JP3365885B2 - Assay method for human oxidized lipoprotein - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring human oxidized lipoprotein. More specifically, the present invention is characterized by contacting a blood component with an antibody that recognizes oxidized phospholipid, and measuring the reactivity of the antibody with respect to a sample to measure the oxidized lipoprotein contained in the blood component. The present invention relates to a method for measuring human oxidized lipoprotein in blood. The present invention also uses the above-mentioned measurement method to coronary artery disease such as myocardial infarction and angina, cerebral artery disease such as cerebral infarction and cerebrovascular dementia, or nephropathy, renal artery system such as diabetic nephropathy. The present invention relates to a method for diagnosing various cardiovascular diseases such as diseases and peripheral arterial diseases such as peripheral arterial occlusion.

[0002]

BACKGROUND ART Arteriosclerosis frequently occurs in muscular arteries such as aorta, coronary artery, cerebral artery and carotid artery and is a main cause of angina, myocardial infarction, cerebral infarction and the like. Elevated serum cholesterol, platelet aggregation, endothelial damage, etc. have been proposed as the cause, but the cause is hardly analyzed at present.

Since the association between oxidized lipoprotein, which is one of denatured lipoproteins, and the development of atherosclerotic lesions was pointed out by Steinberg et al., The problem of oxidized lipoproteins in the progression of arteriosclerosis has been highlighted. Bathing (eg Steinberg, D., Partha
sarathy, S., Carew, TE, Khoo, JC, and Witztum, J.
L., (1989) N. Engl. J. Med. 320: 915).

The existence of receptors for oxidized lipoproteins such as scavenger receptors has been clarified, and oxidized LDL is taken up into cells through these receptors to become foam cells and initiate plaque formation. The hypothesis is that oxidative LDL damages endothelial cells, causing platelet adhesion and leukocyte aggregation,
A hypothesis has been proposed that plasma components infiltrate into blood vessels, which trigger the migration and proliferation of smooth muscle cells.

Whether or not oxidized LDL was indeed accumulated in the lesion was examined by, for example, 1988 in Haberland (Haber).
antibody to LDL modified with malondialdehyde; showing that anti-MDA-LDL antibody stains atherosclerotic lesions (Herberland, ME, Fong, D., and Chen)
g, L., (1988) Science 241: 215), and in 1989 YLa-Herttuala et al. also had anti-MDA-ap.
By immunoblotting with oB antibody, we searched for apoB extracted from the lesion and received oxidative modification.
It is reported that LDL was indeed extracted from the lesion (Yl
a-Herttuala, S., Parinski, W., Rosenfeld, ME, P
arthasarathy, S., Carew, TE, Butler, S., Witztu
m, JL, and Steinberg, D., (1989) J. Clin. Inves
t. 84: 1086). However, the antibody used here was obtained by using LDL artificially modified with malondialdehyde as an antigen, but it crosses not only the oxidation product of LDL but also other oxidized proteins such as albumin oxide. It has the property of showing a reaction.

[0006] However, a hybridoma was prepared by using a homogenate of atherosclerotic lesion as an antigen, and a hybridoma was prepared from the hybridoma.
It has been disclosed that a highly specific monoclonal antibody can be obtained by selecting a hybridoma that produces an antibody that specifically recognizes LDL (Itabe, H., Takeshima, E., Iwasa
ki, H., Kimura, J., Yoshida, Y., Imanaka, T., Taka
no, T., (1994) J. Biol. Chem. 269 (21): 15274). Since this antibody is produced by clone FOH1a / DLH3, FO
Although it is named H1a / DLH3, the antibody specifically reacts with oxidized lipoprotein and does not cross-react with normal lipoprotein, malondialdehyde-modified LDL, acetylated LDL, etc. It is disclosed that an epitope recognized by the antibody is generated when phosphatidylcholine, which is a component of lipoprotein, is oxidized. It is also disclosed that the antibody is an antibody that specifically recognizes foam cells in a human atherosclerotic lesion.

[0007] On the other hand, in previous studies, the oxidation of LDL was
It is thought to be caused by secondary chemical modification after deposition in vascular tissue, but there is a possibility that lipoproteins that have undergone oxidative modification are present in circulating blood due to active enzymes etc. generated at inflammatory sites. . In fact, it has been reported that lipids are extracted from human blood or its LDL fraction, the presence of peroxidized phospholipids in them is verified, and that it is elevated during diseases such as cardiac ischemia, diabetes and hepatitis (Miyazawa, T., (198
9) Free Radical Biology 7: 209, Hodis, HN, Krams
ch, DM, Avogaro, P., Bittolo-Bon, G., Cazzolat
o, G., Hwang, J., Peterson, H., and Sevanian, A.,
(1994) J. Lipids Res. 35: 669). However, the measurement method is complicated and unsuitable for measuring a large number of clinical specimens to clarify the clinical diagnostic significance of the oxidized lipoprotein in blood. The relationship is still unclear. If oxidized LDL is deeply involved in the development of atheromatous lesions, highly sensitive and quantitative detection of oxidized lipoproteins such as oxidized LDL in circulating blood would be useful for early diagnosis of disease progression. Obviously, the development of such a method was highly desired.

[0008]

Therefore, the object of the present invention is to provide a novel method for measuring human oxidized lipoprotein. It is still another object of the present invention to provide a method for measuring human oxidized lipoprotein that detects oxidized lipoprotein such as oxidized LDL in circulating blood with high sensitivity and quantitatively by a relatively simple procedure. is there. The present invention also measures human oxidized lipoprotein in blood, coronary artery disease such as myocardial infarction and angina, cerebral artery disease such as cerebral infarction and cerebrovascular dementia, or nephropathy,
An object is to provide a method for diagnosing various cardiovascular diseases mainly caused by atherosclerosis, including renal arterial diseases such as diabetic nephropathy and peripheral arterial diseases such as peripheral arterial occlusion. To do.

[0009]

The above objects are achieved by a method for measuring human oxidized lipoprotein, which comprises measuring an oxidized lipoprotein in plasma using an antibody that recognizes an antigen produced by the oxidation of phospholipids.

The present invention is also a method for measuring human oxidized lipoprotein in which the antibody recognizes an antigen produced by the oxidation of phosphatidylcholine in the presence of the peptide. The present invention also provides that an antibody that recognizes an antigen produced by the oxidation of phospholipids immunizes a suitable mammal and / or an antibody-producing lymphocyte of the mammal with a homogenate of atherosclerotic lesions to produce antibody of the animal. Fused lymphocytes and myeloma cells, isolated anti-human atheroma antibody-producing fused cell group formed, fusion cells selected as those that react specifically with oxidized human lipoprotein from the cell group It is a method for measuring human oxidized lipoprotein that is produced. The present invention is also a method for measuring human oxidized lipoprotein, wherein the antibody is produced by the hybridoma cell line FOH1a / DLH3 (accession number FERM P-14153). The present invention further provides that after diluting the plasma and / or the lipoprotein fraction separated therefrom to an optimum concentration,
A method for measuring human oxidized lipoprotein, which comprises contacting an antibody that recognizes an antigen produced by the oxidation of phospholipids, and then contacting the oxidized lipoprotein bound to the antibody with an antibody that recognizes the lipoprotein. .
The present invention further provides a method for measuring human oxidized lipoprotein, characterized in that an antibody that recognizes an antigen produced by the oxidation of phospholipids is immobilized on a carrier.

[0011]

In order to solve the above-mentioned problems, it is necessary to have a method of separately quantifying various lipoproteins having a clear epitope and high specificity for oxidized LDL. The present inventors have revealed that the antibody FOH1a / DLH3 obtained using an atherosclerotic lesion as an antigen recognizes an epitope generated by the oxidation of phospholipids and has a property of specifically binding to an oxidized lipoprotein. (Patent Application No. 6-51,209, Itabe, H., Takeshima, E., Iwas
aki, H., Kimura, J., Yoshida, Y., Imanaka, T., Tak
ano, T., (1994) J. Biol. Chem. 269 (21): 15274)).

As a result of intensive studies on such an antibody, the present inventors have found that such an antibody is the most excellent antibody that can provide the required assay method described above. It was the invention.

That is, as described above, highly sensitive and quantitative detection of oxidized lipoprotein can be performed by using an antibody that recognizes an antigen produced by the oxidation of phospholipids. In particular, the antibody
When the atherosclerotic lesion is actually obtained by sensitizing an appropriate animal, and when the epitope recognized by it is derived from the structure generated by the oxidation of phosphatidylcholine in the coexistence of the peptide, the effect is Become more certain.

The fact that such an antibody is excellent in solving the above problems is due to the fact that an antibody having the following properties can be obtained. The antigen of such an antibody is highly likely to be expressed by the production of lipoprotein that actually occurs in human tissues, and is very likely to be generated by the oxidation of lipoprotein having both peptides and phospholipids in plasma proteins. The antibody produced by the hybridoma FOH1a / DLH3 has exactly that property.

When measuring with such an antibody, the object can be achieved by directly contacting plasma and / or serum with the antibody.
When using the SA method or the like, in order to prevent nonspecific adsorption and the like due to the measurement method, the sample is fractionated in advance to a lipoprotein fraction by an appropriate method (for example, ultracentrifugation) and then used. Good.

Second, since the epitope recognized by such an antibody does not depend on the apoprotein, it can provide a method for separately evaluating the oxides of different lipoproteins in blood. For this purpose, it is necessary to combine two types of oxidized phospholipid-specific antibody and the lipoprotein-specific antibody. At this time, it is convenient to use a so-called sandwich ELISA method in which either antibody is immobilized on a flat or spherical carrier such as a plastic plate or glass beads.

At this time, there is no limitation on which of the antibodies is immobilized, but when the antibody titer of the antibody that recognizes the oxidized phospholipid is high, it is preferable to immobilize either of them because the antigen can be concentrated. It is advantageous in that the sensitivity can be increased. In the examples, E prepared by immobilizing the antibody produced by the hybridoma FOH1a / DLH3
An example of the LISA method will be shown, but it goes without saying that the present invention is not limited to this example.

Thirdly, by using such an antibody, as shown in the examples, for example, by using an artificially prepared oxidized LDL as a standard substance, its value can be measured, for example, per 1 μg of LDL protein. It can be quantitatively evaluated in the form of ng amount of oxidized LDL.

As described above, highly specific and highly sensitive, the degree of oxidation of each lipoprotein can be discriminated, and quantifiable oxidizable LDL in blood can be measured by a simple method such as an ELISA method. It is, therefore, the first time that the present invention has provided a measurable form for a large number of clinical specimens.

Hereinafter, the present invention will be described in more detail based on the embodiments.

The assay method of the present invention comprises contacting a blood component of a subject with an antibody that recognizes an antigen produced by the oxidation of phospholipids as described above, and quantifying the amount of the antigen specifically reacted with the antibody. Is characterized by the oxidized lipoprotein contained in blood. Measurement is RIA method, EL
It can be performed based on a known method such as ISA method, immunoblotting method, immunoprecipitation method and the like.

Further, as described above, since the epitope recognized by the antibody recognizing the antigen produced by the oxidation of the phospholipid according to the present invention does not depend on the apoprotein, an antibody specific to the lipoprotein is selected. By combining, the oxides of different lipoproteins in blood can be evaluated separately. Antibodies specific for such lipoproteins include chylomicron, VLDL, LDL
, HDL2, HDL3, Lp (a) and the like can be used. Especially in this, oxide LD
The evaluation of L is important because of its association with oxidized LDL and atherosclerosis, and in addition to this, Lp (a) (eg, Scanu, AM, L
awn, RM, and Berg, K., (1991) Lipoprotein (a) and ath
erogenesis, Ann, Int. Med. 115: 209-218). Antibodies to these lipoproteins can be easily obtained or prepared as commercial products or by known methods. When separately evaluating different lipoprotein oxides in blood in this manner, either the oxidized phospholipid-specific antibody or the lipoprotein-specific antibody as described above, preferably the one with a higher antibody titer, is used as a plate-shaped sample. Alternatively, it is desirable to solidify into a spherical shape or the like, and it is particularly preferable to use the sandwich ELISA method. For example, examples of the carrier used in the solid phase include a plate such as a multi-well plate and beads, which are commonly used in these fields, such as plastic or glass instruments.

A blood component as a sample to be measured is collected from a subject, preferably a blood sample obtained by adding an anticoagulant such as heparin to obtain a blood sample, which is separated by a conventional method such as a centrifugation method. It is the plasma or serum component obtained by Also,
This plasma component may be further separated by ultracentrifugation to obtain a lipoprotein fraction in order to suppress non-specific adsorption due to the measurement method and to perform measurement with higher accuracy.

In the measurement, such plasma and / or lipoprotein fraction is diluted to an optimum concentration. The concentration depends on the measurement conditions and cannot be specified unconditionally, but, for example, 0 to 500 μg / m
Dilute to 1, preferably 0-100 μg / ml. The dilution medium is not particularly limited, but physiological saline, phosphate buffer (PBS) containing EDTA, etc. may be used.

The contact between the plasma and / or lipoprotein fraction at an optimum concentration and the above-mentioned oxidized phospholipid-specific antibody is carried out by contacting the oxidized phospholipid and the oxidized phospholipid-specific antibody contained in such a blood component. It is not particularly limited as long as the specific reaction with the antibody sufficiently proceeds, but for example, at 4 to 30 ° C, more preferably at 25 ° C,
It is desirable to allow the reaction to stand for 1 to 24 hours, more preferably 1 to 2 hours. In addition, the concentration of the oxidized phospholipid-specific antibody at that time may be a sufficiently supersaturated amount than the amount of phospholipid that is considered to be present in the blood sample,
Although it depends on the antibody titer of this antibody and the type of measurement method, for example, oxidized LD present in 1 μg of plasma
In the case where the amount of L is assumed to be about 0 to 1 ng, DLH as described below as an oxidized phospholipid-specific antibody
When 3 antibodies are used, 0.2 ~
It is desirable that the amount is 1.0 μg, and more preferably around 0.5 μg.

Next, the antibody that recognizes the antigen produced by the oxidation of phospholipid used in the assay method of the present invention will be described in detail.

The method for obtaining such an antibody is not particularly limited, but is preferably a method for sensitizing atherosclerotic lesions to an appropriate animal as described below,
It is produced by a hybridoma cell line obtained by the following method based on a general cell fusion method.

The animal species used for preparing the hybridomas are not particularly limited, and conventionally used mice, rats, hamsters and the like can be used. Especially, Balb / c is easy to obtain and handle. Mice are preferred, and splenocytes of these animals are mainly used. In addition, human lymph node cells and peripheral lymphocytes can also be used.

Antigens for immunization against these animals are prepared from atherosclerotic lesions. For example, obtain a diseased blood vessel taken out by autopsy or bypass surgery immediately after the death of an arteriosclerosis patient, cut out a blood vessel part including atherosclerotic lesions from this diseased blood vessel, and exfoliate the adventitia part in a buffer solution. After removal, the intima and media of the lesion (intima and med
ia) is homogenized under cooling with a homogenizer, preferably under ice-cooling, and the supernatant obtained after standing is used as the antigen solution. If necessary, centrifuge after standing,
It is also possible to add a buffer solution to the obtained pellet and perform the same operation, and combine the obtained supernatant with the previous supernatant to obtain an antigen solution. The antigen solution thus prepared is preferably frozen and stored immediately before use after being replaced with an inert gas such as argon.

Next, the above-prepared animal species are immunized with the antigen composed of homogenate of atherosclerotic lesion at a predetermined protein (antigen) concentration. At this time, if necessary, an adjuvant such as Freund's complete adjuvant or Freund's incomplete adjuvant may be added.

The dose depends on the animal species,
In the case of mice, the initial immunization is 2.0-60 μg (protein) /
The number is about 40 μg (protein) / animal, and more preferably about 40 μg (protein) / animal.

Furthermore, after the initial immunization, it is desirable to carry out booster immunization at an interval of about 2 weeks and 4 weeks, for example, with the same or less amount of protein than the initial immunization.

Two to three days after the final immunization, blood was collected from the immunized animals and subjected to ELISA (enzyme-linked immunosorbent assa).
y) The increase in serum antibody titer is confirmed by assay methods such as immunoblotting and immunoblotting, and immunized animals with an increase in antibody titer are screened.

From the immunized animal screened, splenocytes, or antibody-producing cells from lymph nodes, etc. are collected,
The cells are washed and suspended in a maintenance medium such as RPMI medium or DMEM medium heated to about 37 ° C, and the number of viable cells is counted.

On the other hand, HGPRT (hypoxanthine-guanine
Tumor cells of phosphoribosyl transferase) -deficient strain were treated with fetal calf serum (FCS) -added RPMI medium and FCS-added DME.
The cells are grown in a growth medium such as M medium and cultivated in a logarithmic growth phase. The tumor cells of the HGPRT-deficient strain include, for example, P3 / X63-Ag8 (X63) (the names in parentheses are the same below), P3 / NSI-1-Ag4-1 (NS-1), P3 / X63- Ag8.U1
(P3 / U1), Sp2 / O-Ag14 (Sp2 / O), FO, 210.RCY3.Ag 1.2.
3. (Y3), U-266AR1 (SKO-007), LICR-LON-HMy2 (HMy2),
Known tumor cells such as 8226AR / NIP4-1 (NP41) can be used depending on the animal species used. The tumor cells in the logarithmic growth phase are adjusted so that the number of tumor cells is 1: 1 to 1:10 with respect to the number of antibody-producing cells,
FCS components that inhibit cell fusion are removed by washing with a maintenance medium such as RPMI medium and DEME medium heated to about 37 ° C.

Then, the antibody-producing cells whose number of cells has been adjusted and the tumor cells are mixed in a container such as a glass tube and centrifuged to obtain a pellet, and the supernatant is removed as much as possible.
In addition, the following operations including this operation are 20 to 37 ° C.,
More preferably, it is desirable to carry out under a temperature condition of about 37 ° C.

Then, the obtained pellets are
Cell aggregating medium warmed to 37 ° C, more preferably about 37 ° C, is slowly added while loosening the pellet. As the cell aggregating medium, compounds such as polyethylene glycol (PEG), lysolecithin, glycerol oleate, or inactivated Sendai virus (HVJ), measles virus, Paramyxovirus such as Newcastle disease virus, etc. can be used. Of these, PEG is particularly preferable. When PEG is used, it is desirable to dilute it to a concentration of about 45 to 50% by weight in the case of PEG4000, although it depends on the average molecular weight of the medium, such as RPMI medium and DMEM medium.

After the addition of the cell-aggregating medium, the mixture is further stirred for about 1 to 2 minutes, and then a maintenance medium such as RPMI medium is slowly added in 2 to 3 times.

Then, in order to remove the cell aggregating medium such as PEG, centrifugation is performed under weak conditions of, for example, 800 to 1200 × g and 3 to 5 minutes, and the supernatant is removed.

Subsequently, while loosening the obtained pellet, a selective medium such as FCS-added HAT medium was slowly added so that the spleen cell concentration was 1 × 10 ⁶ to 1 × 10 ⁷ cells / ml, Dispense into each well of a multi-well plate such as a 96-well plate, at a temperature of about 37 ° C., a CO ₂ concentration of about 7%,
Incubate under the condition of 100% humidity. During the culture period,
Although it depends on the state of the cells, the liquid is changed at intervals of about 2 to 3 days. The medium conditions are not limited to those exemplified above, but in addition to this, for example, a growth medium such as FCS-added RPMI medium is first added to the pellet, and after the culture is started, selection is performed. A mode in which a medium is added to each well is also possible. The unfused cells begin to die rapidly from around the 3rd day, and completely die in about 7 days. On the other hand, cells that have been successfully fused, that is, hybridomas, start forming colonies around this time. Screening as described below is started from the wells in which formation of hybridoma colonies was observed, and subcultured to a larger plate such as a 24-well plate as needed.

Screening is carried out by RIA method and ELISA.
Method, immunoblot method, etc., of which the ELISA method is preferred. As the antigen, oxidized LDL obtained by reacting with CuSO4 for 3 hours or more is used. Native LD as required
L may be used in combination. Particularly preferred is an antigen produced by the oxidation of phosphatidylcholine, and an antigen that recognizes this in the presence of a peptide is desirable. Based on each assay method, the culture supernatant collected from the wells in which the formation of hybridoma colonies was observed was screened and oxidized LD
Select a cell line that becomes positive in the reaction with L (and negative in the reaction with native LDL).

Then, cloning is immediately carried out from the wells positive in the screening. Cloning can be performed by using limiting dilution, single cell manipulation, etc., but the limiting dilution method is technically easy and is preferable.

When the cloned cells grow again, screening is carried out in the same manner as described above, cloning is repeated again, and a high-producing cell line which does not react with native LDL but reacts only with oxidized LDL is identified.

The method for preserving the obtained hybridomas is not particularly limited, but for example, as many cells as 1 vial for cryopreservation, for example, 1 × 1 may be used.
The 0 ⁷ ~2 × 10 ⁷ or so, were suspended in 90% FCS, 10% dimethyl sulfoxide (DMSO) of about 1 to 2 ml, is suitable method for cryopreservation in liquid nitrogen.

By the cell fusion operation as described above using Balb / c mice, there was no reaction with native LDL, and oxidized LDL
It is possible to obtain a cell line that reacts only with (Itabe, H., Takeshima, E., Iwasaki, H., Kim.
ura, J., Yoshida, Y., Imanaka, T., Takano, T., (19
94) J. Biol. Chem. 269 (21): 15274). In particular, the antibody FOH1a / D described in the same article
LH3 is a particularly preferred monoclonal antibody in that it does not react with malondialdehyde-modified LDL (MDA-LDL) or acetylated LDL (AcLDL). The mouse-mouse hybridoma cell line FOH1a / DLH3 that produces this antibody is
Deposited at the Institute of Biotechnology, Industrial Technology Institute of Industrial Technology, deposit number
Granted FERM P-14153.

Monoclonal antibody FOH1a / DLH3
Reacts with LDL, which is an artificially oxidized LDL using copper ion, but does not react with unmodified LDL, and other methods (eg malondialdehyde, acetylation, etc.)
It does not react to the modified one. It also does not react with other serum proteins such as those obtained by oxidizing albumin or globulin. However, it reacts with oxidized high-density lipoprotein (HDL), which is a lipoprotein different from LDL.

However, the method for obtaining the antibody recognizing the antigen produced by the oxidation of phospholipid according to the present invention is not limited to the method for sensitizing atherosclerotic lesions as described above to an appropriate animal. Other than this, for example, a method of using oxidized LDL as an immunogen, apoprotein, or
A method of using an phospholipid-oxidized substance as an immunogen in the presence of a part of its constituent peptides is considered.

The following conditions can be considered as the conditions for producing the artificial oxidized lipoprotein required for obtaining the assay method of the present invention or the hybridoma producing the antibody used in the assay method. That is,
A lipoprotein fraction is obtained from normal human serum by, for example, a centrifugal precipitation method, and this fraction is purified by dialysis and desalting, if necessary, and then the protein concentration is 0.1 to 1 mg / m 2.
l, more preferably 0.2 mg / ml, CuSO ₄ concentration 2.5～25MyuM, more preferably at a rate of 5 [mu] M, was added CuSO ₄ in lipoprotein fractions, under about 37 ° C., 3 to 24 hours To react.

[0049]

EXAMPLES Next, the sandwich ELISA analysis method for oxidized lipoprotein according to the present invention will be described more specifically with reference to Examples.

Example 1 Sandwich ELISA analysis method of oxidized LDL (1) Preparation of LDL fraction in human plasma Human plasma obtained by heparin blood sampling had a final concentration of 0.25.
Add EDTA to bring the concentration to mM and add 0.75 ml
Collect each into a test tube for ultracentrifugation (1 to 4 ml volume),
2 0.15M NaCl containing 0.3 mM EDTA
1. Overlay 50 μl at 185,000 × g at 10 ° C.
Centrifuge for 5 hours. Discard 150 μl of the upper layer and 750 μm of the lower layer
1 aliquot, 150 μl of KBr solution (50 w / v%)
To give a specific gravity of 1.063. Transfer the specific gravity adjusted plasma to the bottom of the ultracentrifugation test tube (1 to 4 ml volume).
Centrifuge at 4,000 xg for 16 hours at 10 ° C. The upper orange band (about 100-150 μl) was carefully collected and 4 times against PBS containing 0.25 mM EDTA.
Dialyze at 6 ° C. for 6 hours (3 liters are changed twice at 2-hour intervals). The obtained LDL sample is quantified for protein and cholesterol.

(2) DLH3 antibody and non-immunized mouse IgM antibody (0.6 μg / well each) diluted with PBS were added to the sandwich ELISA analysis plate, and the plate was left at room temperature for 2 hours. Subsequently, 200 μl of 1% BSA-TBS solution (pH 7.4) is added, and the mixture is left standing at room temperature for 2 hours for blocking. The blocking solution was discarded, and the oxidized LDL standard product and the human LDL fraction were directly dispensed (0.1 to 20 ng LDL protein / well for the oxidized LDL standard product, 2 μg LDL protein / well for the human LDL fraction), and the mixture was placed at 4 ° C. Leave for 18 hours. After washing three times with 0.05% Tween 20-TBS (pH 7.4),
000-fold diluted sheep anti-human apo B antibody (Bindind Site
100 μl) and left at room temperature for 2 hours. 0.
Alkaline phosphatase-labeled donkey anti-sheep Ig diluted 2,000-fold with 2% skim milk-TBS after washing 3 times with 05% Tween 20-TBS (pH 7.4).
Add 100 μl of G antibody (Chemicon),
After standing at room temperature for 2 hours, 0.05% Tween 20-
It was washed 3 times with TBS (pH 7.4). 100 μl of 0.1% p-nitrophenylphosphate solution (pH 8.8) is added to develop color, and the absorbance at 405 nm after 10 to 60 minutes is measured. (3) Analysis Results Using Human Plasma (Healthy Subjects and Renal Dialysis Patients) Results obtained by analyzing a typical calibration curve using oxidized LDL as a standard and clinical samples are shown in FIGS. 1 and 2, respectively.

Example 2 Sandwich ELISA Assay for Oxidized Lp (a) (1) Preparation of Peroxidase-Labeled Anti-Lp (a) Antibody Human plasma obtained by heparin blood sampling had a final concentration of 0.25 mM.
EDTA was added so that
250 μl of 0.15M NaCl containing
Centrifuge at 105,000 xg for 20 hours at 8 ° C. The upper layer is discarded, KBr powdered in a mortar in advance is added to the lower layer, and the mixture is dissolved at 4 ° C. without bubbling, and the specific gravity is 1.
Adjust to 125 and centrifuge at 105,000 xg for 20 hours at 8 ° C. The orange band in the upper layer was carefully collected, and 1 M NaCl, 2 mM EDTA,
Gel filtration is performed using 10 mM phosphate buffer as a developing solvent. Each obtained fraction is Lp manufactured by Terumo Co., Ltd.
(A) The Lp (a) fraction is collected by measurement with a measurement kit. This fraction was added to Pharmacia Lysine Sepharose 4B.
Then, the adsorbed fraction was eluted with a buffer containing 0.2 Mε-aminocapronsan and dialyzed against PBS containing 0.25 mM EDTA to obtain an Lp (a) fraction.
An antiserum was prepared by immunizing a rabbit with 0.5 mg of the obtained Lp (a). The obtained anti-Lp (a) serum was purified to IgG using a Protein G column manufactured by Pharmacia, and the anti-LDL antibody was removed through a separately prepared LDL column to obtain an anti-Lp (a) antibody. Purified anti-Lp (a)
The antibody was labeled with peroxidase using the maleimide method.

(2) Preparation of Lipoprotein Fraction in Human Plasma Human plasma obtained by heparin blood sampling had a final concentration of 0.25 m.
Add EDTA to give M, and add 0.75 ml of each to a test tube for ultracentrifugation (1 ml volume) to obtain 0.3 mM.
250 μl of 0.15 M NaCl containing EDTA
Overlay and centrifuge at 185,000 xg for 2.5 hours at 10 ° C. 150 μl of the upper layer is discarded, 750 μl of the lower layer is collected, KBr (70.0 mg) powdered in a mortar in advance is added, and the mixture is dissolved at 4 ° C. without foaming. Transfer the specific gravity-adjusted plasma (d = 1.12) to the bottom of the ultracentrifugation test tube (1 ml volume) and transfer it to 244,000 × g for 10
Centrifuge for 16 hours at ° C. The upper orange band (about 100 ~
150 μl) was carefully collected and 0.25 mM EDT
PBS containing A at 4 ° C for 6 hours (3 liters of 2
Change twice at time intervals) Dialyze. The resulting lipoprotein fraction is quantified for protein and cholesterol.

(3) Sandwich ELISA analysis Partially purified FOH1a / diluted with PBS in a plate
DLH3 antibody and non-immunized rat IgM antibody (0.
6 μg / well) and then left at room temperature for 2 hours.
Continuously, 1% BSA-TBS solution (pH 7.4) 200
Add μl and leave at room temperature for 2 hours for blocking.
The blocking solution is discarded, and the oxidized Lp (a) standard and the human lipoprotein fraction are dispensed as they are (oxidized Lp (a)).
(A) The standard product is 0.1 to 10 ng / well, the human lipoptein fraction is a 20-fold diluted PBS solution, and the mixture is allowed to stand at room temperature for 2 hours. 0.05% Tween 20-TBS (pH 7.
After washing 3 times with 4), 20 with 2% skim milk solution
100 μl of peroxidase-labeled anti-Lp (a) polyclonal antibody diluted with 00 times is added, and the mixture is allowed to stand at room temperature for 1 hour. 0.05% Tween 20-TBS (pH 7.
After washing 3 times with 4), o-phenylenediamine 3m
100 μl of 0.03% hydrogen peroxide solution containing g / ml is added to develop color, and after 10 to 15 minutes, the reaction is stopped with 50 μl of 2N sulfuric acid, and the absorbance at 492 nm is measured.

(4) Analysis Results Using Human Plasma (Patients with Renal Vascular Disease) Typical calibration curves using oxidized Lp (a) as a standard and analysis results of clinical samples are shown in FIGS. 3 and 4, respectively. Indicated.

[0056]

Industrial Applicability As described above, the present invention is characterized in that a blood component is brought into contact with an antibody that recognizes oxidized phospholipid, and the reactivity of the antibody with respect to the sample is measured. And a method for diagnosing various cardiovascular diseases mainly caused by atherosclerosis using the detection method. Such cardiovascular diseases include coronary artery diseases such as myocardial infarction and angina,
There are all cardiovascular diseases such as cerebral arterial diseases such as cerebral infarction and cerebrovascular dementia, renal arterial diseases such as nephropathy and diabetic nephropathy, and peripheral arterial diseases such as peripheral arterial occlusion. . As shown in the examples, according to the present invention, a high concentration of oxidization in the blood of a patient who has undergone hemodialysis due to diabetic nephropathy.
LDL and oxidized Lp (a) were detected, and such diseases and oxidation were detected.
The causal relationship of LDL-oxidized Lp (a) was clarified. However, the effect of the present invention is not limited to this, and it is applied to all the diseases in which oxidized lipoprotein is involved, and the range of the disease is the future clinical study using the present invention. It goes without saying that it will be expanded by.

[Brief description of drawings]

FIG. 1 Oxidized LDL obtained in Example 1 of the present invention
Is a standard curve,

FIG. 2 is a graph showing the results of analysis of clinical samples in Example 1 of the present invention,

FIG. 3 Oxidized Lp obtained in Example 2 of the present invention
Calibration curve using (a) as a standard product,

FIG. 4 is a graph showing the results of analysis of clinical samples in Example 2 of the present invention.

─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kyoko Shimamura 3-6-6 Asahimachi, Machida-shi, Tokyo Inside Vessel Research Laboratory, Inc. (72) Inventor Tatsuya Takano 432 Terada-cho, Hachioji-shi, Tokyo Green Hill Terada 44-3 (56) References Hiroyuki Itabet et. al, "A Monoclonal Antibody gains (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/53 JISC file (JOIS)

Claims (12)

(57) [Claims] 1. A method of directly converting oxidized lipoprotein in plasma to a solid phase
A method for measuring oxidized lipoprotein, which comprises measuring oxidized lipoprotein in plasma using an antibody that recognizes an antigen produced by oxidation of phospholipids without immobilization . 2. Oxidation characterized in that plasma and / or a lipoprotein fraction separated therefrom is brought into contact with an antibody that recognizes an antigen produced by the oxidation of phospholipids, without directly immobilizing it on a solid phase. A method for measuring lipoproteins. 3. The oxidized lipoprotein in plasma is directly transferred to a solid phase.
It is possible to measure oxidized lipoproteins in plasma using an antibody that recognizes an antigen produced by oxidation of phospholipids and an antibody that recognizes lipoproteins without immobilization.
Measurement of oxidative lipoprotein characterized. 4. A plasma and / or a lipoprotein fraction separated therefrom are brought into contact with an antibody that recognizes an antigen produced by the oxidation of phospholipids without directly immobilizing it on a solid phase, and the oxidation bound to the antibody. A method for measuring oxidized lipoprotein, which comprises contacting the lipoprotein with an antibody that recognizes the lipoprotein. 5. recognize antigens which are produced by the oxidation of the phospholipid antibody, measurement of oxidation lipoproteins according to any one of claims 1 to 4 is immobilized on a carrier. 6. antigen recognizing antibody produced by the oxidation of phospholipids, measurement of oxidation lipoproteins according to any one of claims 1 to 4 is intended to recognize the antigens produced by the oxidation of phosphatidylcholine . 7. An antibody which recognizes an antigen produced by the oxidation of phospholipids, immunizes a suitable mammal and / or a lymphocyte in charge of antibody production of the mammal with a homogenate of atherosclerotic lesion, and produces the antibody of the animal. Fused lymphocytes and myeloma cells, isolated anti-human atheroma antibody-producing cell fusion cell group formed, the fusion cells selected as those that react specifically with the oxidized lipoprotein from the cell group The method for measuring oxidized lipoprotein according to claim 1 , which is produced. 8. An antibody that recognizes an antigen produced by the oxidation of phospholipids is a hybridoma cell line FOH1a /
The method for measuring oxidized lipoprotein according to any one of claims 1 to 4 , which is produced by DLH3 (accession number FERM BP-7171). 9. An antibody that recognizes the lipoprotein is an anti-antibody.
It is an apo B antibody and oxidized lipoprotein
9. The protein according to claim 3, which is poprotein (LDL).
The method for measuring oxidized lipoprotein according to 1. 10. An antibody that recognizes the lipoprotein is
Anti-Lp (a) antibody, in which oxidized lipoprotein is oxidized L
The oxidized lithium according to any one of claims 3 to 8, which is p (a).
Method for measuring poprotein. 11. Plasma and / or separated therefrom
The lipoprotein fraction is used by diluting it.
The method described in either. 12. The sandwich ELISA method is used for the measurement.
The method according to any one of claims 3 to 11, wherein