JPH09297137A - Antibody bound to denatured or modified lipoprotein, and measurement using this antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To precisely measure a denaturated or modified lipoprotein by use of an antibody which is not bound to a lipoprotein not denaturated or modified, but specifically bonded to the denaturated or modified lipoprotein. SOLUTION: From not more than 50 amino acids containing part or all of amino acid sequence represented by a sequence number 1, peptide is synthesized by liquid phase method or solid phase method. This peptide or its combination with a carrier producing antibody is used as immunogen to immunize a mammal or bird followed by purification to provide a polyclonal antibody or monoclonal antibody. By use of this, a measurement is performed by enzyme immunoassay, fluorescent immunoassay, radiation immunoassay or the like. According to this method, a denaturated or modified lipoprotein can be precisely measured without erroneously measuring lipoprotein not denaturated or modified, LDL and plasminogen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention has been pointed out to be a risk factor for promoting arteriosclerosis, and is specific to denatured or modified lipoprotein (a) which has an important role in the diagnosis of diseases. And a method for measuring denatured or modified lipoprotein (a) using this antibody.

The present invention is particularly useful in the medical field including the clinical laboratory field.

[0003]

2. Description of the Related Art Lipoprotein (a) (lipopro)
tein (a), Lp (a)) was first reported by Berg in 1963 as a mutant form of β-lipoprotein [K. Berg, Acta Pathol. Micr
obiol. Scand. , Volume 59, 369-382
P., 1963].

As a result of various studies thereafter, lipoprotein (a) is a protein unique to low-density lipoprotein (LDL) and lipoprotein (a), which normally plays a major role in transporting cholesterol ester in vivo. Apolipoprotein (a) (apolipoprotein)
(A), apo (a), apo (a)), LDL
B-100, which is the protein part of
It was revealed that the substance was a substance that was bound by a disulfide bond between and apolipoprotein (a).

It has been clinically revealed that many patients with ischemic heart disease such as angina and myocardial infarction have high lipoprotein (a) levels [G. Dahlen
et al. , Acta Med. Scand. (Sup
pl. ), 531, 1-29, 1972, K.S. B
erg et al. , Clin. Genet. , 16
Volume, 347-352, 1979, G. M. Kost
ner et al. , Atherosclerosis
s, 38, 51-61, 1981].

[0006] Lipoprotein (a) has no correlation with known risk factors for ischemic heart disease such as cholesterol, LDL-cholesterol and HDL-cholesterol, and is independent of arteriosclerosis and ischemic heart disease. It was reported to be a novel risk factor [C. Ehnholm e
t al. , Biochim. Biophys. Act
a, 236, 431-439, 1971, H.A. S
chriewer et al. , J. et al. Clin. Ch
em. Clin. Biochem. , Volume 22, 591-
596, 1984].

In 1987, Eaton et al. [D. L. Eat
on et al. Proc. Natl. Acad.
Sci. U. S. A. , 84, 3224-3228
Page, 1987] by biochemical methods, followed by McLean et al. [J. W. McLeanet al. , Natu
re, Vol. 330, pp. 132-137, 1987] is c
The amino acid sequence of apolipoprotein (a) was determined from the DNA base sequence.

From this, it was revealed that most of the molecular structure of apolipoprotein (a) is composed of a portion highly homologous to the plasminogen molecule acting in the fibrinolytic system.

This suggests that lipoprotein (a) is an important key for considering arteriosclerosis, lipoprotein and blood coagulation / fibrinolysis system from the same viewpoint.

Further, lipoprotein (a) is not only a risk factor for arteriosclerosis, but also shows a high value in people with diabetic nephropathy or restenosis after PTCA (percutaneous transluminal coronary angioplasty). There is also a report that there is a tendency and that it behaves similarly to an acute reactive protein such as CRP, and thus measuring lipoprotein (a) in a living body such as blood has clinically significant significance. However, a method capable of accurately measuring lipoprotein (a) has been desired.

The lipoprotein (a) can be measured by simple immunodiffusion method, rocket immunoelectrophoresis method, immunoturbidimetric method, latex nephelometric method, radioimmunoassay (RIA) [J.
J. Albers et al. , J. et al. Lipid R
es. , 18, 331-338, 1977], and enzyme-linked immunosorbent assay (EIA, ELISA) [A. Abe
et al. , Clin. Chim. Acta, 177
, 31-40, 1988], etc., and the antibodies obtained by immunizing animals with lipoprotein (a) as an immunogen are used in these immunoassays. When used as it is, lipoprotein (a) contains LDL in its molecule and has a portion highly homologous to plasminogen, so that it also reacts with LDL and plasminogen contained in the biological sample, and LDL and plus Since it also measures minogen, it is not possible to obtain an accurate measurement value of lipoprotein (a).

Therefore, from lipoprotein (a) to LDL
A method of producing an antiserum (polyclonal antibody) against lipoprotein (a) using apolipoprotein (a) excluding the portion as an immunogen has been reported [G. M. Flats et al. , J. et al. Biol. C
hem. , 261, 8712-8718, 1986.
Year, G. Utermann et al. , J. et al. Bio
l. Chem. 265, pp. 981-986, 199
0 years], the antiserum (polyclonal antibody) obtained by this method does not cross-react with LDL, but since apolipoprotein (a) has a portion highly homologous to plasminogen, this antiserum (Polyclonal antibody) has cross-reactivity with plasminogen.

Therefore, this antiserum (polyclonal antibody) could not be used unless a complicated operation of absorbing the antibody with human plasminogen to remove the antibody that reacts with plasminogen.

By the way, LDL, which is said to be a bad lipoprotein, is an LDL that has undergone denaturation or modification such as oxidation.
It has been pointed out that the pathogenesis of arteriosclerosis is related to.

It is an LD which has not been modified or modified.
In L, the LDL receptor of the cell performs negative feedback regulation according to the amount of cholesterol in the cell, and the amount of LDL in the cell is controlled to a normal value.
However, if LDL undergoes some chemical modification or denaturation in vivo to become a modified or denatured LDL, it is recognized as a foreign substance by macrophages, and is taken up into macrophages indefinitely to be foamed, resulting in arteries. It has a very significant meaning as an initial lesion of hardening [M. S. Brown, J.M. L. Goldstein,
Science, 232, 34-47, 1986.
Year〕.

As in the case of this LDL, when the lipoprotein (a) was subjected to certain oxidation, it did not exist in the unmodified or modified lipoprotein (a). It has been pointed out that these properties are manifested and that they may be a risk factor for promoting arteriosclerosis [M. E. FIG. Haberland et a
l. , J. et al. Biol. Chem. , 267, 4143
Pp. 4151, 1992, J. Am. Galle et a
l. , Europe. J. Pharm. , 265, 11
1-115, 1994].

This means that the denatured or modified lipoprotein (a) is a risk factor for arteriosclerosis in a different aspect from the non-denatured or unmodified lipoprotein (a). To do.

Denatured or modified lipoprotein (a) which is important as a risk factor for this arteriosclerosis
There has been a demand for a method capable of accurately measuring.

However, oxidized LDL and β2
-Glycoprotein I complex and oxidized HD
Regarding the method for measuring the complex of L and β2-glycoprotein I by the sandwich method of enzyme immunoassay, WO95
No. 093633 republished patent publication, but unmodified or unmodified lipoprotein (a)
An antibody that specifically binds to denatured or modified lipoprotein (a), which does not bind to, and a method for measuring denatured or modified lipoprotein (a) have not been known.

[0020]

DISCLOSURE OF THE INVENTION The present inventors do not bind to a lipoprotein (a) that has not been denatured or modified, which did not exist in the past, but do not bind to the lipoprotein (a) that has been denatured or modified. As a result of intensive studies aimed at developing an antibody that specifically binds to glycerin, and a method for measuring denatured or modified lipoprotein (a), as a result, denatured or unmodified lipoprotein (a), and further LDL And an antibody that does not bind to plasminogen and can specifically bind to a modified or modified lipoprotein (a), and a modified or modified lipoprotein (a), We have completed a measuring method that can accurately measure denatured or modified lipoprotein (a) without measuring LDL and plasminogen.

[0021]

The present invention comprises the following inventions.

(1) An antibody which does not bind to a lipoprotein (a) that has not been denatured or modified, but specifically binds to a lipoprotein (a) that has been denatured or modified.

(2) A peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or a conjugate of this peptide and a carrier is an immunogen. The antibody according to (1) above.

(3) The antibody according to (1) or (2) above, wherein the modification or modification is by oxidation, reduction, decomposition, heating or a modifying agent.

(4) The antibody according to (1), (2) or (3) above, which is a monoclonal antibody.

(5) The antibody according to (1), (2) or (3) above, which is a polyclonal antibody.

(6) The above (1), (2), (3),
A method for measuring denatured or modified lipoprotein (a) using the antibody according to (4) or (5).

(7) The denatured or modified lipoprotein (a) is a lipoprotein (a) which has been denatured or modified by oxidation, reduction, decomposition, heating or a denaturing agent.
The measuring method according to (6) above.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Denatured or modified lipoprotein (a) In the present invention, the denatured or modified lipoprotein (a) means oxidation, reduction, hydrolysis or other decomposition, heating,
Or, a denaturing agent such as a protein denaturing agent undergoes a change in primary structure, higher-order structure or three-dimensional structure, decomposition or chemical modification, so that the original natural (native) lipoprotein (a) is denatured or modified. The lipoprotein (a) that has not been subjected to the treatment has a different structure, composition, or steric configuration.

(2) Antibody The antibody of the present invention is an antibody which does not bind to the non-denatured or modified lipoprotein (a) but specifically binds to the denatured or modified lipoprotein (a). However, it also does not bind to LDL and plasminogen and does not cause a cross reaction.

The antibodies of the present invention include any type of polyclonal antibodies, antisera consisting of polyclonal antibodies, or monoclonal antibodies, and fragments of these antibodies (Fab, F (ab ') ₂ , Fab').
Etc.) are also included.

(3) Peptide as Immunogen The antibody of the present invention comprises a peptide composed of 50 or less amino acids containing a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or this peptide and a carrier. It can be obtained by immunizing an animal with the conjugate of the above as an immunogen.

A part of the amino acid sequence in the peptide consisting of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as the immunogen is represented by SEQ ID NO: 1. It is a sequence of arbitrary contiguous amino acids in an amino acid sequence, but it is reported that an antibody can recognize an amino acid sequence consisting of three amino acids [F. Hudecz et al. , J. et al. Imm
unol. Methods, Volume 147, 201-210
Page, 1992], it is preferable that the amino acid sequence shown in SEQ ID NO: 1 is a sequence of 3 or more consecutive amino acids.

A peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as the immunogen means one of the amino acid sequences represented by SEQ ID NO: 1. It is meant to include, in addition to a peptide consisting of a part or the whole, a peptide in which an amino acid or peptide is further bound to the N-terminal side or the C-terminal side or both the N-terminal side and the C-terminal side of the peptide.

The amino acid or peptide bound here is
There is no particular limitation as long as it does not contain an amino acid sequence highly homologous to LDL or plasminogen.

However, since the homology with LDL or plasminogen may occur when the number of amino acids increases and the size of the peptide increases, the amino acid sequence of SEQ ID NO: 1 in the sequence listing as this immunogen is A peptide composed of 50 or less amino acids, including some or all, is
It is preferably composed of 50 or less amino acids, and more preferably 30 or less amino acids.

(4) Preparation of Peptide as Immunogen A peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing as an immunogen can be prepared by the liquid phase method and It can be synthesized by a method of peptide synthesis such as a solid phase method, or an automatic peptide synthesizer may be used. “Biochemistry Experimental Course 1 Protein Chemistry IV” edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 19
1975, Izumiya et al., "Basics and Experiments of Peptide Synthesis," Maruzen,
In 1985, it can be synthesized according to the method described in "Biochemistry Chemistry Laboratory 2 Protein Chemistry," edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1987, etc.

This peptide may be prepared from the DNA having the corresponding sequence by using the recombinant DNA technique, and is described in "Biological Chemistry Experimental Course 1 Gene Research Method I" edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1986. Year, Bioscience Society of Japan, "Sequel Biochemistry Experiment Course 1 Gene Research Method II", Tokyo Kagaku Dojin, 1986, Biochemistry Society of Japan, "Sequence Biochemistry Experiment Course 1
It may be prepared by referring to “Gene Research Method III”, Tokyo Kagaku Dojin, 1987, etc.

(5) Conjugate of peptide and carrier as immunogen A peptide and carrier composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing as an immunogen The binding product with is a product in which a peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the above sequence listing is bound to a carrier for antibody production. Is.

As the carrier, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, tetanus toxoid. Alternatively, a known carrier such as a polysaccharide can be used.

Then, the method of binding the peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing to the carrier is the glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, N-succimidyl-3- (2-pyridyldithio) propionic acid method,
A known coupling method such as a bisdiazotized penzidine method or a dipalmityl lysine method can be used.

Further, a carrier such as nitrocellulose particles, polyvinylpyrrolidone or liposomes to which the above peptides are adsorbed may be used as an immunogen.

When the immunogen is a low molecular weight substance, it is common to immunize a substance bound to a carrier,
Reported that a peptide with 5 amino acids was used as an immunogen to produce a specific antibody against it [Kiyama et al.
It is not essential to use a carrier, since the 12th Annual Meeting Abstracts 3 ”, p. 122, 1992] is also available.

(6) Preparation of polyclonal antibody or antiserum In the antibody of the present invention, the polyclonal antibody and antiserum can be prepared by the following procedure.

First, a peptide comprising 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the above sequence listing or a conjugate of this peptide and a carrier is used as an immunogen to give a mammal. Immunize (mouse, rabbit, rat, sheep, goat, horse, etc.) or bird (chicken, etc.).

The immunizing amount of this immunogen is appropriately determined depending on the type of animal to be immunized, the injection site of immunization, etc. For example, in the case of mice, it is 0 per mouse at about 5 to 10 weeks of age. 0.1 μg to 5 mg, preferably 50 μg
Immunize with an amount of immunogen containing ˜1 mg of the above peptide. Further, in the case of rabbits, it is preferable to immunize each rabbit once with an immunogen in an amount containing 10 μg to several tens of mg of the above peptide.

It is preferable that the immunogen is admixed with an adjuvant for immunization injection. As the adjuvant, known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or B. pertussis adjuvant can be used.

Immunization may be performed subcutaneously, intravenously, intraperitoneally, or on the back.

After the initial immunization, booster injection of the immunogen is carried out at a site such as subcutaneously, intravenously, intraperitoneally or on the back at intervals of 2 to 3 weeks. Also in this case, it is preferable that the immunogen is mixed with an adjuvant for booster injection.

After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by the ELISA method or the like, and when the antibody titer reaches a plateau, whole blood is collected and the serum is separated to obtain the antiserum of the present invention. .

The antiserum is purified by a method such as salting out with ammonium sulfate or sodium sulfate, ion exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods to purify the antibody. Can be obtained.

When human serum albumin or BSA is used as a carrier for the immunogen, the obtained antibody or antiserum may contain an antibody that causes a cross reaction with human serum albumin. It is preferable to carry out a treatment for removing various antibodies. As this removal processing method,
Human serum albumin or BSA used as a carrier is added to the obtained antibody or antiserum solution to remove purified aggregates, or human serum albumin or BSA used as a carrier is immobilized on an insolubilized carrier. A method of removing by affinity chromatography or the like can be used.

(7) Preparation of Monoclonal Antibody The method for preparing the monoclonal antibody in the antibody of the present invention will be described below.

Monoclonal antibodies were prepared by the cell fusion method of Keller et al. [G. Koehler et al. , Natur
e, 256, 495-497, 1975], or antibody-producing cells such as tumorigenic cells caused by a virus such as Epstan-Bar virus.

The monoclonal antibody can be prepared by the cell fusion method by the following procedure.

First, a peptide composed of 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the above sequence listing, or a conjugate of this peptide and a carrier is used as an immunogen to give a mammal. (Mouse, nude mouse, rat, etc., such as BALB / inbred mouse
c) or immunize with birds (chicken, etc.).

The immunizing amount of this immunogen depends on the type of immunized animal,
It may be appropriately determined depending on the site of immunization injection. For example, in the case of a mouse, 0.1 μ / mouse
It is preferred to immunize with an immunogen in an amount comprising g to 5 mg of the peptide.

It is preferable that the immunogen is mixed with an adjuvant and injected for immunization.

As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and pertussis adjuvant can be used.

Immunization may be performed subcutaneously, intravenously, intraperitoneally, or on the back.

After the initial immunization, booster injection of the immunogen is carried out at a site such as subcutaneously, intravenously, intraperitoneally or on the back at intervals of 1 to 2 weeks. The number of times of this booster injection is generally 2 to 6 times. Also in this case, it is preferable that the immunogen is mixed with an adjuvant for booster injection.

After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by the ELISA method or the like, and when the antibody titer reaches a plateau, the immunogen is added to physiological saline (0.9% sodium chloride aqueous solution). The dissolved substance is injected intravenously or intraperitoneally to give the final immunization.

Three to five days after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of the immunized animal are obtained.

The cells having antibody-producing ability obtained from this immunized animal and myeloma cells (myeloma cells) of a mammal (mouse, nude mouse, rat, etc.) are fused with each other. Xanthine guanine phosphoribosyl transferase (H
A cell line lacking an enzyme such as GPRT) or thymidine kinase (TK) is preferable, and examples thereof include BALB / c.
P3-X6, a mouse-derived HGPRT-deficient cell line
3-Ag8 strain (ATCC TIB9), P3-X63-
Ag8-U1 strain (Research Source Bank for Cancer Research (JCR
B) 9085), P3-NS1-1-Ag4-1 strain (JCRB0009), P3-X63-Ag8.65.
3 strains (JCRB0028) or SP2 / O-Ag-1
4 strains (JCRB0029) and the like can be used.

Cell fusion can be carried out by using a fusion promoter such as polyethylene glycol (PEG) having various molecular weights, liposomes or Sendai virus (HVJ), or by an electrofusion method.

The myeloma cell is a HGPRT-deficient strain or T
In the case of K-deficient strain, hypoxanthine / aminopterin / thymidine-containing selection medium (HAT medium)
By using, it is possible to selectively culture and proliferate only fused cells (hybridomas) of cells capable of producing antibodies and myeloma cells.

By measuring the culture supernatant of the hybridoma thus obtained by an immunological assay such as ELISA or Western blotting, it is bound to the unmodified or unmodified lipoprotein (a). Hybridomas that produce an antibody that specifically binds to denatured or modified lipoprotein (a) without any modification, and this method should be combined with known cloning methods such as limiting dilution. Thus, the cell line producing the monoclonal antibody of the present invention can be isolated and obtained.

This monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the monoclonal antibody of the present invention can be obtained from the culture supernatant. A serum-free medium or a low-concentration serum medium can be used as the medium. Also, in this case, it is preferable because the antibody can be easily purified, and DMEM medium,
A medium such as RPMI164 medium or ASF medium 103 can be used.

In addition, the monoclonal antibody producing cell line is
The monoclonal antibody of the present invention can be obtained by injecting into the abdominal cavity of a mammal which is compatible with this and has been stimulated in advance with pristane or the like, and after a certain period of time, ascites accumulated in the abdominal cavity.

The monoclonal antibody thus obtained is subjected to a method such as a salting-out method using ammonium sulfate or sodium sulfate, an ion exchange chromatography, a gel filtration method, or an affinity chromatography, or a combination of these methods. A purified monoclonal antibody of the present invention can be obtained.

(8) Assay Method The assay method of the present invention does not bind to the non-denatured or modified lipoprotein (a) and is specific to the denatured or modified lipoprotein (a). A method for measuring denatured or modified lipoprotein (a) using an antibody that binds to, but without measuring the denatured or modified lipoprotein (a), LDL and plasminogen, It is a measurement method capable of accurately measuring denatured or modified lipoprotein (a).

In the assay method of the present invention, not only one kind of the above-mentioned antibodies may be used but a plurality of kinds of antibodies may be used in combination.

The assay method of the present invention can be carried out in any assay method using an antibody, that is, an immunological assay method, by using the above-mentioned antibody as the antibody used in the assay method. Which has the effect of
For example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay, radioimmunoassay (RIA), luminescence immunoassay, enzyme antibody method, fluorescent antibody method, immunoturbidimetric method, latex agglutination method, latex ratio This measurement method can be carried out by a turbidity method, a hemagglutination method, a particle agglutination method, a Western blotting method, or the like.

(9) Measurement sample As a sample in the measurement method of the present invention, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tear, ascites or amniotic fluid, stool, stool, Denatured or modified lipoprotein (a) such as organs, tissues, cells such as blood vessels or livers, or extracts of organs, tissues or cells
Any biological sample that may contain is targeted.

(10) Immunoassay Method Using Labeled Antibody The assay method of the present invention comprises enzyme immunoassay, fluorescence immunoassay,
When performing an immunoassay using a labeled antibody such as a radioimmunoassay or a luminescent immunoassay, the sandwich method or the competitive method can also be used. In the case of the sandwich method, a solid-phased antibody or a labeled antibody is used. At least one kind of antibody that directly binds to the denatured or modified lipoprotein (a) such as the above may be the above antibody.

As the solid phase carrier, polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose, glass, metal, ceramics, or magnetic material can be used. Solid phase carriers in the form of beads, microplates, test tubes, sticks, test pieces or the like made of a material can be used.

The solid-phased antibody can be prepared by a known method such as a physical adsorption method, a chemical binding method or a combination thereof with the solid-phase carrier and the antibody.

In the case of the physical adsorption method, the antibody and the solid phase carrier are mixed and brought into contact with each other in a solution such as a buffer solution or the antibody dissolved in the buffer solution and the solid phase carrier are brought into contact with each other according to a known method. Can be done by.

In the case of using the chemical coupling method, "Clinical Pathology Extra Number Special Issue No. 53, Immunoassay for Clinical Testing -Technology and Application-", edited by Japan Society for Clinical Pathology, Clinical Pathology Society, 1983, Japan. Biochemical Society, "Shinsei Chemistry Laboratory 1 Protein IV", Tokyo Kagaku Dojin, 1991
According to a known method described in the year etc., the antibody and the solid phase carrier are mixed with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, and maleimide, and brought into contact with each amino group of the antibody and the solid phase carrier, It can be carried out by reacting with a carboxyl group, a thiol group, an aldehyde group, a hydroxyl group or the like.

In the case of enzyme-linked immunosorbent assay, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase or the like is used as the labeling substance for fluorescence. In the case of immunoassay,
Fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate or dichlorotriazine isothiocyanate and the like, and in the case of radioimmunoassay, tritium, iodine 125 or iodine 131 and the like can be used.
For the luminescence immunoassay, a NADH-FMNH ₂ -luciferase system, a luminol-hydrogen peroxide-POD system, an acridinium ester system, a dioxetane compound system, or the like can be used.

The binding method of a labeling substance and an antibody is described in "Special Issue on Clinical Pathology Special Issue No. 53, Immunoassay for Clinical Testing-Technology and Application-", edited by Japan Society for Clinical Pathology, Clinical Pathology Publication Society, 1983, Nippon Birth. Academic Society "Shinsei Chemistry Laboratory 1
Protein IV ”, Tokyo Kagaku Dojin, 1991, etc., and the antibody and the labeling substance are mixed with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide, etc. and contacted to label the antibody. The bond can be formed by reacting with each amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the substance.

The operation method of the measurement is a known method [Journal of the Japanese Society for Clinical Pathology, "Special Issue on Clinical Pathology, Special Issue No. 53, Immunoassay for Clinical Testing-Technology and Application-", Clinical Pathology Publishing Association, 1983, Eiji Ishikawa et al. "Enzyme-linked immunosorbent assay", 3rd edition, Medical Institute, 1987, Tsunehiro Kitagawa et al. "Protein nucleic acid enzyme separate volume No. 31 enzyme-linked immunosorbent assay", Kyoritsu Publishing,
1987].

For example, the immobilized antibody and the sample are reacted with the labeled antibody at the same time, or the labeled antibody is reacted after washing to obtain the immobilized antibody-denatured or modified lipoprotein (a). -Form a complex of labeled antibody. Then, the unbound labeled antibody can be washed and separated, and the amount of denatured or modified lipoprotein (a) in the sample can be measured from the amount of the bound labeled antibody or the amount of unbound labeled antibody.

Specifically, in the case of the enzyme immunoassay, the substrate is reacted with the labeled enzyme under the optimum conditions, and the amount of the reaction product is measured by an optical method or the like. In the case of the fluorescent immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of the luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

(11) Aggregation reaction method This measurement method is used for the permeation of the formation of immune complex aggregates such as immunonephelometry, latex agglutination, latex nephelometry, erythrocyte agglutination or particle agglutination. When light or scattered light is measured by an optical method, or when it is carried out by a visually measuring method, a phosphate buffer solution, a glycine buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used as a solvent. Further, a reaction accelerator such as polyethylene glycol or a non-specific reaction inhibitor may be included.

When the antibody is sensitized to a solid phase carrier, the solid phase carrier may be polystyrene, styrene-butadiene copolymer, (meth) acrylic acid ester polymer, latex, gelatin, liposome, microcapsule. , Red blood cells, silica, alumina, carbon black,
Particles made of a material such as a metal compound, a metal, a ceramic, or a magnetic material can be used.

As the sensitization method, a physical adsorption method,
It can be performed by a known method such as a chemical bonding method or a combination thereof.

In the case of the physical adsorption method, the antibody and the solid phase carrier are mixed and contacted in a solution such as a buffer solution or the antibody dissolved in the buffer solution and the solid phase carrier are contacted according to a known method. Can be done by.

In the case of using the chemical coupling method, "Clinical Pathology Extra Edition Special Issue No. 53: Immunoassay for Clinical Testing-Technology and Application-", edited by Japan Society for Clinical Pathology, Clinical Pathology Press, 1983, Japan. Biochemical Society, "Shinsei Chemistry Laboratory 1 Protein IV", Tokyo Kagaku Dojin, 1991
According to a known method described in the year etc., the antibody and the solid phase carrier are mixed with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, and maleimide, and brought into contact with each amino group of the antibody and the solid phase carrier, It can be carried out by reacting with a carboxyl group, a thiol group, an aldehyde group, a hydroxyl group or the like.

The operation method of the measurement can be carried out by a known method. For example, in the case of measuring by an optical method, the sample and the antibody or the sample and the antibody sensitized to the solid phase carrier are reacted, The transmitted light and scattered light are measured by the end point method or the rate method.

In the case of visual measurement, the sample and the antibody sensitized to the solid phase carrier are allowed to react in a container such as a plate or a microtiter plate, and the state of aggregation is visually determined.

Instead of the visual measurement, a device such as a microplate reader may be used for the measurement.

[0093]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] (Synthesis of peptide represented by SEQ ID NO: 2 in Sequence Listing) Synthesis of peptide represented by SEQ ID NO: 2 in Sequence Listing which is a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in Sequence Listing I went.

First, Applied Biosystems (A
Model 430 of Applied Biosystems)
A peptide automatic synthesizer (Model 430A pe
The peptide was synthesized by the solid-phase t-butoxycarbonylamino acid solid phase method according to the instruction manual by Ptide Synthesizer). In order to suppress the side reaction, the synthesized peptide was released from the resin by the hydrogen fluoride method in the presence of dimethyl sulfide, p-thiocresol, m-cresol, and anisole as scavengers.

Then, the scavenger was extracted with dimethyl ether, and the peptide synthesized with 2N acetic acid was extracted.

Dowex 1-anion exchange resin
Anion exchange column chromatography was performed with X2 (DOWEX 1-X2) for purification, and high performance liquid chromatography (H) was performed on an octadecyl (ODS) column.
The main peak pattern was confirmed by PLC).

Then, it was freeze-dried by an evaporator and concentrated, and then purified by HPLC and fractionated. In addition, the apparatus and conditions at the time of this HPLC purification are the reverse-phase ODS column YMC-D-ODS- of Yamamura Chemical Laboratory.
5 (20 mm × 300 mm), TWINCLE pump manufactured by JASCO Corporation and GP-A4 manufactured by JASCO Corporation
0.1% trifluoroacetic acid (TF type 0
A gradient of 0 to 70% of acetonitrile in A) was performed at a flow rate of 7.0 ml / min, and UVI manufactured by JASCO Corporation was used.
DEC-100V type detector (210nm, 1.28AU
Detection was carried out by FS).

The purified and separated synthetic peptide was freeze-dried by an evaporator and concentrated.

The purity of the obtained synthetic peptide was analyzed by HPLC. Equipment and conditions are reverse phase O of Yamamura Chemical Laboratory
DS column YMC-R-ODS-5 (4.9 mm x 30)
0 mm) and a gradient of 0% to 70% of acetonitrile in 0.1% trifluoroacetic acid (TFA) with a TWINCLE pump of JASCO Corporation and a GP-A40 type gradient of JASCO Corporation, and a flow rate of 1.0 ml. /
Minutes, 25 minutes, JASCO Corporation UVIDEC-
Detection was performed with a 100V type detector (210 nm, 1.28 AUFS).

The results of this analysis are shown in FIG. In this figure, PKNO is the peak number in the chart, TIM
E is the elution time, AREA is the peak area, and CONC is the ratio of that peak area in the total peak area (ie, percent concentration).

[0102]

FIG. 1 shows that the synthetic peptide obtained from this has a purity of almost 1.
It can be seen that it is 00%.

Further, the amino acid composition analysis of the obtained synthetic peptide was carried out by using Waters Pico-Tag (Millipore) Pico-Tag.
An amino acid analyzer was used according to the instruction manual.
The peptide sample was hydrolyzed in 6N hydrochloric acid containing 1% phenol at 150 ° C. for 1 hour.

The results of this amino acid analysis are shown in Table 1.
(Since cysteine cannot be quantified by the hydrochloric acid hydrolysis method,
The analytical value was omitted. )

[Table 1] In Table 1, Asx represents asparagine or aspartic acid.

It was confirmed that the synthetic peptide thus obtained had the same composition as the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and was the peptide represented by SEQ ID NO: 2 in the sequence listing. The isoelectric point of the obtained synthetic peptide was 5. The mass spectrum of this synthetic peptide is shown in FIG.

[0106]

[Example 2] (Preparation of immunogen consisting of carrier bound to peptide represented by SEQ ID NO: 2 in the sequence listing) Carrier limpet hemocyanin (KLH) [manufactured by Calbiochem] or bovine serum albumin ( BSA) [Seikagaku Corporation] 10mg 1
2.5% maleimidobenzoyl-N-hydroxysuccinimide ester dissolved in 0 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) and dissolved in N, N-dimethylformamide. (MBS)
[Pierce Co.] 150 μl of solution was added and reacted at room temperature for 30 minutes with stirring.

This was stored in a 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH
It was applied to a Sephadex G-25 (Sephadex G-25) column [Pharmacia-LCB Co., Ltd.], which was a gel filtration column equilibrated with 7.0).
The MBS-carrier-bound component was separated by monitoring the absorbance at 0 nm.

The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide represented by SEQ ID NO: 2 in the sequence listing synthesized in Example 1 was added and mixed and reacted for 150 minutes. .

After the reaction, the mixture was dialyzed against water three times and then freeze-dried to obtain an immunogen consisting of a carrier bound to the peptide represented by SEQ ID NO: 2 in the sequence listing.

When the carrier was KLH, the yield of the obtained immunogen was 3.0 mg (yield 75%), and the amount of the peptide represented by SEQ ID NO: 2 in the sequence listing in the obtained immunogen was The amount was 0.9 mg, and the ratio (weight ratio) of the peptide represented by SEQ ID NO: 2 in the sequence listing in this immunogen was 30.
It was 3% (W / W).

When the carrier was BSA, the yield of the obtained immunogen was 7.1 mg (yield 52%), and the peptide of SEQ ID NO: 2 in the sequence listing in the obtained immunogen was obtained. The amount was 1.5 mg, and the ratio (weight ratio) of the peptide represented by SEQ ID NO: 2 in the sequence listing in this immunogen was 21.
It was 7% (W / W). Therefore, from this, it is understood that the immunogen in which the carrier is BSA is one in which 16 to 17 molecules of the peptide represented by SEQ ID NO: 2 in the sequence listing are bound to one molecule of BSA which is the carrier.

[Example 3] (Preparation of an antibody that specifically binds to denatured or modified lipoprotein (a)) A monoclonal antibody that specifically binds to denatured or modified lipoprotein (a) was prepared. Prepared as follows.

[1] Immunization of animals (1) Immunogen obtained in Example 2 (carrier is KLH)
Physiological saline (0.9%
It was dissolved in an aqueous solution of sodium chloride) and mixed in an equal amount with Freund's complete adjuvant to form an emulsion, and 0.5 ml of an 8-week-old female BALB / c mouse (Charles River Japan Co., Ltd.) was subcutaneously injected into the abdomen by immunization. .

(2) Two weeks after the first immunization, the above immunogen was dissolved in physiological saline so as to have a concentration of 200 μg / ml, and this was mixed with Freund's incomplete adjuvant in equal amounts to form an emulsion. A booster injection was made with 0.5 ml.

This booster injection was given every two weeks.

(3) The antibody titer in the serum of this immunized mouse was determined by enzyme immunoassay (ELISA, EIA).
At 6 weeks after the first immunization, the measurement was performed every week.

The operation of this ELISA method is shown below.

The immunogen (having BSA carrier) obtained in Example 2 consisting of the peptide represented by SEQ ID NO: 2 in the sequence listing, which contains the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, becomes 5 μg / ml. As saline (0.9%
96 well-
10 per well on a microplate (Nunc)
0 μl of each was added, and the mixture was allowed to stand at 37 ° C. for 2 hours to solidify the peptide.

The microplate was washed with a washing solution (0.
Phosphate buffered saline (5.59 mM disodium hydrogen phosphate, containing 05% Tween 20),
1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM potassium chloride (pH 7.
2))) and then washed with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH) containing 1% BSA
7.2) Add 300 μl / well and add 37
Blocking was carried out by leaving still at 2 ° C. for 2 hours, and then washing was performed again with a washing solution.

To the wells of this microplate, 100 wells of the supernatant to be tested for the production of the above-mentioned antibody are added.
μl each, and let stand at 37 ° C. for 2 hours to carry out reaction,
Then, it was washed with a washing liquid.

As a control, 100 μl of HAT medium was added to each well of the above microplate,
It was left standing at 37 ° C. for 2 hours and then washed with a washing solution.

A peroxidase (POD) -labeled anti-mouse IgG antibody (manufactured by Amersham) was diluted 2000 times with phosphate buffered saline containing 3% BSA, and then diluted.
100 μl per well in and microplate
Each of them was added and allowed to react at 37 ° C. for 2 hours.

After washing this with a washing solution, a peroxidase reaction solution (3 mM 2,2′-azino-bis (3
-Ethylbenzthiazoline-6-sulfonic acid) [ABT
S] containing 50 mM disodium hydrogen phosphate-24 mM
2 μl of 1.7% hydrogen peroxide added to 1 ml of citrate buffer just before use) 1 per well
Each 100 μl was added and the reaction was carried out at room temperature. After 15 minutes, the reaction was stopped by adding 50 μl of 6N sulfuric acid per well.

The absorbance at 415 nm was measured using an EIA plate reader (manufactured by Bio-Rad).

(4) From the 18th week after the initial immunization, it was confirmed that the antibody titer reached a plateau. Therefore, 800 μg of physiological saline was subcutaneously injected into the abdomen of this immunized mouse.
/ Ml of the immunogen obtained in Example 2 (carrier is KLH) was injected.

On day 3 thereafter, spleens were obtained from the mice of this immunized animal.

[2] Proliferation of myeloma cells Hypoxanthine / guanine / derived from BALB / c mouse
Phosphoribosyl transferase-deficient myeloma cell line P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank 9085) was added to fetal bovine serum at 10%.
Proliferation was carried out in RPMI1640 tissue culture medium (Biocell) supplemented with glutamine, penicillin and streptomycin.

The myeloma cells were grown in a medium-sized bottle for cell culture (Nunc, 200 ml volume) until the cells occupied about 80% of the bottom surface of the bottle. The number of cells was counted by trypan blue dye exclusion method and hemacytometer.

[3] Cell Fusion (1) The spleen obtained from the mouse of the above-mentioned immunized animal was
It was thoroughly disintegrated using stainless steel mesh # 200 and filtered while washing with serum-free RPMI 1640 medium.

Then, centrifugation was performed at 200 g to separate spleen cells.

Furthermore, again RPMI164 free of serum
The spleen cells were washed 3 times with 0 medium solution.

(2) The spleen cells and the proliferated P3-X63-Ag8-U1 strain myeloma cells were mixed at a ratio of 5: 1 and then centrifuged.

The mixed cells were slowly suspended in RPMI1640 medium solution containing 50% of polyethylene glycol 1500 (PEG 1500, Boehringer Mannheim).

Then, this was gradually diluted with RPMI1640 medium solution so that the final polyethylene glycol concentration would be 5%.

(3) From this, the cells were separated by centrifugation and gradually dispersed in a growth medium consisting of S-Clone medium (manufactured by Sanko Junyaku Co., Ltd.) containing 5% hybridoma cloning factor (manufactured by Origen). It was

10 wells were added to the wells of a flat bottom 96-well microtiter plate (Nunc).
Cells with a cell number of ⁶ cells / 100 μl were seeded and cultured at 37 ° C. in 5% carbon dioxide.

(4) On the first day after cell fusion, 100 μl of HAT medium (0.01 m in the above growth medium was added to each well).
M hypoxanthine, 1.6 μM thymidine and 0.04 μ
M-aminopterin supplemented with each of them was added, both of which were manufactured by Tokyo Kasei.

For the next 3 days, about half of the HAT medium was replaced with fresh HAT medium every day, and 2-3 days thereafter.
Similar exchanges were made daily.

(5) The cells were observed with a microscope.

Hybridoma (fused cell) clones appeared from day 10 onward, and from day 14 onward, the supernatant of the wells was subjected to ELISA in order to test the production of an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Screened by method.

The operation of this ELISA method was performed in the same manner as in (3) of [1] above.

(6) In the screening of (5) above, a hybridoma in a well found to produce an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was plated on a plate having 24 wells. The cells were cultured in a small bottle, a medium-sized bottle, and a large scale as the cell density increased.

(7) Then, the hybridoma was cultured and maintained in HT medium (HAT medium containing no aminopterin and hybridoma cloning factor).

(8) The production of an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was examined by the ELISA method in the same manner as in (5) above, and the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was found. Bound to the immunogen obtained in Example 2 consisting of a peptide containing
Moreover, four hybridomas that produce an antibody that does not bind to BSA were confirmed.

[4] Hybridoma Subcloning (1) The above four hybridomas were subcloned by the limiting dilution method.

The cell numbers of these hybridomas were counted by the trypan blue dye exclusion method and a hemacytometer.

Then, these hybridomas were treated with 10
Suspension was carried out at a ratio of two kinds of the number of viable cells of 0.5 and the number of viable cells per 0 μl of HT medium.
100μ per well of 6-well flat bottom microplate
1 was dispensed.

The medium was exchanged every 2-3 days to grow hybridomas.

(2) After 2 weeks, the number of colonies in each well was examined under a microscope, and the immunogen obtained in Example 2 consisting of a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing (carrier is BSA Wells that produce antibodies that bind to BSA but not BSA in the same manner as above.
It was examined by the ISA method.

There was one colony in one well and four wells producing such antibodies were obtained.

(3) This was transferred to a 24-well plate and cultured for 2 weeks until the cell growth became good.

(4) Next, the reactivity of the antibodies produced by these hybridomas with the denatured or modified lipoprotein (a) was examined by the ELISA method.

The operation was carried out on the culture supernatant of the hybridoma in the same manner as in Example 4 described later.

As a result, it was revealed that one hybridoma was a cell line producing an antibody against denatured or modified lipoprotein (a).

(5) This hybridoma was cloned again in the same manner as in (1) and (2) above, and the production of antibody in each well was examined.
There was one colony of hybridoma in the well, and it was bound to the immunogen (having BSA carrier) obtained in Example 2 and consisting of a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and with BSA. Obtained a total of 9 clones that produced antibodies that did not bind.

(6) The reactivity of the antibodies produced by these hybridoma clones with the denatured or modified lipoprotein (a) was determined by ELI again as in (4) above.
It investigated by SA method.

From this, it was confirmed that all these hybridoma clones are cells producing an antibody that specifically binds to denatured or modified lipoprotein (a).

(7) A hybridoma cell line [322H which produces a monoclonal antibody that specifically binds to the denatured or modified lipoprotein (a) of the present invention.
4P2 strain].

This hybridoma cell line [322H4P
2 shares] has been deposited as FERM P-15611 on May 7, 1996 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

[5] Production of Monoclonal Antibody (1) The resulting monoclonal antibody-producing cell line against the denatured or modified lipoprotein (a) was placed in a medium-sized bottle (Nunc) at about 8 Culturing was carried out in HT medium until the cells occupied the percentage.

(2) Then, these hybridomas were scraped off, and centrifuged at 200 g for 5 minutes to collect them.

Next, this was added to RPMI16 containing no serum.
After washing 3 times with 40 medium solution, 2 ml of RPMI164
It was suspended in 0 medium solution.

(3) Male BAL previously treated with 2,6,10,14-tetramethylpentadecane.
1 ml of the hybridoma suspension obtained in (2) above was injected into the abdominal cavity of a B / c mouse (Japan Charles River).

When no swelling of the abdomen was observed within 2 weeks after the injection, this was repeated again.

(4) Ascites was collected when swelling of the abdomen of this mouse was observed.

This was centrifuged at 200 g for 5 minutes, and a supernatant containing a monoclonal antibody against denatured or modified lipoprotein (a) was separated from the hybridoma and obtained.

[6] Purification of Monoclonal Antibody (1) 10 ml of supernatant containing a monoclonal antibody against denatured or modified lipoprotein (a)
To the above, 1.8 g of sodium sulfate was added with stirring at 22 ° C., and after the sodium sulfate was completely dissolved, stirring was continued for another 1 hour for salting out.

(2) This was centrifuged at 22 ° C. (700
(0 g, 15 minutes), and the precipitate obtained by separating from the supernatant was dissolved in 2 ml of 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride.

(3) Next, this was added to a 40 mM sodium phosphate buffer solution (pH 8.
After sufficiently dialyzing against 0), the insoluble matter was removed by centrifugation at 1000 g for 20 minutes.

(4) A DEAE-cellulose ion exchange column (manufactured by Selva) [1 × 10 cm], which had been equilibrated with 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride, had a flow rate of 0. 4 ml /
The eluate was collected by 2 ml per minute.

(5) It was confirmed from the absorbance at 280 nm that immunoglobulin G (IgG) was contained in the flow-through fraction of the eluate, which was collected and concentrated to 2 ml.

(6) Further, this was purified by subjecting it to Protein A-Sepharose CL-4B affinity chromatography (Pharmacia-LCB) to specifically bind to the denatured or modified lipoprotein (a). An antibody was obtained.

The amount of the obtained monoclonal antibody was 7 mg in terms of protein amount.

The antibody class and subtype of the monoclonal antibody specifically binding to the denatured or modified lipoprotein (a) obtained here are commercially available specific anti-mouse immunoglobulin antiserum (manufactured by Dako). Was determined to be IgG ₁ and λ chain by the Octalonii immunodiffusion method.

Example 4 (Measurement of Lipoprotein (a) Denatured or Modified by Oxidation) Specific binding to the denatured or modified lipoprotein (a) obtained in Example 3 An antibody was used to establish a measurement system for denatured or modified lipoprotein (a).

[1] Preparation of denatured or modified lipoprotein (a) as a sample (1) Human serum having high lipoprotein (a) concentration
Ultracentrifugation was performed to separate a portion having a specific gravity of 1.05 or more and 1.12 or less, and further subjected to lysine-Sepharose 4B affinity chromatography (Pharmacia-LCB) to obtain purified lipoprotein (a).

(2) This purified lipoprotein (a)
In a 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH: 0.01% sodium azide).
It was dialyzed thoroughly with 7.4).

(3) The purified lipoprotein (a) thus dialyzed was added to 50 mM potassium dihydrogen phosphate-dihydrogen phosphate containing 0.01% sodium azide to a concentration of 0.56 mg / ml. Potassium buffer (pH 7.
After diluting with 4), 1.8 ml each of
0, 1, 2, 5, 10, 20, 40, 60, 100 μg
/ Ml of copper (II) chloride (CuCl ₂ ) [manufactured by Wako Pure Chemical Industries, Ltd.] was added to and mixed with 0.2 ml of an aqueous solution.
0, 0.1, 0.2, 0.5, 1.0, 2.0, 4.
A 0.5 mg / ml purified lipoprotein (a) solution with a copper (II) chloride concentration of 0, 6.0, 10.0 μg / ml was prepared.

(4) Each of the purified lipoprotein (a) solutions prepared in (3) above was heated at 37 ° C. for 12 hours and oxidized with copper (II) ions, and the lipoproteins denatured or modified by the oxidation were treated. A sample of protein (a) was prepared.

(5) It should be noted that 9 of the lipoprotein (a) modified or modified by oxidation prepared in (4) above was used.
The degree of oxidation of each kind of sample was measured by "Lipid peroxide test Wako" (Yagi method, fluorescence method) [manufactured by Wako Pure Chemical Industries, Ltd.] which is a reagent for measuring lipid peroxide.

The results are shown in Table 2.

[0182]

[Table 2] From this, it can be seen that the TBARS value [nmol (converted to malondialdehyde) / ml], which indicates the degree of oxidation, increases proportionally as the concentration of copper (II) chloride used for oxidation increases.

Thus, it was confirmed that the lipoprotein (a) of the above sample was oxidized according to the concentration of copper (II) chloride used for oxidation.

[2] Measurement by ELISA method (1) 3% B of the antibody that specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3 was used.
After adjusting to 15 μg / ml with phosphate buffered saline containing SA (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM potassium chloride (pH 7.2)) ,
Add 100 μl per well to 96-well microplate (Nunc) and leave at 37 ° C. for 2 hours to immobilize antibody that specifically binds to denatured or modified lipoprotein (a). Was performed (immobilized antibody).

(2) This microplate was washed with a washing solution (phosphate buffered saline (pH 7.2) containing 0.05% Tween 20), and then 1
10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) containing 10% BSA was added in an amount of 300 μl per well, left standing at 37 ° C. for 2 hours for blocking, and then washed again with a washing solution. did.

(3) In (4) of the above [1], 0, 0.1, 0.2, 0.5, 1.0, 2.
Nine kinds of samples of lipoprotein (a) which had been denatured or modified by oxidation with copper (II) chloride at a concentration of 0, 4.0, 6.0, 10.0 μg / ml were used as physiological saline in 100 samples.
After doubling the dilution, 100 μl per well was dispensed into the microplate prepared in (2), and the mixture was allowed to stand at 37 ° C. for 2 hours for antigen-antibody reaction.

(4) After washing this with a washing liquid, 50
An antibody that specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3 dissolved in phosphate buffered saline containing 3% BSA at a concentration of μg / ml was 100 μl was added to each well, and the mixture was allowed to stand at 37 ° C. for 2 hours to carry out the reaction (primary antibody), and then washed with a washing solution.

(5) A peroxidase (POD) -labeled anti-mouse IgG antibody (manufactured by Amersham) was diluted 2,000-fold with phosphate buffered saline containing 3% BSA, and then the microplate of (4) above. 1 per well
Aliquots of 00 μl were dispensed, and the mixture was allowed to stand at 37 ° C. for 2 hours to carry out the reaction (secondary antibody).

(6) After washing this with a washing solution, a peroxidase reaction solution (3 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) in 50 mM disodium hydrogen phosphate-24 mM citrate buffer solution) 1 ml
2 μl of 1.7% hydrogen peroxide was added immediately before use), 100 μl was added per well, and the reaction was carried out at room temperature.

After 15 minutes 100 μl of 6 per well
The reaction was stopped by adding N-sulfuric acid.

(7) This was measured for absorbance at 450 nm with an EIA microplate reader (manufactured by Bio-Rad).

FIG. 3 shows a calibration curve obtained by measuring 9 kinds of samples of lipoprotein (a) which have been denatured or modified by oxidation. In this figure, the horizontal axis represents the concentration of copper (II) chloride used for the oxidation of lipoprotein (a), and the vertical axis represents the measured absorbance at 450 nm. However, the measured value of the absorbance is 0 (μ) concentration of copper (II) chloride.
The value obtained by subtracting the absorbance of the g / ml sample as a blind value is shown.

[0193]

FIG. 3 shows that the higher the concentration of copper (II) chloride and the higher the degree of oxidation of the sample of lipoprotein (a), the higher the measured absorbance value, which was proportional to the degree of oxidation of lipoprotein (a). It can be seen that the measured values are obtained.

Accordingly, in the denaturation or modification by oxidation, the antibody of the present invention specifically binds to the denatured or modified lipoprotein (a), and the assay method of the present invention uses the denatured or modified lipoprotein. It was confirmed that the protein (a) can be quantitatively measured.

[Example 5] (Measurement of denatured or modified lipoprotein (a) by reduction) Specific binding to the denatured or modified lipoprotein (a) obtained in Example 3 An antibody was used to establish a measurement system for denatured or modified lipoprotein (a).

[1] Preparation of denatured or modified lipoprotein (a) as a sample (1) Human serum having high lipoprotein (a) concentration
Ultracentrifugation was performed to separate a portion having a specific gravity of 1.05 or more and 1.12 or less, and further subjected to lysine-Sepharose 4B affinity chromatography (Pharmacia-LCB) to obtain purified lipoprotein (a).

(2) This purified lipoprotein (a)
In a 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH: 0.01% sodium azide).
It was dialyzed thoroughly with 7.4).

(3) The purified lipoprotein (a) thus dialyzed was diluted with physiological saline to a concentration of 0.56 mg / ml, and 1.8 ml of the diluted lipoprotein (a) was diluted with 0. It was added to 0.2 ml of an aqueous solution of 1, 1, 5, 10, 50% 2-mercaptoethanol [manufactured by Tokyo Chemical Industry Co., Ltd.], and mixed to give 0, 0.01, 0.1, 0.
A 0.5 mg / ml purified lipoprotein (a) solution having a concentration of 2-mercaptoethanol of 5, 1.0 and 5.0% was prepared.

(4) Each purified lipoprotein (a) solution prepared in (3) above was heated at 37 ° C. for 3 hours, reduced with 2-mercaptoethanol, and denatured or modified lipoprotein by reduction. The sample of (a) was prepared.

[2] Measurement by ELISA method The measurement operation was carried out according to the above-mentioned Example 4, [2] (1) to (7).
It carried out similarly to the process of. However, the samples were respectively 0, 0.01, 0.1, 0.5, from 9 kinds of samples of lipoprotein (a) which had been denatured or modified by oxidation.
The measurement was carried out by changing to 6 kinds of samples of lipoprotein (a) which had been denatured or modified by reduction with 2-mercaptoethanol having a concentration of 1.0 or 5.0%.

A calibration curve obtained by measuring 6 types of samples of lipoprotein (a) that have been denatured or modified by these reductions is shown in FIG. In this figure, the horizontal axis represents the concentration of 2-mercaptoethanol used for the reduction of lipoprotein (a), and the vertical axis represents the measured value of absorbance at 450 nm. However, the measured value of the absorbance was obtained by subtracting the absorbance of the sample having a 2-mercaptoethanol concentration of 0% as a blind value.

[0202]

FIG. 4 shows that the higher the concentration of 2-mercaptoethanol and the more strongly reduced the sample of lipoprotein (a), the higher the measured absorbance value, and the higher the degree of reduction of lipoprotein (a). It can be seen that proportional measurement values are obtained.

Accordingly, even in the case of denaturation or modification by reduction, the antibody of the present invention can specifically bind to the denatured or modified lipoprotein (a), and the assay method of the present invention uses the denatured or modified lipoprotein. It was confirmed that the protein (a) can be quantitatively measured.

Example 6 (Measurement of Denatured or Modified Lipoprotein (a) Due to Degradation) [0204] It specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3. An antibody was used to establish a measurement system for denatured or modified lipoprotein (a).

[1] Preparation of denatured or modified lipoprotein (a) as a sample (1) Human serum with high lipoprotein (a) concentration
Ultracentrifugation was performed to separate a portion having a specific gravity of 1.05 or more and 1.12 or less, and further subjected to lysine-Sepharose 4B affinity chromatography (Pharmacia-LCB) to obtain purified lipoprotein (a).

(2) This purified lipoprotein (a)
Was extensively dialyzed against 50 mM sodium acetate buffer (pH 4.8) containing 0.1 M sodium chloride, 0.04% EDTA and 0.01% sodium azide.

(3) The purified lipoprotein (a) thus dialyzed was adjusted to a concentration of 0.5 mg / ml with 0.1 M sodium chloride, 0.04% EDTA and 0.
After diluting with 50 mM sodium acetate buffer (pH 4.8) containing 01% sodium azide, 2.0 ml of each was dissolved in the above buffer, 0, 500, 1, respectively.
0.01 ml of 2,000 and 2,000 ng / ml of cathepsin D (manufactured by Sigma) was added and mixed to give 0 and 0, respectively.
5.0, 10.0, 20.0 ng / ml cathepsin D
A concentrated 0.5 mg / ml purified lipoprotein (a) solution was prepared.

(4) Each of the purified lipoprotein (a) solutions prepared in (3) above was heated at 37 ° C. for 90 minutes to hydrolyze the lipoprotein (a) with cathepsin D which is a protease (proteolytic enzyme). After degradation, a sample of lipoprotein (a) that had been denatured or modified by degradation was prepared.

[2] Measurement by ELISA method The measurement operation was carried out according to (1) to (7) of [2] of Example 4 above.
It carried out similarly to the process of. However, the samples were respectively 0, 5.0, 10.0, 20.0 from 9 types of samples of lipoprotein (a) that had been denatured or modified by oxidation.
The measurement was carried out by changing to four types of samples of lipoprotein (a) that had been denatured or modified by decomposition with cathepsin D at a concentration of ng / ml.

FIG. 5 shows a calibration curve obtained by measuring four kinds of samples of lipoprotein (a) that have been denatured or modified by these decompositions. In this figure, the horizontal axis represents the concentration of cathepsin D used for the decomposition of lipoprotein (a), and the vertical axis represents the measured absorbance at 450 nm.
However, the measured value of the absorbance is 0 ng / m for the cathepsin D concentration.
The value obtained by subtracting the absorbance of 1 sample as a blind value is shown.

[0211]

FIG. 5 shows that the higher the cathepsin D concentration and the more strongly degraded lipoprotein (a), the higher the measured absorbance value, which was proportional to the degree of lipoprotein (a) degradation. It can be seen that the measured values are obtained.

Therefore, even in denaturation or modification by decomposition, the antibody of the present invention specifically binds to the denatured or modified lipoprotein (a), and the assay method of the present invention is denatured or modified. It was confirmed that the lipoprotein (a) can be quantitatively measured.

From the above, the antibody of the present invention which specifically binds to the modified or modified lipoprotein (a) and the method for measuring the modified or modified lipoprotein (a) are Change in secondary structure or three-dimensional structure,
If it is a denatured or modified lipoprotein (a) that has undergone decomposition or chemical modification and has a different structure, composition, or steric configuration from the original natural (native) lipoprotein (a), Can bind specifically,
It was confirmed that the measurement can be performed accurately.

[Reference Example 1] (Study of reactivity of the antibody of the present invention with non-denatured or modified lipoprotein (a), LDL and plasminogen) Denatured or modified obtained in Example 3 The reactivity of the antibody that specifically binds to the received lipoprotein (a) with plasminogen, LDL, and unmodified or unmodified lipoprotein (a) was confirmed by Western blotting.

[1] Preparation of plasminogen, LDL and lipoprotein (a) (1) Human plasma having high plasminogen concentration is subjected to ultracentrifugation to separate a portion having a specific gravity of 1.21 or more, Lysine-Sepharose 4B affinity chromatography (manufactured by Pharmacia-LCB) and affinity chromatography in which an anti-lipoprotein (a) antibody (manufactured by Immuno) was bound as a ligand to fractionate the flow-through fraction, and purified plasminol Got gen.

(2) Human serum having a high LDL concentration is subjected to ultracentrifugation to separate a portion having a specific gravity of 1.006 or more and 1.063 or less, and an anti-lipoprotein (a) antibody (manufactured by Immuno Co.) The flow-through fraction was collected by affinity chromatography with binding as a ligand to obtain purified LDL.

(3) Human serum having a high lipoprotein (a) concentration is subjected to ultracentrifugation to separate a portion having a specific gravity of 1.05 or more and 1.12 or less, and further lysine-Sepharose 4B affinity chromatography ( (Pharmacia-LCB Co., Ltd.) to obtain purified denatured or unmodified lipoprotein (a).

[2] Measurement of Reactivity by Western Blotting (1) An antigen to be electrophoresed in Western blotting was prepared as follows.

Purified plasminogen antigen: 0.5 mg of the purified plasminogen in (1) of [1] above
/ Ml and diluted with physiological saline.

Purified LDL and Purified Lipoprotein (a) Antigen: The purified LDL of (2) of [1] above and the purified unmodified or modified lipoprotein (a) of (3) of [1] above. Respectively in the mixture.
The mixture was diluted with physiological saline so as to be 5 mg / ml and mixed.

Purified lipoprotein (a) antigen: The purified undenatured or modified lipoprotein (a) of (3) of [1] above was treated with physiological saline to give a concentration of 0.5 mg / ml. Diluted.

(2) 2 μl each of the purified plasminogen antigen, purified LDL and purified lipoprotein (a) antigen, and purified lipoprotein (a) antigen prepared in the above (1) were titan gel lipoprotein. Electrophoresis was performed using an electrophoresis kit (Helena Laboratories). The support was an agarose gel, and the running buffer was a barbital buffer (pH 8.8), and the voltage was 90 V for 75 minutes for energization.

(3) Transcription was carried out by a dry method using a Nova Blot Electrophoretic Transfer Kit (Pharmacia-LCB) according to the instruction manual.

(4) A 9 cm × 9 cm nitrocellulose membrane (manufactured by Bio-Rad) was placed on the agarose gel of (2) placed on the transfer device, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine,
0.0375% (W / V) sodium dodecyl sulfate (S
Transfer was performed for 2 hours at a current of 65 mA using a transfer buffer consisting of DS) and 20% (V / V) methanol.

(5) The transferred nitrocellulose membrane was treated with phosphate-buffered saline containing 1% BSA (5.59).
mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM
It was immersed in 20 ml of potassium chloride (pH 7.2) at 4 ° C. overnight for blocking.

(6) Next, this was washed with shaking in 20 ml of a washing solution (phosphate buffered saline containing 0.05% Tween 20) for 10 minutes. This operation was performed three times.

(7) 80 μg of the antibody that specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3 was dissolved in 20 ml of phosphate buffered saline, and the solution ( The nitrocellulose membrane subjected to the operation of 6) was immersed and reacted at room temperature for 2 hours.

(8) As a control, instead of the antibody that specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3, the same concentration of sheep anti-lipoprotein (a) antibody was used. (Manufactured by Immuno Co., Ltd.), goat anti-apolipoprotein B antibody of the same concentration against apolipoprotein B-100 which is a constituent of LDL (manufactured by International Enzyme), and goat anti-plasminogen antibody of the same concentration (Medical and Biological Research Institute). The above-mentioned operation (7) was carried out using a mixture (produced by Mitsui Chemicals, Inc.).

As a negative control, the nitrocellulose membrane obtained in (6), an antibody that specifically binds to the modified or modified lipoprotein (a) obtained in Example 3, and sheep anti-lipoprotein. A protein (a) antibody, a goat anti-apolipoprotein B antibody, and a goat anti-plasminogen antibody that did not act were prepared.

(9) The nitrocellulose membrane subjected to the above operation (7) or (8) was washed by shaking with 20 ml of the washing liquid for 10 minutes. This was done 3 times.

(10) Next, 3% of peroxidase-labeled anti-mouse IgG antibody (manufactured by Dako), peroxidase-labeled anti-sheep IgG antibody (manufactured by Dako) and peroxidase-labeled anti-goat IgG antibody (manufactured by Dako) were used. A 20-ml solution was prepared by diluting 500-fold with phosphate-buffered saline containing BSA, and the nitrocellulose membrane was immersed in this for 2 hours at room temperature for reaction.

(11) This nitrocellulose membrane was washed with 20 ml of the washing solution by shaking for 10 minutes. This operation was performed three times.

(12) 20 ml of phosphate buffered saline containing 0.025% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at room temperature for 15 minutes (1
The nitrocellulose membrane of 1) was dipped to develop the color.

[3] Measurement Results The results of this Western blotting method are shown in FIG.

In this figure, "3" is a negative control and "4" is a control, in which purified plasminogen antigen is electrophoresed and then transferred, and the membrane is reacted with the antibody mixture.
"5" is also a control and purified LDL and purified lipoprotein (a) antigen are electrophoresed, and then the antibody mixture is allowed to act on the membrane, and "6" is also a control and purified lipoprotein (a) antigen is electrophoresed. The antibody mixture was allowed to act on the post-transferred membrane, and "7" was the denatured or modified lipoprotein obtained in Example 3 on the post-transferred membrane of purified plasminogen antigen ( "8" is a lipoprotein which was treated with an antibody that specifically binds to a), and was subjected to denatured or modified lipoprotein obtained in Example 3 on the membrane to which purified LDL and purified lipoprotein (a) antigen were transferred after electrophoresis. Those which are reacted with an antibody that specifically binds to protein (a),
“9” is the membrane obtained by transcribing the purified lipoprotein (a) antigen after the migration, and the antibody that specifically binds to the modified or modified lipoprotein (a) obtained in Example 3 was allowed to act on the membrane. .

[0236]

FIG. 6 shows that commercially available sheep anti-lipoprotein (a) antibody, goat anti-apolipoprotein B antibody, and goat anti-plasminogen antibody developed in the controls “4”, “5” and “6”, respectively. Example 3 at the position
Modified or modified lipoprotein (a) obtained in
It can be seen that no color was observed in "7", "8", and "9" treated with an antibody that specifically binds to.

Therefore, the antibody that specifically binds to the denatured or modified lipoprotein (a) of the present invention includes any of plasminogen, LDL, and non-denatured or modified lipoprotein (a). It was confirmed that they did not bind.

An antibody that specifically binds to the modified or modified lipoprotein (a) obtained in Example 3,
Nonspecific color development occurred because no color was observed in the negative control "3" which was not treated with sheep anti-lipoprotein (a) antibody, goat anti-apolipoprotein B antibody or goat anti-plasminogen antibody. It was shown that not.

[Reference Example 2] (Study of reactivity of antibody of the present invention with peptide represented by SEQ ID NO: 2 in Sequence Listing) The modified or modified lipoprotein (a) obtained in Example 3 was used. The reactivity of the antibody that specifically binds with the peptide represented by SEQ ID NO: 2 in the sequence listing, which is a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, was confirmed by the ELISA method.

[1] Preparation of Sample Comprising Peptide Represented by SEQ ID NO: 2 in Sequence Listing [0240] The peptide obtained in Example 2 and containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is an immunogen. The conjugate of the peptide represented by SEQ ID NO: 2 in the sequence listing and the carrier (BSA) was dissolved in physiological saline to 100 ng / ml, 200 ng / ml, and 300 ng / ml,
Three types of samples were prepared.

[2] Measurement by ELISA method The measurement operation was carried out according to (1) to (7) of [2] of Example 4 above.
It carried out similarly to the process of. However, the samples were 100 ng / ml and 2 prepared from the 9 kinds of samples of lipoprotein (a) which had been denatured or modified by oxidation, in the above [1].
The measurement was performed by changing to three kinds of samples of the conjugate of the peptide represented by SEQ ID NO: 2 in the sequence listing and the carrier (BSA) at the concentrations of 00 ng / ml and 300 ng / ml.

The calibration curves obtained by measuring these three types of samples are shown in FIG. In this figure, the horizontal axis represents the concentration of the bound substance of the peptide represented by SEQ ID NO: 2 in the sequence listing and the carrier (BSA), which is a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and the vertical axis represents It represents the measured value of absorbance at 450 nm. However, the measured value of the absorbance represents the value obtained by subtracting the absorbance when the physiological saline was measured as a sample as a blind value.

[0243]

FIG. 7 shows that the origin of the graph of the measurement of the binding product of the peptide represented by SEQ ID NO: 2 in the sequence listing, which is a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and the carrier (BSA). It is a straight line that passes through and can be quantitatively measured, and it can be seen that the antibody of the present invention specifically binds to the conjugate of this peptide and the carrier.

Thus, it was confirmed that the antibody of the present invention specifically recognizes the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

[Reference Example 3] (Study of influence of serum sample in assay method of the present invention) Subjected to denaturation or modification using an antibody that specifically binds to the denatured or modified lipoprotein (a) of the present invention It was confirmed by an addition recovery test by the ELISA method that the measurement method for lipoprotein (a) was not affected by the serum sample.

[1] Preparation of Sample for Addition Recovery Test Two types of sera (A, B), which were confirmed not to contain denatured or modified lipoprotein (a), were prepared,
The following samples were prepared based on this.

2 ml of physiological saline was added to and mixed with 0.9 ml of two kinds of serum (A and B), respectively.
Types of samples. [Sample] Example 2 in which saline was adjusted to 3,000 ng / ml
SEQ ID NO: 2 in the sequence listing, which is a peptide containing the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, which is the immunogen obtained in
A conjugate of the peptide represented by
0.9 ml of each of two types of serum (A, B)
The concentration of the conjugate of the peptide represented by SEQ ID NO: 2 in the sequence listing of the two types of serum samples prepared in (3) was increased by 300 ng / ml by
Types of samples. [Sample] A conjugate of the peptide represented by SEQ ID NO: 2 in the sequence listing, which is a peptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, which is the immunogen obtained in Example 2, and a carrier (BSA) was prepared. Sample diluted to 300 ng / ml with physiological saline. [Sample] [2] Measurement by ELISA method The measurement operation was carried out according to the above-mentioned Example 4, [2] (1) to (7).
It carried out similarly to the process of. However, the measurement was performed by changing the samples from nine types of lipoprotein (a) samples that had been denatured or modified by oxidation to the five types of samples prepared in [1] above.

The absorbance values obtained by measuring these 5 types of samples are summarized in Table 3.

[0249]

[Table 3] From this table, it was found that the measurement method of the denatured or modified lipoprotein (a) of the present invention can obtain a measured value almost in accordance with the theoretical value even in the measurement of a serum sample.

Therefore, the assay method of the present invention was found to be a method that allows accurate measurement without being affected by non-specific reactions and the like by serum samples, and it was confirmed that there is no practical problem in clinical tests and the like. It was

[0251]

The antibody which specifically binds to the denatured or modified lipoprotein (a) of the present invention does not bind to the non-denatured or modified lipoprotein (a), and the original natural (native The reason why it is possible to specifically bind to denatured or modified lipoprotein (a) whose structure, composition or steric configuration is different from that of lipoprotein (a) is not clear at present. Absent.

It is speculated that when the lipoprotein (a) is denatured or modified, a portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing existing inside the molecule appears on the surface of the molecule, or The present invention capable of recognizing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, for example, due to the change in the three-dimensional structure and also the portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, due to the configuration change It is thought to be because it becomes possible to bind to the antibody of.

That is, the above is possible because the portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing of lipoprotein (a) is a site characteristically showing denaturation or modification of lipoprotein (a). It is supposed that it will become.

[0254]

INDUSTRIAL APPLICABILITY The antibody of the present invention which specifically binds to denatured or modified lipoprotein (a) binds to non-denatured or modified lipoprotein (a), LDL and plasminogen. The lipoprotein (a), which is not modified and has been modified or modified, has the characteristics and effects that it can specifically bind.

The method for measuring the denatured or modified lipoprotein (a) of the present invention can measure any of the denatured or unmodified lipoprotein (a), LDL and plasminogen. It has the characteristics and effects of being able to accurately measure the lipoprotein (a) that is absent and that has been denatured or modified.

[0256]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: Amino acid Topology: Unknown Sequence type: Peptide SEQ ID NO: 2 Sequence length: 10 Sequence type: Amino acid Topology: Unknown Sequence type: Peptide

[Brief description of drawings]

FIG. 1 is a diagram showing the results of HPLC analysis of the peptide represented by SEQ ID NO: 2 in the sequence listing obtained in Example 1.

FIG. 2 is a diagram showing a mass spectrum of the peptide represented by SEQ ID NO: 2 in the sequence listing obtained in Example 1.

FIG. 3 is a diagram showing a calibration curve obtained by measuring nine types of samples of lipoprotein (a) that have been denatured or modified by oxidation by the measurement method of the present invention.

FIG. 4 is a diagram showing a calibration curve obtained by measuring 6 types of samples of lipoprotein (a) that have been denatured or modified by reduction by the assay method of the present invention.

FIG. 5 is a diagram showing a calibration curve obtained by measuring four types of samples of lipoprotein (a) that have been denatured or modified by decomposition by the measurement method of the present invention.

FIG. 6 shows that the antibody that specifically binds to the denatured or modified lipoprotein (a) obtained in Example 3 was converted into plasminogen, LDL and undenatured or modified lipoprotein (a). 3 is a photograph showing an electrophoretic pattern in which the reactivity of is confirmed by Western blotting.

FIG. 7: EL using an antibody that specifically binds to the modified or modified lipoprotein (a) obtained in Example 3
FIG. 3 is a diagram showing a calibration curve obtained by measuring three kinds of samples of a conjugate of the peptide represented by SEQ ID NO: 2 in the sequence listing and a carrier by the ISA method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 G01N 33/531 A G01N 33/531 33/577 B 33/577 9282-4B C12N 15 / 00 C // (C12P 21/08 C12R 1:91) (72) Inventor Nobuhiko Kubo 1-847 Amanumacho Omiya-shi, Saitama Saitama Prefecture Omiya Medical Center (72) Inventor Ikunosuke Sakurabayashi Saitama 1-847 Amanuma-cho, Omiya-shi, Oita Prefectural Medical College Omiya Medical Center

Claims (7)

[Claims] 1. An antibody which does not bind to a non-denatured or modified lipoprotein (a) but specifically binds to a denatured or modified lipoprotein (a). 2. A peptide comprising 50 or less amino acids including a part or the whole of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or a conjugate of the peptide and a carrier, which is an immunogen. The antibody according to claim 1, which comprises: 3. The method according to claim 1 or 2, wherein the modification or modification is by oxidation, reduction, decomposition, heating, or a modifying agent.
The antibody according to. 4. The method according to claim 1, which is a monoclonal antibody.
The antibody according to claim 3. 5. The method according to claim 1, which is a polyclonal antibody.
The antibody according to claim 3. 6. A method for measuring denatured or modified lipoprotein (a), which uses the antibody according to any one of claims 1 to 5. 7. The measurement according to claim 6, wherein the denatured or modified lipoprotein (a) is a lipoprotein (a) denatured or modified by oxidation, reduction, decomposition, heating or a denaturing agent. Law.