JPH08319298A - Monoclonal antibody against anti-human aging marker protein and measurement using the same - Google Patents

Abstract

PURPOSE: To obtain the subject new monoclonal antibody specifically recognizing a human aged marker protein, useful for monitoring a hepatocellular disorder and breakdown of uriniferous tubule and observing development degree of liver and kidney of newborn and an aging ratio with age. CONSTITUTION: An RNA is extracted from a human hepatic cell by a conventional procedure and an mRNA is isolated from an oligo dT cellulose column. The mRNA is used as a template to synthesize a cDNA, which is amplified by PCR method to prepare a cDNA library. A gene of a human SMP30 is selected by using a probe comprising a gene encoding a human aged marker protein (SMP) and incorporated into a vector and manifested in a host cell to give a recombinant human SMP. Then the human SMP is emulsified and administered to a BALB/C mouse. The mouse is immunized and a spleen cell collected after the final immunization is fused with a mouse myeloma cell. After selective culture, the fused cell is cloned and the prepared hybridoma is cultured to give the objective monoclonal antibody specifically recognizing the human SMP.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody (hereinafter referred to as an anti-human antibody) that recognizes a human senescence marker protein (hereinafter referred to as human SMP (senescence marker protein)) present in human organs, tissues, blood, urine, cerebrospinal fluid, etc. Human SMP
Monoclonal antibody) and a method for measuring human SMP using this monoclonal antibody.

[0002]

2. Description of the Related Art SMP was found in rat liver as a protein whose amount changes with aging. This S
MP has a molecular weight of 30 kDa which decreases with age and correlates with its age, for example, androgen-independently.
SMP30 is known (Biochimica Biophisica
Acta 1116: 297-305 (1992)). Regarding human SMP30, rat cDNA was used as a probe.
Was isolated and the nucleotide sequence was determined (Biochemistry, No. 66).
Vol. 4, p. 359 (1994)), a method for producing the monoclonal antibody has not been established, and measurement using the antibody is not known.

[0003]

[Problems to be Solved by the Invention] Human SMP or SMP
The blood level of 30 increases due to liver damage and renal damage. Therefore, by measuring human SMP or SMP30 in blood, it can be used as monitoring of hepatocellular injury and monitoring of renal tubular destruction. By measuring this, the degree of liver and kidney development of the newborn can be observed. Furthermore, human SMP or SMP
Since the concentration of 30 decreases as it ages, it is possible to observe the aging rate against age by measuring this and monitor the index of aging and the health condition. An object of the present invention is to provide a method for producing a monoclonal antibody that recognizes human SMP and measuring human SMP by immunological means using this antibody.

[0004]

Means for Solving the Problems As a result of intensive studies by the present inventors, an anti-human SMP monoclonal antibody that reacts with human SMP and an anti-human SMP3 that reacts with human SMP30
The present invention was completed by discovering 0 monoclonal antibodies and developing an immunoassay using these antibodies.

That is, the present invention relates to human SMP or human SMP.
By providing an antibody that specifically reacts with MP30 and measuring human SMP or human SMP30 using this antibody, liver damage, renal damage, and health status can be monitored.

The present invention will be further described below with reference to human SMP30. The anti-human SMP30 monoclonal antibody of the present invention can be produced by using human SMP30, which is a polypeptide, as an immunogen. Human SMP30 used as this immunogen can be obtained, for example, by isolating it as a polypeptide having a molecular weight of 30 KDa from human liver by combining isoelectric focusing, gel filtration and the like (see Reference Example below). Larger amounts of human SMP3
As a method of obtaining 0, human SMP30 was obtained by cloning the cDNA of this human SMP30 and incorporating it into a plasmid vector and culturing.

The cloning can be performed as follows. RNA was extracted from liver tissue, and DNA was extracted from Poly (A) ⁺ RNA using an oligo dT cellulose column.
Is synthesized and amplified by the PCR method. This is the restriction enzyme E
A cDNA library cleaved with coRI is obtained. Further T4
Treat with ligase and ligate to EcoRI-Not I adaptor. Excess adapter is removed by gel filtration and λZap II
Religate to the vector. Next, human SMP30 cDNA
Human SMP3 already isolated to obtain the A fragment
An oligonucleotide primer is synthesized from a partial amino acid sequence of 0. SMP30 was cloned from the liver cDNA library using a part of SMP30 amplified by the PCR method by the DNA amplification kit as a probe.

After amplifying the DNA fragment by the PCR method, a part of SMP30 was cleaved from the cloning vector with the restriction enzymes BamH1 and Sac1 and introduced into the expression vector by gene recombination, and the vector was used to transform the host. The PCR method is a commercially available PCR amplification kit (PCR
Amplification Kit (Takara) is used as a vector for gene recombination, such as pET-22b (+).
(Novagen) or the like as a host, for example, E. coli,
It can be easily performed by a known method using Bacillus subtilis, yeast and the like.

The polypeptide produced by the host is treated with salt,
After crushing the cells by ultrasonic treatment or the like, the cells can be purified by a known separation and purification means such as gel chromatography and ion exchange chromatography.

Human SMP obtained by such an operation
Anti-human SMP30 monoclonal antibody was prepared using a polypeptide encoded by 30 genes as an immunogen. The anti-human SMP30 monoclonal antibody of the present invention may be a monoclonal antibody that recognizes any part of human SMP30.

The monoclonal antibody of the present invention can be produced, for example, according to the following method. In the production of this monoclonal antibody, the polypeptide encoded by the human SMP30 gene produced by the above-mentioned method or a portion thereof is used as an antigen for animals, such as mice,
Immunize rats. This polypeptide can be used alone or together with an adjuvant to immunize an animal, but if necessary, after binding to a suitable carrier such as KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin). , Can also be administered.

Next, antibody-producing cells of the immunized animal,
For example, a splenocyte and a tumor cell line such as a myeloma cell are fused with a fusion agent such as polyethylene glycol (PEG) to prepare a hybridoma. The hybridomas are then selected using a selection medium such as HAT medium,
Monoclonalization is performed by an appropriate method such as the limiting dilution method, and the cells are cultured. The culture supernatant is analyzed by an appropriate immunoassay, for example, an enzyme immunoassay, to check whether or not the desired anti-human SMP30 antibody is being produced, and to select a clone producing the anti-human SMP30 antibody. . These steps are carried out in a known manner, for example Koehler and Milstein, Nature.
256, 495, (1975), Scheller, N.
It can be carried out by the method described in Nature 285, 446, (1980) and the like.

The monoclonal antibody of the present invention can be separated from the culture supernatant thereof by a conventional method such as salting out, ion exchange chromatography, gel filtration chromatography, etc.
It can be recovered by a purification means. In the case of obtaining a large amount, if the hybridoma is transplanted into the abdominal cavity of a histocompatibility animal or athymic nude mouse or the like and proliferated, and the monoclonal antibody contained in the produced ascites fluid is separated and purified. Good.

Furthermore, the monoclonal antibody of the present invention can be widely used in immunoassays for detecting human SMP30. This monoclonal antibody is a fragment of Fab, Fab ', which is a fragment produced by proteolytic enzyme such as pepsin as it is as the produced antibody.
It may be F (ab ') ₂ or the like. Immunoassays using these antibodies are well known in the art and may be used in any immunoassay. That is, examples of the immunoassay method include a radioimmunoassay method (RIA), an enzyme immunoassay method (EIA), an agglutination immunoassay method, an immunoturbidimetric assay method, and a Western blot method. Even if the method is adopted, human SMP30 can be sufficiently measured.

The human SMP30 measured in the present invention can be measured by a sandwich method using a label, a competitive method or the like. This method is carried out by immobilizing the monoclonal antibody obtained by the present invention at a concentration of 0.001 to 0.1 mg / ml at 4 ° C. overnight on a solid phase such as a microtiter plate or polystyrene beads, and then immobilizing it with a physiological saline solution. It is washed and post-coated with 2% BSA to obtain an immobilized antibody.

The enzyme immunoassay by the competitive method using this immobilized antibody is carried out by combining an enzyme-labeled antibody obtained by binding an enzyme that recognizes an antigenic determinant different from that of the immobilized antibody with an enzyme, and human S.
The immobilized antibody can be further reacted at the same time or after 10 minutes to 3 hours by reacting with a sample containing MP30. The reaction temperature at the time of implementation is 4 ° C to 40 ° C, preferably 25 ° C to 38 ° C. After completion of the reaction, the solid phase is washed, the amount of the enzyme bound to the solid phase is added to an enzyme substrate, and its activity is measured, whereby human SMP30 in the sample can be quantified. Enzymes that can be used in the present invention are, for example, peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase and the like. At this time, a substrate corresponding to the enzyme can be used, for example, ABTS, luminol-H ₂ O.
₂ (for peroxidase), 3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), p-nitrophenyl Phosphate, methyl umbelliferyl phosphate (for alkaline phosphatase), p-nitrophenyl-β-D-galactose, methyl umbelliferyl-β-D-galactose,
3- (2'-spiroadamantane) -4- (3-β-D
-Galactopyranosyl) phenyl-1,2-dioxetane (AMGPD) (for β-D-galactosidase) and the like. Enzyme reaction, measurement from 4 ℃ to 4
The rate method or the reaction for 1 minute to 18 hours while heating at 0 ° C. can be performed to measure the generated color, fluorescence or luminescence.

In the case of radioimmunoassay, a radioisotope such as ¹²⁵ I is labeled instead of the above enzyme label. The measurement operation for carrying out this method is the same as that for the enzyme immunoassay method. Generally, a 1/4 inch polystyrene bead, a polystyrene tube having a diameter of about 1 cm, or a solid phase in which the monoclonal antibody is bound to 0.2 to 10 μm ferrite particles can be prepared and used. For example, if this solid phase is a carboxylated solid phase, 0.1-1 mg of antibody solution (pH
It is prepared by adding water-soluble carbodiimide to 3.5 to 7) and reacting for 1 to 5 hours. Further, the radiolabel of the antibody can be easily prepared by using a Bolton-Hunter reagent which is already commercially available. For this sign, for example, 0.1
It can be prepared by adding this Bolton-Hunter reagent to an antibody solution dissolved in M sodium hydrogen carbonate solution and removing unreacted Bolton-Hunter reagent by using a desalting column of G-25 after 1-2 hours. In addition to this, the chloramine T method, the iodogen method, etc. can be easily adopted.
¹²⁵ I radiolabelling can be performed. To perform an immune reaction, add a sample to the antibody-immobilized solid phase described above, and
℃ ~ 40 ℃, preferably 1 ~ 1 at 25 ℃ ~ 38 ℃
The reaction is carried out for 8 hours. After that, washing with physiological saline or distilled water is performed, the radiolabeled antibody is added to this solid phase, and the temperature is further 4 ° C to 40 ° C, preferably 25 ° C to 3 ° C.
The reaction is carried out at 8 ° C. for 1 to 18 hours, followed by washing with physiological saline or distilled water, and the radioactivity thereof is measured. A scintillation counter is used for the measurement.

In addition to this, the assay method using the label of the present invention may be, for example, a chemiluminescence assay method labeled with isoluminol, acridine ester or the like, or a fluorescence immunoassay method labeled with fluorescein, rhodamine or the like. At this time, the labeling can be easily carried out by adopting the active ester method, the isocyanate method or the like (see “Enzyme-linked immunosorbent assay”, Medical Shoin, 1987).

The other competitive method is that human SMP contained in a sample using human SMP30 labeled with the above-mentioned antibody-bound solid phase.
Thirty measurements can be made. Labeling of human SMP30 can be carried out by reacting in the same manner as the labeling of the above-mentioned antibody. This measurement can be performed by a competitive method well known to those skilled in the art.

Furthermore, the anti-human SMP30 antibody can be used in an agglutination immunoassay. The reagent used for this measurement can be produced by binding one or more kinds of the above-mentioned monoclonal antibodies to a carrier. Examples of the carrier include erythrocytes of animals such as chickens, sheep, goats, cattle, horses, etc., gelatin particles containing gelatin as a main component and water-soluble polysaccharides and metaphosphates (JP-B-63-2).
9223), gelatin magnetic particles containing a magnetic substance in these gelatin particles (Japanese Patent Publication No. 3-17103), and the like. The antibody can be bound to blood cells or particles by the tannic acid treatment method or the glutaraldehyde method ("Introduction to Immunology Experiments", Academic Publishing Center, 19
1981). The measurement is performed using a reagent for agglutination immunoassay and human S
It can be carried out by reacting with a sample containing MP30 and by using the resulting agglutination image. The reaction can be carried out by observing the agglutination image produced by leaving it at room temperature for 1 to 5 hours with a pattern analyzer or visual observation.

Further, the monoclonal antibody of the present invention can be used in an immunoturbidimetric method. The reagent used for this measurement can be produced by binding a monoclonal antibody to a carrier as in the agglutination immunoassay. Examples of the carrier include latex particles such as polystyrene having a particle size of 0.05 to 5 μm, preferably 0.1 to 0.5 μm. In order to produce the antibody-bonded particles, the antibody can usually be physically adsorbed onto the particles. The measurement by the immunoturbidimetric method uses, for example, a wavelength of 550n.
Antibody binding particles and human SMP in the spectrophotometer set to m
It can be carried out by adding a sample containing 30 and mixing, and immediately measuring the change in the absorbance.

Furthermore, the anti-human SMP30 antibody of the present invention can also be used in Western blotting. In this method, after a sample containing human SMP30 is fractionated by gel electrophoresis, the fractionated protein is transferred while maintaining the electrophoretic pattern and transferred onto a carrier such as a nitrocellulose membrane.
This can be performed by reacting the antibody of the present invention and detecting the antibody bound to human SMP30 on the carrier. For the detection of the antibody, a method of using a labeled antibody as an antibody that reacts with a carrier, and an anti-human SMP3 that reacts with the bound antibody
It can be carried out by a method of reacting a labeled antibody against the 0 antibody. The measurement can be performed by the same operation using the above-mentioned enzyme, radioisotope, luminescent substance, fluorescent substance or the like as the labeled substance.

The sample used for the above immunoassay method is
Examples thereof include human organs, tissues, blood, urine, cerebrospinal fluid, and the like, but are not limited thereto.

EXAMPLES The present invention will be described in more detail below with reference to Reference Examples and Examples. However, the present invention is not limited to the following examples.

Reference Example 1 Purification of human SMP30 100 g of human liver was added to 1000 ml of 10 mM Tris.
-HCl / 1mM DTT, 1mM phenylmethyl sulfonyl fluoride, 2mM ATP, 2mM NaF solution (pH = 8.0) It homogenized and it centrifuged for 60 minutes (35000xg). The supernatant was salted out with 50-70% ammonium sulfate to precipitate the protein. Centrifuge for 20 minutes (35
The precipitated protein was recovered, redissolved in 100 ml of the above extract and dialyzed 3 times with the same. This solution was subjected to sucrose concentration gradient isoelectric focusing, and a pI = 4.9 (4.5-5.5) fraction was collected and dialyzed. After further concentration, Sephadex G-75 (Tris-hydrochloric acid, 1 mM DTT, 100 mM NaCl, p
(H = 8.0) was subjected to gel filtration to collect a fraction having a molecular weight of 30 kDa to obtain 1.0 mg of the target human SMP30. The substance purified in this way is human SMP30.
It was confirmed by the Western blot method using the crossability of the anti-rat SMP30 rabbit antibody that the band had a molecular weight of 30 kDa.

Reference Example 2 Cloning of human SMP30 cDNA Cloning of human SMP30 cDNA was carried out by incorporating in a plasmid vector and culturing a large amount of human SMP30.
I got 30. This was done as follows.

RNA was extracted from the liver tissue to obtain oligo dT.
Poly with a cellulose column (Pharmacia)
(A) ⁺ RNA. DNA was synthesized from this poly (A) ⁺ RNA and amplified by the PCR method. The sequence of the primer used in the PCR method is 5'-GGGAGGCCCCTTTTTTTTT
It was TTT-3 'and 5'-AAGGAATTCCCCCCCCCCCCCCC-3'. A cDNA library is obtained by cleaving this with the restriction enzyme EcoRI. Further, it was treated with T4 ligase and ligated to EcoRI-NotI adapter (Invitrogene). Excess adapter was removed by gel filtration and the λZapII vector (Strategen
Company).

Next, in order to obtain a cDNA fragment of human SMP30, an oligonucleotide primer was synthesized from the partially isolated amino acid sequence of human SMP30. The sequence of the synthesized primer is 5'-AGGCTATGTTGCCAC.
CATTGGAA-3 'and 5'-TTCCTCAGCCATGGTACCAGCAAA-3'. CDNA prepared previously using these primers
The A library was used as a template and amplified by the PCR method using a DNA amplification kit, and the obtained DNA was purified by electrophoresis and recovered. This recovered DNA is used as the plasmid SmaI.
Inserted at the site.

DN of the product thus obtained
The A sequence was confirmed and labeled with ³² P for use as a probe for screening. The λZapII bacteriophage plaques (1 × 10 ⁵ ) prepared above are screened with this probe. The plaques were fixed with Hybond-N, prehybridized, and then hybridized with the ³² P-labeled probe. Autoradiography was performed by exposing to X-ray film. The plasmid, which was confirmed to be positive, was amplified with E. coli XL1-BLUE. As a result, DN encoding human SMP30
A plasmid containing A was obtained. This was cleaved with several kinds of restriction enzymes and the sequence was analyzed. Based on this data, computer analysis was performed to determine the total nucleic acid sequence and amino acid sequence. The result of the amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

Example 1 Preparation of Anti-Human SMP30 Monoclonal Antibody-Producing Hybridoma 50 μg of recombinant human SMP30 produced by the method of Reference Example 2 was emulsified using Freund's complete adjuvant and 8-10 weeks old BALB / C
It was intraperitoneally administered to male mice. Two weeks apart,
2 more with Freund Complete Adjuvant
Repeated dose was given. For the final immunization, 20 μg of recombinant human SMP30 diluted with physiological saline was injected from the tail vein, and the spleen was extracted 3 days later. The isolated spleen cells and mouse myeloma cells P3-x63-Ag8-U1 were 3: 1.
50% polyethylene glycol 15
00 was used to perform cell fusion. The cells were suspended in RPMI1640 medium supplemented with HAT (hypoxatin, aminopterin, thymidine) and 10% fetal calf serum (FCS) and dispensed into a 96-well microculture plate,
Cultured. After 10 days, the culture supernatant in which the hybridoma had grown was examined by the following method, and wells containing clones producing a specific monoclonal antibody were selected. That is, 1 μg / ml of human recombinant SMP30 antigen was added to PBS.
Prepared at, and dispensed into a micro assay plate at room temperature 1
It was left to stand for a time for adsorption. After washing with PBS containing 0.05% Tween 20, the hybridoma culture supernatant was added to each well and reacted at room temperature for 1 hour. After washing, peroxidase-labeled anti-mouse immunoglobulin (DAKO
Co., Ltd.) was diluted 1000 times, dispensed, reacted at room temperature for 1 hour, washed, and washed with a substrate (0.1% citrate buffer containing 0.1% ABTS and 0.031% hydrogen peroxide (pH 4. 0))
Was added. Stop the reaction by adding 1% oxalic acid, and
The absorption at 05 nm was measured. Cells in positive wells were selected and cloned by the limiting dilution method. The antibody activity of the culture supernatant of the well containing a single colony was examined and selected by the method described above. Hybridomas SMP3.1 and SMP10.1 producing antibodies specific for human SMP30 were established. These two types of hybridomas SMP3.1 and SMP10.1 have been deposited at the Institute of Biotechnology,
The deposit numbers are FERMP-14845 and F, respectively.
ERM P-14846.

Example 2 Determination of Subclass of Antibody The subclass of these antibodies was determined by the Ouchterlony method. That is, the culture supernatant of the hybridoma was added to the holes formed on the agarose gel plate, and various rabbit antisera (Cap
pl) and the sedimentation line was examined.
It was gG 1 (κ).

Example 3 Preparation of anti-human SMP30 monoclonal antibody Hybridoma SMP3.1 and SMP10.1 cells 1
x10 ⁷ cells were each inoculated intraperitoneally into BALB / C mice after two weeks after pristane 0.5ml dose. One week later, ascites containing a high concentration of antibody was collected. 2 this ascites
Dilute 4 times or more with 5 mM morpholine ethanesulfonic acid (MES) buffer solution (pH 4.0) to obtain 20 mM MES
Adsorbed on a carbon dioxide ABx column equilibrated with (pH 5.6) and containing 500 mM ammonium sulfate 20
Elution with a gradient of mM sodium acetate (pH 7.0) gave anti-SMP30 monoclonal antibodies 3.1 and 10.1, respectively.

Example 4 Confirmation of Specificity by Immunoblotting Human liver homogenate was centrifuged at 105,000 × g for 60 minutes, and the supernatant fraction was heat-treated at 60 ° C. for 10 minutes. 1 more
A supernatant of 20,000 xg was used as a sample of SMP30. Human liver SMP30 sample was mixed with an equal volume of sample diluent (40% glycerin, 2% SDS, 2% 2-mercaptoethanol, 20%).
mM Tris-HCl buffer), heated in boiling water, migrated with 15% SDS PAGE and transferred to a nitrocellulose membrane. The transfer membrane was blocked with 3% BSA-PBS, and reacted with anti-SMP30 monoclonal antibodies 3.1 and 10.1 at room temperature for 1 hour. Tween20-
After washing with PBS, POD-labeled anti-mouse immunoglobulin was reacted at room temperature for 1 hour, and after washing, POD-insoluble substrate (4-
Chloronaphthol-hydrogen peroxide system) was added to develop the color. As a result, a band was generated near the molecular weight of 30 kDa,
This coincided with the protein band of Coomassie Brilliant Blue which was performed at the same time, and it was confirmed that this monoclonal antibody specifically recognizes human SMP30.

Example 5 Determination of Optimal Antibody Sensitization Amount of antibody sensitization to 96-well plate was determined by the following method. That is, the anti-SMP30 monoclonal antibody 3.1 was diluted 2 ⁿ from 20 μg / ml with 0.1 M phosphate buffer and added to a 96-well plate at a rate of 50 μl / well. After standing overnight at 4 ° C, PBS-Tween
And washed three times. After that, 200 μl / well of 3% BSA was added, and the mixture was left at 4 ° C. overnight for blocking, and PBS-
It was washed 3 times with Tween and used for measurement. Recombinant human S
MP30 was diluted with a buffer solution to a concentration of 50 ng / ml, added at a rate of 50 μl / well, and reacted at room temperature for 2 hours. After washing three times with PBS-Tween, anti-mouse immunoglobulin-POD labeled antibody (DAKO) was diluted 1000 times with 1% BSA-PBS buffer, and 50
μl / well was added and the mixture was reacted at room temperature for 2 hours. PB
After washing 4 times with S-Tween, 50 μl / well of substrate solution (ABTS) was added and the absorbance after 15 minutes (wavelength 405
/ 492 nm) was measured. The result is shown in FIG. As shown in Fig. 1, the amount of antibody sensitized to the solid phase was 5 μg.
If the concentration is not less than / ml, human SMP30 can be efficiently measured.

Example 6 Preparation of Enzyme-Labeled Antibody Anti-human SMP30 monoclonal antibody 10.1 was labeled with horseradish-derived peroxidase using the Nakane method. The concentration of labeled anti-human SMP30 monoclonal antibody was determined to be 360.9 μg / ml. The number of enzyme molecules introduced per molecule of the antibody was one.

Example 7 Measurement of human SMP30 Recombinant human SMP30 was diluted with 1% BSA-0.1% phosphate buffer (pH 7.0) to give 0.7 each.
8, 1.56, 3.13, 6.25, 12.5, 50n
A standard recombinant human SMP30 solution at g / ml was prepared. This was used as a sample and measured by the following method. 50 μl / well of standard recombinant human SMP30 was added to a plate sensitized with anti-human SMP30 monoclonal antibody 3.1, and the mixture was reacted at 4 ° C. overnight. After washing 3 times with PBS-Tween buffer, it was prepared in Example 6 and prepared with 1% BSA-0.
Enzyme-labeled anti-human SMP30 diluted with 1M PBS buffer
Add 50 μl / well of monoclonal antibody 10.1,
The reaction was carried out for 2 hours at room temperature. After washing 4 times with PBS-Tween buffer, 50 μl / well of substrate solution (ABTS) was added, and color was developed for 15 minutes. The reaction was stopped by further adding 100 μl / well of oxalic acid, and the absorbance (wavelength 405 /
492 nm) was measured. The calibration curve thus obtained is shown in FIG. As you can see from this figure, 50 ng / m
It showed linearity up to 1. Recombinant human SMP3
The lowest detected concentration of 0 was 0.78 ng / ml.

[0036]

INDUSTRIAL APPLICABILITY The present invention provides an anti-human SMP monoclonal antibody that specifically reacts with human SMP. The measurement using this antibody has made it possible to detect and quantify human SMP present in human tissues or body fluids. Therefore, the method of the present invention can be used to monitor hepatocellular injury and renal tubular destruction, observe the degree of liver and kidney development in newborns, and observe the age-related aging rate to determine the health status. It is considered that it will contribute to monitoring the

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 299 Sequence type: Amino acid Topology-: Linear sequence Met Ser Ser Ile Lys Ile Glu Cys Val Leu Pro Glu Asn Cys Arg Cys 1 5 10 15 Gly Glu Ser Pro Val Trp Glu Glu Val Ser Asn Ser Leu Leu Phe Val 20 25 30 Asp Ile Pro Ala Lys lys Val Cys Arg Trp Asp Ser Phe Thr Lys Gln 35 40 45 Val Gln Arg Val Thr Met Asp Ala Pro Val Ser Ser Val Ala Leu Arg 50 55 60 Gln Ser Gly Gly Tyr Val Ala Thr Ile Gly Thr Lys Phe Cys Ala Leu 65 70 75 80 Asn Trp Lys Glu Gln Ser Ala Val Val Leu Ala Thr Val Asp Asn Asp 85 90 95 Lys Lys Asn Asn Arg Phe Asn Asp Gly Lys Val Asp Pro Ala Gly Arg 100 105 110 Tyr Phe Ala Gly Thr Met Ala Glu Glu Thr Ala Pro Ala Val Leu Glu 115 120 125 Arg His Gln Gly Ala Leu Tyr Ser Leu Phe Pro Asp His His Val Lys 130 135 140 Lys Tyr Phe Asp Gln Val Asp Ile Ser Asn Gly Leu Asp Trp Ser Leu 145 150 155 160 Asp His Lys Ile Phe Tyr Tyr Ile Asp Ser Leu Ser Tyr Ser Val Asp 165 170 175 Ala Phe Asp Tyr Asp Leu Gln Thr Gly Gln Ile Ser Asn Arg Arg Ser180 185 190 Val Tyr Lys Leu Glu Lys Glu Glu Gln Ile Pro Asp Gly Met Cys Ile 195 200 205 Asp Ala Glu Gly Lys Leu Trp Val Ala Cys Tyr Asn Gly Gly Arg Val 210 215 220 Ile Arg Leu Asp Pro Val Thr Gly Lys Arg Leu Gln Thr Val Lys Leu 225 230 235 240 Pro Val Asp Lys Thr Thr Ser Cys Cys Phe Gly Gly Lys Asn Tyr Ser 245 250 255 Glu Met Tyr Val Thr Cys Ala Arg Asp Gly Met Asp Pro Glu Gly Leu 260 265 270 Leu Arg Gln Pro Glu Ala Gly Gly Ile Phe Lys Ile Thr Gly Leu Gly 275 280 285 Val Lys Gly Ile Ala Pro Tyr Ser Tyr Ala Gly 290 295

[Brief description of drawings]

FIG. 1 shows the human S when the amount of antibody binding was examined by binding the monoclonal antibody of the present invention to a 96-well plate.
It is a figure which shows the MP30 measurement result.

FIG. 2: Human S using the monoclonal antibody of the present invention
It is a figure which shows the result of the calibration curve when measuring MP30 standard solution.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location C07H 21/04 C07H 21/04 B C12N 5/10 9453-4B C12Q 1/68 A 15/02 9281-4B C12N 5/00 B C12Q 1/68 9162-4B 15/00 C (C12P 21/08 C12R 1:91) (C12N 5/10 C12R 1:91) (72) Inventor Masahisa Okada 2-chome Nishishinjuku, Shinjuku-ku, Tokyo 7-1 Fuji Rebio Co., Ltd. (72) Inventor Naoharu Maruyama 18-5-501 Sakaemachi, Itabashi-ku, Tokyo (72) Inventor Keiko Fujita 7-4-7 Higashi-Oizumi, Nerima-ku, Tokyo

Claims (6)

[Claims] 1. A human aging marker protein (human SM
A monoclonal antibody that recognizes P). 2. The monoclonal antibody according to claim 1, wherein the human SMP is human SMP30. 3. The monoclonal antibody according to claim 2, wherein human SMP30 is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 4. The monoclonal antibody according to claim 3, wherein the antibody is obtained from a hybridoma which is FERM P-14845. 5. The monoclonal antibody according to claim 3, wherein the antibody is obtained from a hybridoma that is FERM P-14846. 6. A method for measuring human SMP using the monoclonal antibody according to any one of claims 1 to 5.