusion] Four days after the final immunization, spleen lymphocyte cells collected from the spleen of this mouse were used as mouse myeloma cells (P3-X63-Ag
8-U1). The fusion method uses a 50% polyethylene glycol 4000 solution as a fusion accelerator, and the fusion time, which consists of adding, mixing and diluting the fusion accelerator, is 10 minutes.
Minutes at 37 ° C. Next, the fused cells after completion of the fusion were dispersed in a HAT medium (RPMI medium containing hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum), and 200 μl was added to each well of 10 96-well microplates. The mixture was poured and cultured at 37 ° C. in the presence of 5% carbon dioxide.

[Selection and Unification of Hybridoma Producing Anti-Human High Polymer α1 Antitrypsin Monoclonal Antibody]
After about one week, 100 μl of the HAT medium in each well was aspirated, and HT medium (RPMI medium containing hypoxanthine / thymidine / 10% fetal bovine serum) was dispensed into each well.
〜3 days later, antibody-producing hybridomas were selected. The selection method was to fix the polymer α1 antitrypsin 9
100 μl of the culture supernatant of the hybridoma-forming colony of each well was dispensed into a 6-well microplate and reacted.
Then, after washing, 100 μl of a peroxidase-labeled anti-mouse immunoglobulin antibody was added, and the mixture was washed after the reaction, and the antibody of interest was detected by peroxidase activity. Next, colonies showing antibody production were collected and cloned by a limiting dilution method so as to obtain single hybridoma colonies. In this method, the collected colonies were diluted with an HT medium, and spread on each well of a 96-well microplate together with feeder cells such that the number of hybridomas was one or less per well. The above operation was performed twice to obtain a monocloned anti-human macromolecule α1 antitrypsin antibody-producing hybridoma. [Ascites of anti-human high-molecular α1 antitrypsin monoclonal antibody] Pristane (immunosuppressant) was intraperitoneally administered to 8-week-old mice (Balb / C mice). After 3 to 7 days, the antibody-producing hybridoma was intraperitoneally administered, and after about 7 days, the ascites-purified antibody was recovered from the abdominal cavity of the mouse. [Purification of Antibody] The antibody obtained by ascites was subjected to salt separation twice with 50% ammonium sulfate, and purified by dialyzing with phosphate buffered saline.

Next, a method of measuring an object to be measured by enzyme immunoassay (ELISA) will be described. 2. Measurement of denatured LDL-α1 antitrypsin polymer complex [Immobilization of antibody on microplate] Each well of a microplate (SUMILON, Japan) contains 5 μg / ml of anti-human macromolecule α1 antitrypsin monoclonal antibody at 5 μg / ml. M
100 μl of Tris buffer is dispensed at a time, and the solution is allowed to stand at 4 ° C. to adsorb the antibody. [Preparation of biotin-labeled antibody] Separately, anti-human α1 antitrypsin antibody (DAKOPATTS, Demmark) or anti-human Apo
The B antibody is converted to Fab 'with pepsin and 2 mercaptoethanolamine, and the Fab' is prepared by labeling this Fab 'with BiotinMaleimide.

[Determinated LDL-α1 in serum or plasma
Measurement of antitrypsin polymer complex] 100 per well
After dispensing 1 μl of Tris buffer (0.1 M, pH 8.0) containing 1% casein, adding 50 μl of serum or plasma, mixing and reacting at 37 ° C. for 2 hours. Next, Tween20 is set to 0.05
3 times with a phosphate buffer (0.02M, pH 7.4) containing 3%. Thereafter, a biotin-labeled anti-human α1 antitrypsin Fab ′ antibody solution or a biotin-labeled anti-human Apo B Fab ′ antibody solution (Tris buffer containing 1% casein) was added to each well at 100 μl.
After adding and mixing, the mixture is reacted at 37 ° C. for 1 hour, and washed three times as before. An avidinated peroxidase solution (Tris buffer containing 1% casein) was further added to each well for 10 times.
After adding 0 μl and mixing, the mixture was reacted at 37 ° C. for 30 minutes.
Wash 3 times as before. Substrate coloring solution is 1.66 mmol / l TMB
After dissolving Z (Dojin Kagaku) in methanol, mix the same amount of a substrate solution containing 0.2 mol / l Tris solution to a methanol concentration of 50% and a 35 mmol / l citric acid solution containing 0.02% H202. 100 μl of the prepared solution was added to each well, and allowed to stand at room temperature for 10 minutes. Then, a reaction stopping solution (2.5 mol / l phosphoric acid solution) 10
Add 0 μl to each well. Using a colorimeter for microplate, colorimetry is performed at a wavelength of 450/630 nm and absorbance is calculated.

[Brief description of the drawings]

FIG. 1 is a drawing showing the reaction characteristics of a modified LDL-α1 antitrypsin polymer complex measurement system using each sample.

FIG. 2 shows the formation of a modified LDL-α1 antitrypsin polymer complex by denatured LDL and the modified LDL-α1 antitrypsin polymer complex in the serum of a patient with arteriosclerosis.

FIG. 3 is a graph showing the value of the method of the present invention in a hemoglobin A1c high diabetes group or a high cholesterol group.

Claims (2)

[Claims] 1. Denatured LDL-α in serum or plasma
(1) A kit for measuring arteriosclerosis, wherein an antitrypsin polymer complex is measured. 2. The kit for measuring arteriosclerosis according to claim 1, wherein an immunological measurement method such as an enzyme immunoassay, a latex agglutination reaction, or an immunochromatography method, or an immunological tissue staining method is used. .