JP3194762B2 - Monoclonal antibodies, their production and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a useful and novel antibody having binding specificity to human big endothelin-2 (sometimes abbreviated as big ET-2). More specifically, human big endothelin based on antigen-antibody reaction
Of the assay method for human 2 and human big endothelin-2
Alternatively, the present invention relates to an antibody useful for diagnosing a disease associated with endothelin-2.

[0002]

2. Description of the Related Art Endothelin-1 is a peptide consisting of 21 amino acids found in the culture supernatant of vascular endothelial cells and has an extremely potent and sustained vascular smooth muscle contracting activity and a blood pressure increasing activity. In addition, endothelin-1
From the analysis of the cDNA of the above, the existence of big endothelin-1 consisting of about 40 amino acids is also assumed as an intermediate in the biosynthesis process. To date, monoclonal antibodies with binding properties to endothelin-1 and big endothelin-1 have been produced, and a highly sensitive enzyme immunoassay has been developed to elucidate the physiological role of endothelin-1 and its relationship to disease states. (Yanagisawa et al., Nature, 332 , 44)
1, (1988), Ito et al., FEBS Letters
s), 231 , 440 (1988), JP-A-1-206997, JP-A-2-72877, JP-A-2-238894.

On the other hand, analysis of chromosomal DNA has revealed that endothelin-2, endothelin-3 and their cD
NA was found, and it was revealed that endothelin forms a gene family (Yanagisawa et al., Procesings of National Academy of Sciences (Pro. Natl. Acad. Sci. USA.)). , 85 , 6964 (198
8), Inoue et al., The Prossings of National Academy of Science (Pro.Natl.Acad.Sci.US)
A.), 86 , 2863 (1989), Japanese Patent Application No. 1-274990, Japanese Patent Application No. 2-257492). As shown in the following amino acid sequence (see underline), endothelin-1 and endothelin-2 differ by 2 residues, and endothelin-1 and endothelin-3 differ by 6 residues. Endothelin-1 (derived from pigs, humans, dogs, rats, mice, and cows; sometimes called endothelin-α) Cys Ser Cys Ser Ser Leu Met
Asp Lys Glu Cys Val Tyr Phe Cys His
Leu Asp Ile Ile Trp (SEQ ID NO: 1) Endothelin-2 (derived from human and dog) Cys Ser Cys Ser Ser Trp Leu
Asp Lys Glu Cys Val Tyr Phe Cys His
Leu Asp Ile Ile Trp (SEQ ID NO: 2) Endothelin-3 (from rat, human, pig, rabbit).
It was sometimes called endothelin-γ. ) Cys Thr Cys Phe Thr Tyr Lys
Asp Lys Glu Cys Val Tyr Tyr Cys His
Leu Asp Ile Ile Trp (SEQ ID NO: 3) Since a monoclonal antibody having a binding property to endothelin-3 has already been prepared and a specific assay method has been developed (Japanese Patent Application No. 2-194081), −
Research on the physiological and pathophysiological roles of 3 has also become possible.

[0004]

On the other hand, endothelin-
1 and endothelin-3 compared to endothelin-2
Research on the physiological and pathophysiological roles of has little progress to date. The main cause of this is that no monoclonal antibody specifically recognizing endothelin-2 or endothelin-2 precursor has been produced so far, and endothelin-2 or endothelin-2 precursor is measured specifically and with high sensitivity. Immunological assays have not been developed. These immunological approaches have been used to study endothelin-2, especially metabolic pathways,
It is considered to be one of the most effective means for comprehensively conducting research on the secretory mechanism, receptor system, association with a disease state, and the like, and the establishment of such a method has been eagerly desired by various fields.

[0005]

Means for Solving the Problems The present inventors have found that the amino acid sequence of a human endothelin-2 precursor that has recently been identified (Japanese Patent Application No. 3-143127) is a direct precursor of human endothelin-2. Focusing on the sequence of human big endothelin-2, which is the body, as a result of diligent efforts, a monoclonal antibody having specific binding properties to human big endothelin-2 could be produced. That is, human big endothelin-1 (human big endothelin-
Homology to 2 30 residues out of 38 residues: 79%) and human big endothelin-3 (human big endothelin-
Homology with 3 residues 41 or 26 out of 42 residues: 6
2 or 63%) and a monoclonal antibody having a binding property to human big endothelin-2 having a cross-reactivity of not more than 0.5%. Human Big Endothelin-1 Cys Ser Cys Ser Ser Leu Met
Asp Lys Glu Cys Val Tyr Phe Cys His Leu
Asp Ile Ile Trp Val Asn Thr Pro Glu His
Val Val Pro Tyr Gly Leu Gly Ser Pro Arg
Ser (SEQ ID NO: 4) Human Big Endothelin-2 Cys Ser Cys Ser Ser Trp Leu
Asp Lys Glu Cys Val Tyr Phe Cys His Leu
Asp Ile Ile Trp Val Asn Thr Pro Glu Gln
Thr Ala Pro Tyr Gly Leu Gly Asn Pro Xaa
(However, Xaa = Pro or Xaa = Pro Ar
g) (SEQ ID NO: 5) Hereinafter, Xaa = P for human big endothelin-2.
ro of the human big endothelin-2 (1
-37) or bigET-2 (1-37) and those having the structure of Xaa = ProArg were compared with human big endothelin-2 (1-38) or bigET-2.
It may be expressed as (1-38).

[0006] Human Big Endothelin-3 CysThr CysPhe Thr Tyr Lys 
Asp Lys Glu Cys Val TyrTyr Cys His Leu
Asp Ile Ile TrpIle Asn Thr Pro Glu Gln
ThrVal Pro Tyr Gly LeuSer AsnTyr Arg 
Gly Ser Phe Arg Xaa (Xaa = Gly-OH or Xaa =
NH_Two) (SEQ ID NO: 6) Further increasing human big endothelin-2 using the antibody
An immunoassay that can be detected sensitively and specifically has been developed.
That is, the present invention relates to human big endothelin-2 or
Is a new monochrome that binds to its C-terminal peptide
Null antibody, hybrid producing the monoclonal antibody
Antibody, a method for producing the antibody, and a competitive method using the antibody.
Or human big endothelin by sandwich method
2 and its C-terminal peptide. Human
As a big endothelin-2 C-terminal peptide,
For example, the formula Val Asn Thr Pro Glu Gln Thr
Ala Pro Tyr Gly Leu Gly Asn Pro Xaa
And Xaa = Pro or Xaa = Pro Arg
Human big endothelin-2 C-terminal pepti represented by (SEQ ID NO: 7)
(22-37) (Xaa = Pro) or (22-37)
38) (Xaa = Pro Arg).

In preparing the monoclonal antibody of the present invention, a complex of human big endothelin-2 or its C-terminal peptide and a carrier protein is prepared, and this is inoculated into an animal for immunization. From 2 to 5 days after the final immunization, the spleen or lymph node is collected, and the antibody-producing cells contained therein are fused with myeloma cells to produce a hybridoma stably producing a high-titer antibody. To obtain a monoclonal hybridoma. As the immunizing antigen, any of a natural purified product, a synthetic product and the like can be used, and the above-mentioned human big endothelin-2 or its C-terminal peptide is used. As an immunizing antigen, human big endothelin-
In some cases, bacterial or cell products into which a plasmid containing the gene sequence 2 has been introduced may be used. The various peptides used in the present invention can be produced by known conventional means of peptide synthesis. Either solid phase synthesis or liquid phase synthesis may be used. For example, when human big endothelin-2 is synthesized by a solid phase method, a solid phase peptide synthesis method of Maryfield [Journal of the American Chemical Society (J. Am. Chem. Soc.), 85 , 2149 (1963)]
It is preferable to use The insoluble resin may be any of those known in the art, for example, chloromethylated styrene-divinylbenzene copolymer, phenacylacetic methylated styrene-
A polystyrene type resin such as a divinylbenzene copolymer and a polyamide type resin such as a polydimethylacrylamide resin are exemplified. After binding the C-terminal N-protected amino acid to the insoluble resin, human big endothelin-2
, The protected amino acids are sequentially bonded from the C-terminal side according to a conventional method, and then treated with hydrogen fluoride, thereby forming a disulfide bond to synthesize the desired human big endothelin-2. As N-protected amino acids, α-
All amino groups are protected with Boc groups, the hydroxyl groups of serine and threonine are Bzl groups, the ω-carboxylic acids of glutamic acid and aspartic acid are OBzl groups, the ε-amino group of lysine is Cl-Z group, and the thiol group of cysteine is Acm
Group, MeBzl group, hydroxyl group of tyrosine is Br-Z group, imidazole group of histidine and guanide group of arginine are Tos group, and indole group of tryptophan is CHO.
It is preferred to protect with a group.

[0008] As a means of synthesis by the liquid phase method, for example, “The Peptides”, Vol.
Year), Schroder and Lubke, Academic Press, New Y
ork, USA or "Peptide Synthesis", a method described by Izumiya et al., Maruzen Co., Ltd. (1975), for example, azide method, chloride method, acid anhydride method, mixed acid anhydride method, DC
C method, active ester method, method using Woodward reagent K, carbodiimidazole method, redox method, DCC / additive (eg, HONB, HOBt, HOSu) method and the like. In addition, as the various peptides used in the present invention, bacterial or cell products into which a plasmid containing the human big endothelin-2 gene sequence has been introduced may be used. As a method for producing human big endothelin-2-related peptide by genetic manipulation, for example, a method using a product of a transformed CHO-K1 cell into which an expression vector for ET-2 precursor cDNA has been introduced (for example, Japanese Patent Application No. 3-143127). No.).

Regarding the protein complex of the immunizing antigen and the carrier protein used for immunizing mammals, the type of the carrier protein and the mixing ratio of the carrier and the hapten are determined based on the hapten immunized by coupling to the carrier. As long as the antibody can be efficiently produced, whatever may be coupled at any ratio, for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of 0.1 to 20 with respect to 1 hapten, preferably A method of coupling at a rate of 1 to 5 is used. Further, various coupling agents can be used for coupling of the hapten and the carrier, but glutaraldevito, carbodiimide,
Maleimide active esters and the like are conveniently used. The condensation product is administered to a warm-blooded animal itself or together with a carrier and a diluent at a site where the antibody can be produced by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks. The warm-blooded animals used include, for example, mice, rats, rabbits and the like.

[0010] Next, from the immunized warm-blooded animal, for example, a mouse, an individual having an antibody titer is selected and subjected to the final immunization 2 to 5
After 5 days, the spleen or lymph node is collected, and the antibody-producing cells contained therein are fused with myeloma cells,
An anti-human big endothelin-2 antibody-producing hybridoma can be prepared. The fusion operation can be performed by known methods, for example, the method of Kohler and Milstein [Nature
re), 256 , 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Myeloma cells include, for example, NS-1, P3U
1, SP2 / 0, etc., and P3U1 is particularly preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells used is 1: 1 to 20: 1.
PEG (preferably PEG 1000 to PEG 60
00) is added at a concentration of about 10 to 80%, and the cell fusion can be carried out efficiently by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.

[0011] Anti-human big endothelin-2 antibody producing
Various methods can be used to screen for hybridomas.
But, for example, human big endothelin-2,
C-terminal peptide of egg endothelin-2 or their
Adsorbed on a complex in which is bound to a carrier protein
Add the hybridoma culture supernatant to the phase (e.g., microplate).
And then with horseradish peroxidase (HRP)
Labeled anti-immunoglobulin antibodies (used for cell fusion
If the cells are mice, use anti-mouse immunoglobulin antibodies
Or protein A was added and bound to the solid phase.
ELISA for detecting monoclonal antibodies (Enzyme-link
ed immunosorbent assay), anti-immunoglobulin antibody
Or hybridoma culture on a solid phase to which protein A has been adsorbed.
Add the culture supernatant and add HRP-labeled human big endothese.
C-terminal of phosphorus-2 or human big endothelin-2
Peptide is added and monoclonal antibody bound to solid phase
EIA (Enzyme immunoassay) method to detect
You. Selection and breeding of hybridomas are usually performed using HAT (Hipoki
Santin, aminopterin, thymidine) and add 10
Medium for animal cells containing ~ 20% fetal calf serum (FCS)
(Eg, RPMI 1640). Hybridoma culture
The antibody titer in the supernatant is the same as the above-mentioned antibody titer in antiserum.
Can be measured. The hybrid obtained in this way
To human big endothelin-2 and its C-terminal pepti
As a method for producing an antibody having a binding property to
Cell culture method and ascites formation method are used. Cell culture
In the method, the hybridoma is, for example, 10-15
% FCS-containing RPMI 1640 medium, serum-free medium, etc.
Culture in animal cell culture medium, and
You can get the body. On the other hand, using ascites formation method
Hybridomas and major tissues can be recovered from ascites
Pistane (2, 4, 6, 1)
0,14-tetramethylpentadecane)
After administration into the ascites fluid, about 10 hybridomas ⁷Individual abdominal cavity
Intravenous administration. Hybridomas have ascites in about 10 to 18 days
Forms tumors and produces high concentrations of antibodies in serum and ascites
I do.

The separation and purification of the anti-human big endothelin-2 antibody can be performed by separating and purifying immunoglobulin from the culture supernatant of the hybridoma or ascites of the animal to which the hybridoma has been administered (eg, salting out, alcohol precipitation, etc.).
Isoelectric point precipitation, electrophoresis, ion exchanger (eg, DEA
E) Adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method of collecting only an antibody with an antigen-binding substance or an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody] Done. A hybridoma producing a monoclonal antibody that reacts with a partial region of human big endothelin-2 (eg, N-terminal portion, C-terminal portion, intermediate portion);
Selection of a hybridoma that produces a monoclonal antibody that reacts with 2, but does not react with a partial region thereof can be performed, for example, by measuring the binding between a peptide corresponding to the partial region and an antibody produced by the hybridoma. it can.

In the present invention, a monoclonal antibody which binds to human big endothelin-2 (SEQ ID NO: 5) and does not react with endothelin-2 (SEQ ID NO: 2) and a human big endothelin-2 C-terminal peptide (SEQ ID NO: 2) A monoclonal antibody having the binding property to SEQ ID NO: 7) is obtained.

For the measurement of human big endothelin-2, the competition method described below is usually used, but the sandwich method described later is more preferably used. In the competition method, the anti-human big endothelin-2 obtained by the present invention is used.
Antibody, test solution and labeled human big endothelin
2 or its C-terminal peptide, and by measuring the ratio of the labeling agent bound to the antibody, the human big endothelin-2 or the C-terminal of human big endothelin-2 in the test solution is measured. Quantify the peptide.

Examples of the labeling agent or the labeling agent for the antibody described below include radioisotopes, enzymes, fluorescent substances, luminescent substances and the like. As a radioisotope, for example,
¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C and the like include
Stable and large specific activities are preferable, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., and as a fluorescent substance, fluorescamine, fluorescein isothiocyanate, etc. emit light. Examples of the substance include luminol, luminol derivatives, luciferin, lucigenin and the like. Furthermore, a biotin-avidin system can be used for binding the antibody or human big endothelin-2 or its C-terminal peptide to a labeling agent.

In measuring the activity of the above-mentioned labeling agent, the labeling agent bound to the antibody and the free labeling agent are separated (hereinafter, B / B).
F), but when an enzyme is used as a labeling agent, the reagent for this may be an insoluble antibody to the antibody used for measurement, or an active adsorbent such as insolubilized protein A. Is advantageously used. For example, an anti-IgG antibody (corresponding to an antibody against anti-human big endothelin-2 antibody) is used as a solid phase, and labeled human big endothelin-2 is immobilized on the solid phase through the antibody reactive therewith. And measuring the labeling agent on the solid phase.
When an enzyme is used as a labeling agent, an ordinary colorimetric method or a fluorescent method is used for measuring the enzyme activity on the insolubilized carrier. When a non-proteinaceous substance such as a radioisotope is used as a labeling agent, other than the above reagents for B / F separation, for example, an antibody having a binding property to anti-human big endothelin-2 which is not insolubilized, sodium sulfate, dextran charcoal Finally, a reagent such as polyethylene glycol is used. In any method, the activity of the labeling agent in the supernatant or the precipitate is measured. For the above insolubilization, physical adsorption may be used, and usually insolubilize proteins or enzymes, etc.
A method using a chemical bond used for immobilization may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the competition method, for example, when measuring human big endothelin-2, an anti-human big endothelin-2 antibody, a test solution, and labeled human big endothelin-2 are used.
2 and the B / F separation reagent can be reacted in any order, and all or a part of the reagents can be reacted simultaneously. It is preferably added to the reaction system simultaneously with or after the reaction of the solution with the anti-human big endothelin-2 antibody. However, B such as sodium sulfate, dextran charcoal powder, polyethylene glycol, etc.
The / F separation reagent is mainly used at the end of the reaction system. On the other hand, in the sandwich method, the test solution is brought into contact (reaction) with the insolubilized anti-human big endothelin-2 antibody (1).
Next reaction), and labeled anti-human big endothelin-2
After reacting the antibody (secondary reaction), human big endothelin-2 in the test solution can be quantified by measuring the activity of the labeling agent on the insolubilized carrier. The primary reaction and the secondary reaction may be performed at the same time or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above.

As the human big endothelin-2 antibody used in the secondary reaction, the anti-big endothelin-2 antibody used in the primary reaction is human big endothelin-2.
Antibodies having different sites for binding to the antibody are preferably used. For example, when the antibody used in the primary reaction has a binding property at the C-terminal of human big endothelin-2,
In the subsequent reaction, an anti-human big endothelin-2 antibody that binds preferably to a site other than the C-terminal (eg, N-terminal) is used,
When the antibody used in the primary reaction has a binding property at the N-terminal of human big endothelin-2, in the secondary reaction,
Preferably, an anti-human big endothelin-2 antibody that binds to a moiety other than the N-terminus (eg, the C-terminus) is used. In the immunoassay for human big endothelin-2 by the sandwich method, it is particularly preferable to react with endothelin-2 but to react with the C-terminal peptide of endothelin-2, Cys-His.
-Anti-endothelin-2 monoclonal antibody which does not react with Leu-Asp-Ile-Ile-Trp (SEQ ID NO: 8) and human big endothelin-2 C-terminal peptide (22-37)
Alternatively, a monoclonal antibody against (22-38) is used. In the immunoassay by the sandwich method, both the solid phase antibody and the labeling antibody may be of any class or subclass. If the antibody activity is retained, Fc 'or Fc region is removed from them. (ab ') ₂ fractions, Fab' fraction or F
The ab fraction may be used. When a monoclonal antibody is used in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one type, but may be used for the purpose of improving measurement sensitivity and specificity. A mixture of the above antibodies may be used.

The immunoassay using the antibody obtained in the present invention can be used for diagnosis and prognosis management of human big endothelin-2 or a disease associated with endothelin-2. As test samples, body fluids such as plasma, serum, urine, cerebrospinal fluid, ascites, pleural effusion, amniotic fluid, sputum, stool and the like can be used. These samples can be used as they are or as samples for immunoassay after dilution or extraction with various buffers and concentration. Any buffer or organic solvent may be used as the solvent used for diluting or extracting the sample, but preferably a buffer for immunoassay, water, saline, acetate buffer, acetone, chloroform-methanol or These solutions containing surfactants are used. In addition, concentration is performed by directly reducing the sample under reduced pressure, or normal pressure,
The sample may be concentrated under a stream of nitrogen, or after the sample is added to a carrier for ion exchange or reverse phase chromatography or a carrier binding to anti-human big endothelin-2 antibody, and then eluted under appropriate elution conditions, and then under reduced pressure or normal pressure. Alternatively, it may be concentrated under a stream of nitrogen. Particularly preferably as a carrier for concentration,
C2, C8 or C as a carrier for reversed phase chromatography
18 cartridges are used. The concentrate is dissolved in an immunoassay buffer, and then used as a sample for immunoassay. Furthermore, the anti-human big endothelin-2 antibody obtained in the present invention can be used for immunohistochemical staining of human big endothelin-2. The method can be based on, for example, a direct method using a labeled anti-human big endothelin-2 antibody, an indirect method using a labeled anti-human big endothelin-2 antibody and an antibody against the antibody, or the like.

In the specification of the present invention, when amino acids and the like are represented by abbreviations, IUPAC-IUB Commission on Bioc
Abbreviations based on chemical nomenclature or conventional abbreviations in the field, examples of which are given below. When there is an optical isomer for an amino acid, the L-form is indicated unless otherwise specified. PAM: phenylacetamidomethyl BHA: benzhydrylamine Boc: t-butyloxycarbonyl Cl-Z: 2-chloro-benzyloxycarbonyl Bг-Z: 2-bromo-benzyloxycarbonyl Bzl: benzyl OBzl: benzyl ester Tos: p-toluenesulfonyl MeBzl: 4-methylbenzyl Acm: acetamidomethyl HOBt: 1-benzotriazole DCC: N, N'-dicyclohexylcarbodiimide PBS: phosphate buffered saline PMSF: phenylmethylsulfonyl fluoride Gly: glycine Ala : Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Met: Methionine Asp: Aspartic acid Lys: Lysine A g: arginine His: histidine Phe: phenylalanine Tyr: tyrosine Asn: asparagine Gln: glutamine Cys: Cysteine Glu: glutamic acid Trp: tryptophan Pro: proline

[0021]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention.

The hybridoma cell bET-20 producing the mouse monoclonal antibody bET-20a used in the following Examples was deposited with the Fermentation Research Institute (IFO) under the accession number IFO 50256 from September 21, 1990. Have been. In addition, the hybridoma was awarded the accession number FERM to the Institute of Microbial Industry (FRI) of the Ministry of International Trade and Industry.
It has been deposited under the Budapest Treaty on October 16, 1990 as BP-3132 and is stored at the FRI.

Example 1 Synthesis of I peptide (I-1) bigET-2 (22-37): H-Val-A
sn-Thr-Pro-Glu-Gln-Thr-Al
a-Pro-Tyr-Gly-Leu-Gly-Asn
-Pro-Pro-OH (SEQ ID NO: 7, Xaa = Pr
Synthesis of o) Using a commercially available Boc-Pro-OCH ₂ -PAM resin (manufactured by Applied Biosystems) 0.77 g (0.5 nmole), a peptide synthesizer (Model 430A manufactured by Abride Biosystems)
And synthesized. After treating the Boc group on the resin with 50% trifluoroacetic acid / methylene chloride to release the amino group, the amino group is added to Boc-Pro, Boc-Asn, Boc-Gly, Boc
-Leu, Boc-Tyr (Br-Z), Boc-Ala, Boc-Thr (Bzl), Boc-Gl
n, Boc-Glu (OBzl) and Boc-Val were activated and condensed with HOBt / DCC in the order of the amino acid sequence of big ET-2 (22-37). Further DC
After condensing again with the same amino acid derivative activated with C or HOBt / DCC, unreacted amino groups are acetylated with acetic anhydride to obtain 2.35 g of bigET-2 (22-37) -OCH ₂ -PAM resin. Was.

After treating 0.30 g of this resin with 5 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 0.55 g of p-cresol, hydrogen fluoride is distilled off under reduced pressure, and the residue is diluted with 5 ml of ethyl ether.
After washing twice, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insolubles were removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing solution were combined, concentrated under reduced pressure to 2-3 ml, and Sephadex LH was added.
It was applied to a -20 (2 × 90 cm) column and eluted with 50% acetic acid. The main fractions are collected and concentrated to 0.1% aqueous trifluoroacetic acid 100
The mixture was dissolved in a 0.1 ml solution and applied to a YMC-ODS AM120 S-50 resin column (2.6 × 7 cm), and 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid)
Was eluted with a linear concentration gradient. The main fractions were combined and lyophilized to give 77 mg of a white powder. Amino acid analysis value Asp 1.92 (2), Thr 1.76 (2), Glu 2.04 (2), Gly 2.00
(2), Ala 1.02 (1), Val 1.00 (1), Pro 3.90 (4), Leu 0.
99 (1), Tyr 0.95 (1), by mass spectrometry (M + H) +1654.776 HPLC elution time 20.0 min Column conditions Column: WAKOSIL 5C18-200 (4.6 × 250) Eluent: Solution A (0.1% -trifluoro Aqueous acetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min (I-2) big ET-2 (1- 37); H-Cys-Ser-Cys-Ser-Ser-Trp-
Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Il
e-Ile-Trp-Val-Asn-Thr-Pro-Glu-Gln-Thr-Ala-Pro-Tyr-
Gly-Leu-Gly-Asn-Pro-Pro-OH (SEQ ID NO: 5, Xaa =
Synthesis of Pro) Using a commercially available Boc-Pro-OCH ₂ -PAM resin (manufactured by Applied Biosystems) 0.78 g (0.5 nmole), a peptide synthesizer (Applied Biosystems model 430A)
And synthesized. After treating the Boc group on the resin with 50% trifluoroacetic acid / methylene chloride to release the amino group, the amino group is added to Boc-Pro, Boc-Asn, Boc-Gly, Boc
-Leu, Boc-Tyr (Br-Z), Boc-Ala, Boc-Thr (Bzl), Boc-Gl
n, Boc-Glu (OBzl), Boc-Val, Boc-Trp (CHO), Boc-Ile,
Boc-Asp (0Bzl), Boc-His (DNP), Boc-Cys (MeBzl), Boc-Ph
e, Boc-Lys (Cl-Z) and Boc-Ser (Bzl) were activated and condensed with HOBt / DCC according to the amino acid sequence of big ET-2 (1-37). After another condensation with the same amino acid derivative activated with DCC or HOBt / DCC, the unreacted amino group is acetylated with acetic anhydride, and the protected big-ET-2 (1-37) -OCH _2- PAM resin was obtained. This was suspended in N, N'-dimethylformamide (30 ml), thiophenol (3 ml) was added, and the mixture was gently stirred at room temperature for 2 hours, and then the resin was filtered on a glass filter.
After washing with N'-dimethylformamide and dichloromethane, drying was performed to obtain 3.36 g of a resin.

0.71 g of this resin was added to 1.50 g of p-cresol,
1,4-butanedithiol 1.0 ml anhydrous hydrogen fluoride in the presence of 10 ml
After treatment with 0 for 60 minutes at 0 ° C, hydrogen fluoride was distilled off under reduced pressure. The residue was washed twice with 5 ml of ethyl ether, and the residue was extracted with 4 ml of trifluoroacetic acid. Filter off insolubles,
Washed twice with 2 ml of trifluoroacetic acid. Pour this into 750 ml of a 2.5 M aqueous urea solution, adjust to pH 8.25 under ice cooling,
The mixture was stirred overnight while gently blowing air. After confirming that the Ellman test was negative, all solutions were
-ODS AM120 S-50 resin column (2.6 x 7c
m) and eluted with a linear concentration gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). The main fractions were combined, concentrated, applied to a column of Sephadex LH-20 (2 × 90 cm) and eluted with 50% acetic acid. Lyophilize the main fraction, 35mg white powder
I got Dissolve this in 10 ml of 20% acetonitrile and add DEAE
The mixture was applied to a Cellulofine column (2.6 × 6 cm) and eluted with a linear concentration gradient of the same solvent and 0.5 M ammonium acetate containing 20% acetonitrile.
The mixture was applied to a DS AM120 S-50 resin column (2.6 × 7 cm), eluted with 29% acetonitrile (containing 0.1% trifluoroacetic acid), and the main fraction was lyophilized to give 7 mg of a white powder.

Amino acid analysis values Asp 3.84 (4), Thr 1.76 (2), Ser 2.87 (3), Glu 3.04
(3), Pro 4.22 (4), Gly 2.11 (2), Cys 1.20 (2), Val 1.8
3 (2), Ile 1.77 (3), Leu 3.00 (3), Tyr 1.90 (2), Phe
0.95 (1), Lys 0.93 (1), His 0.88 (1) Mass spectrometry (M + H) + 4181.731 HPLC elution time 26.5 min Column conditions Column: WAKOSIL 5C18-200 (4.6 × 250) Eluent: Solution A ( 0.1% -trifluoroacetic acid aqueous solution Using solution B (0.1% -trifluoroacetic acid-containing acetonitrile), linear concentration gradient elution from solution A to solution B (50 minutes) Flow rate: 1.0 ml / min II. Preparation of anti-bigET-2 (22-37) monoclonal antibody (II-1) Preparation of immunogen The bigET-2 (22-37) obtained in the above I and bovine thyroglobulin (TG) are crosslinked with maleimide as described below. It was condensed by the method to obtain an immunogen. That is, polypeptide 1.9
μmole is dissolved in 400 μl of 0.1 M phosphate buffer, pH 6.8, and a DMF solution containing 28.2 μmole of N- (γ-maleimidobutyryloxy) succinimide (hereinafter referred to as GMBS) 100
, and reacted at room temperature for 60 minutes. On the other hand, TG 20
mg (40 nmole) was dissolved in 1.4 ml of 0.02 M phosphate buffer containing 0.15 M salt, pH 6.8, and 2.5 mg of N-succinimidyl-3- (2-pyridyldithio) propionate (hereinafter abbreviated as SPDP) (8.0 mg). μmole)
After mixing 100 μl of the F solution, the mixture was reacted at room temperature for 40 minutes.
After the reaction, dithiothreitol 24.6 mg (160 μmole)
And 0.5 ml of 0.1 M acetate buffer solution containing pH 4.5 and reacted at room temperature for 20 minutes, fractionated on a Sephadex G-25 column, and added with 10 mg (20 nm) of TG into which SH groups were introduced.
ole). Next, maleimide group-introduced polypeptide 95
0 nmole and SH group-introduced TG 12 nmole
After reacting at 4 ° C. for 2 days, it was dialyzed against physiological saline at 4 ° C. for 2 days.

(II-2) Immunization BALB / C female mice 6 to 8 weeks old were immunized subcutaneously with 100 μg / animal of the immunogen described in II-1 together with an adjuvant. Thereafter, booster immunization was performed two to three times every three weeks. (II-3) Preparation of horseradish peroxidase (HRP) labeled bigET-2 (22-37) bigET-2 (22-37) 650 nmole was added to 400 μl of 0.1 M
Dissolved in phosphate buffer, pH 6.8, GMBS 2.7mg
(9.8 μmole) was mixed with 100 μl of a DMF solution and reacted at room temperature for 60 minutes. After the reaction, Sephadex G-25
Fractionation was performed using a column to obtain a polypeptide 390 nmole into which the maleimide group was introduced. On the other hand, HRP 8mg (200nm
le) in a 0.02 M phosphate buffer containing 0.15 M salt, pH 6.
8. Dissolve in 1.14 ml and SPDP 1.56 mg (5.0 μmole)
Was mixed with 60 μl of a DMF solution containing, and reacted at room temperature for 40 minutes. After the reaction, dithiothreitol 10.5 mg (68 μmol
After adding 0.4 ml of 0.1 M acetate buffer containing e), pH 4.5, and reacting at room temperature for 20 minutes, fractionation was carried out on a Sephadex G-25 column, and 4.8 mg of the enzyme into which the SH group was introduced.
(120 nmole) was obtained. Next, a maleimide group-introduced big
390 nmole of ET-2 (22-37) and 120 nmole of SH group-introduced peroxidase were mixed and reacted at 4 ° C. for 16 hours. After the reaction, fractionation was carried out using an Ultrogel AcA44 (manufactured by LKB-Pharmacia) column, and peroxidase-labeled b
igET-2 was obtained.

(II-4) Cell fusion The mice immunized with bigET-2 (22-37) were
The final immunization was performed by inoculating intravenously a solution prepared by dissolving 50 μg of the immunogen in 0.25 ml of physiological saline. The spleen was removed from the mouse three days after the final immunization, pressed with a stainless steel mesh, filtered, and suspended in Eagle's minimum E. sensual medium (MEM) to obtain a spleen cell suspension. BALB /
C mouse-derived myeloma cells P3-x63. Ag8. U1
(P3U1) [Current Topics in Microbiology and Immunology, 81 , 1 (19
78)]. Cell fusion was performed using the original method [Nature, 256 , 495 (197
5)]. That is, spleen cells and P3U1
Was washed three times with MEM containing no serum, and the spleen cells and the P3U1 number were mixed at a ratio of 5: 1.
The cells were precipitated by centrifugation at 800 rpm for 15 minutes. After sufficiently removing the supernatant, the precipitate is loosened gently, and 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite).
Was added thereto, and the mixture was allowed to stand in a 37 ° C. hot water bath for 7 minutes to perform fusion. After fusion, MEM was added to the cells at a rate of 2 ml / min, and after adding a total of 12 ml of MEM, the supernatant was removed by centrifugation at 600 rpm for 15 minutes. This cell sediment was subjected to GIT medium (Wako Pure Chemical) containing 10% fetal calf serum (GIT-1).
(0FCS), P3U1 was suspended at 2 × 10 ⁶ cells / ml, and seeded into 120 wells of 1 well per 24 well multi-dishes (manufactured by Limbro). After seeding, remove the cells
The cells were cultured at 37 ° C. in a 5% carbon dioxide gas furan apparatus. 24 hours later H
AT (hypoxanthine 1 × 10 ~ ⁴ M, amino Buriteri down 4
^{× 10 ~ 7 M, GIT-} 10 containing thymidine 1.6 × 10 ~ ³ M)
HAT selective culture was started by adding 1 ml of FCS medium (HAT medium) per well. HAT
The selective culture was continued by discarding 1 ml of the old solution 3, 6 and 9 days after the start of the culture, and then adding 1 ml of HAT medium. Hybridoma growth was observed 9 to 14 days after cell fusion, and when the culture broth turned yellow (about 1 × 10 ⁶ cells / m 2).
l) The supernatant was collected, and the antibody titer was measured by the EIA method described later.

(II-5) Hybridoma Screening The antibody titer in the hybridoma culture supernatant was measured by the following method. First, an anti-mouse immunoglobulin antibody-bound microplate was prepared. Contains 20 µg / ml anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel)
100 μl of a 0.1 M carbonate buffer, pH 9.6 solution was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. Next, the plate was washed with PBS, and then 300 μl of PBS containing 25% Block Ace (Snow Brand Milk Products) was dispensed to cover excess binding sites in the wells and treated at least at 4 ° C. for 24 hours. Next, buffer E (10% Block Ace, 2 mg / ml BSA,
0.4 M NaCl, 2 mM EDTA and 0.1% NaN ₃
Was added, and 50 μl of the hybridoma culture supernatant were added, and the mixture was reacted at room temperature for 4 hours. After the reaction, after washing with PBS, HRP-labeled bigET-2 (22-37) prepared in II-3 above [1% BS
A 0.02 M phosphate buffer containing 0.4 M NaCl and 2 mM EDTA, diluted 100-fold with pH 7 (buffer C)] 100 μl
Was added and reacted at 4 ° C. for 16 hours. After the reaction, the plate is washed with PBS, and the enzyme activity on the solid phase is measured using a TMB microwell peroxidase substrate system (KIRKEGAARD & PERRY LA).
B, INC. Funakoshi Pharmaceutical Co., Ltd.) (100 μl) and reacted at room temperature for 20 minutes. After stopping the reaction by adding 100 μl of 1 M phosphoric acid, the absorbance at 450 nm was measured with a plate reader (MTP-32, manufactured by Corona). In this way, the supernatants of all 240 wells in which hybridoma growth was observed were examined. Strong antibody titers were detected in wells 70, 96, 101 and 196.

(II-6) Cloning No. 1 showing a positive antibody activity. Each hybridoma in wells 70, 96, 101 and 196 was subjected to cloning by limiting dilution. That is, the hybridoma was suspended in RPMI1640-20FCS at 1.5 cells / ml, and 0.2 ml per well was dispensed into a 96-well microplate (manufactured by Nunc). When dispensing, use BALB /
C mouse thymocytes were added at 5 × 10 ⁵ cells per well. After about one week, cell proliferation began to be observed, and the antibody titer in the supernatant was examined by the EIA method described in II-5 above. As a result, 12 out of 30 clones of No. 70 hybridoma were No. 70 hybridomas. 7 in 96 hybridomas
Six out of the 0 clones produced antibodies, and 25 out of 30 clones produced antibody in the hybridoma of No. 101. Of these clones, bET-20 (derived from No. 70 clone) and bET-22 (derived from No. 96 clone)
And bET-24 (derived from the clone of No. 101) were selected. Further, from the hybridoma of No. 196, after cloning three times, a bET-25 clone was selected.

(II-7) Class of monoclonal antibody
Subclass determination Next, using the culture supernatant, the monoclonal antibodies, bET-20a, bET-22 produced by these clones
The classes and subclasses of a, bET-24a and bET-25a were examined. First, the bigE
T-2 (22-37) was immobilized by physical adsorption. That is, bi
gET-2 (22-37) was dissolved at a concentration of 5 μg / ml in 0.1 M carbonate buffer, pH 9.6, and 100 μl was dispensed into each well. After leaving at 4 ° C. for 1 day, the well was washed with PBS, and the excess binding site of the well was covered with Block Ace according to the method described in II-5 above. Using the bigET-2 (22-37) binding microplate thus prepared, an isotype typing kit (Mouse-Typer
The class and subclass were examined by enzyme-linked immunosorbent assay (ELISA) using (registered trademark) Sub-Isotyping Kit Bio-Rad Inc.).
As a result, bET-20a, bET-22a, bET-2
4a and bET-25a were all found to belong to the IgG1, kappa class.

(II-8) Preparation of Large Amount of Monoclonal Antibody Hybridoma bET-20, bET-22 or bET-24 1 was administered to mice to which 0.5 ml of mineral oil was intraperitoneally administered or untreated mice (BALB / C).
After intraperitoneal injection of ３3 × 10 ⁶ cells / animal, antibody-containing ascites was collected 10 to 30 days later. Next, a Protein A column (IPA-300, ReA) was obtained from the ascites of bET-20 according to a conventional method.
pligen) to purify the monoclonal antibody.
As a result, 5.8 mg of the antibody was purified from 1 ml of ascites.

Embodiment 2 FIG. Competition method-EIA The anti-mouse immunoglobulin antibody-bound microplate described in II-5 above was added to the anti-bigE diluted with buffer C.
After adding 50 μl of the culture supernatant containing the T-2 monoclonal antibody and the C-terminal peptide of each bigET (Japanese Patent Application Laid-Open No. 2-238894, Japanese Patent Application No. 2-1944081), the mixture was reacted at room temperature for 1 hour, and then described in II-3 above. HRP-labeled bigET-2 (22-3
7) (100-fold dilution with buffer C) was added, and the mixture was reacted at 4 ° C for 16 hours. The dilution factor of the culture supernatant containing the anti-bigET-2 monoclonal antibody was bET-20 (× 75),
bET-22 (x20), bET-24 (x15) and b
ET-25 (x5) was used. The results are shown in FIG. In the figure,-●-is bigET-2 (22-37),-○-is bi
gET-1 (22-38),-△-is bigET-3 (22-4
2) and-□-indicate bigET-3 (22-41) NH ₂ . From FIG. 1, about 3
It can be seen that ~ 12 ng / ml (about 0.15 ng-0.6 ng / well) of bigET-2 (22-37) can be measured.
In addition, these antibodies are either bigET-1 or bigE.
It was revealed that there was almost no cross-reactivity with the C-terminal peptide of T-3.

Embodiment 3 FIG. Sandwich method-EIA (1) Preparation of F (ab ') ₂ fraction of bET-20a The bET-20a described in Example II-7 above was concentrated to 7.5 mg / ml with a collodion bag (manufactured by MS Instruments). Then, 0.1 M acetate buffer containing 0.1 M NaCl (pH
Dialysis was performed against 4.5). To 1 ml of the antibody solution, 0.19 mg of pepsin (Sigma, twice crystal) was added, reacted at 37 ° C. for 20 hours, and then FPLC using a Superose 12 column equilibrated with 0.1 M phosphate buffer, pH 6.8. (Pharmacia) to purify the F (ab ') ₂ fraction. (2) Preparation of HRP-labeled bET-20a F (ab ') ₂ bET-20a F (ab') ₂ described in (1) of Example 3 above
Fraction 1.6mg (16n mole) / ml, 1ml GMBS, 225n
50 μl of DMF containing mole was added and reacted at room temperature for 60 minutes. The reaction solution was applied to a Sephadex G-25 column (1 × 30 cm,
Separation with an eluent, 0.1 M phosphate buffer, pH 6.8), and the obtained F (ab ') ₂ fraction into which a maleimide group was introduced, prepared by the method described in II-3 of Example 1 above Then, 5.5 mg of the introduced HRP-introduced HRP was mixed, concentrated to about 0.3 ml in a collodion bag, and then left at 4 ° C. for 16 hours. The reaction solution was applied to an Ultrogel AcA44 column (10 mmφ × 40 mm) using 0.1 M phosphate buffer, pH 6.5 as an eluent to purify the F (ab ′) ₂ -HRP complex fraction.

(3) Sandwich method-EIA As a solid-phase antibody for sandwich-EIA, a monoclonal antibody recognizing the N-terminal part of ET-1.
AwETN40 [Journal of Immunol. Meth, showing 160% cross-reactivity]
ods), 118 , 245 (1989), Biochemical and Biophysical Research Communication (Bioche
m. Biophys. Res. Commun., 164 , 74 (1989)]. 0.1M containing 20 µg / ml of purified AwETN40
A carbonate buffer solution (pH 9.6) was dispensed at 100 μl into a 96-well microplate and left at 4 ° C. for 24 hours. Excess binding sites in the wells were inactivated by adding 300 μl of Block Ace (manufactured by Snow Brand Milk Products Co., Ltd., sold by Dainippon Pharmaceutical Co.) diluted 4-fold with PBS. The bigET-2 (1-37) and bigET diluted with buffer E were added to the plate prepared as described above.
-1 (1-38) (human), bigET-3 (1-41) NH
₂ (purchased from Peptide Research Laboratories) and 100 μl of ET-2 (purchased from Peptide Research Laboratories) standard solution were added, and reacted at 4 ° C. for 24 hours. After washing with PBS, the bigET-2 (1-37) F (ab ') prepared in (2) of Example 3 above was used.
Add 100 μl of _2- HRP (diluted 150-fold with buffer C),
The reaction was performed at 4 ° C. for 24 hours. After washing with PBS, the enzyme activity on the solid phase was measured by the method described in II-5 of Example 1 above. The results are shown in FIG. In the figure,-●-is bigE
T-2 (1-37),-○-is bigET-1 (1-38) (human),-△-is bigET-3 (1-41) NH ₂ ,
▲-indicates the standard curve of ET-2. By using this measurement method, 1 pg / ml (0.1 pg / well) b
iget-2 and (1-37) was found to be capable of detecting at bigET-1 (1-38) or bigET-3 (1-41) NH ₂ 0.1% or less cross-reactivity. That is, the big of the present invention
The bigET-2 (1) was obtained by an immunological assay using the sandwich method using a monoclonal antibody against ET-2 (22-37) (human) and a monoclonal antibody recognizing the N-terminal of bigET-2 (1-37). -37) to big
ET-1 (1-38), bigET-3 (1-41) NH ₂ , E
The measurement can be performed with extremely high sensitivity without cross-reacting with T-2.

Embodiment 4 FIG. Detection and Identification of Big ET-2 Immunoreactivity in Culture Supernatant of Human Kidney Cancer Cell ACHN It is known that human kidney cancer cell ACHN produces ET-2 (FEBS Lett.), 274, 13
6-140, 1990). Therefore, using the sandwich-EIA prepared in Example 3, the bigET-
2. Detection and identification of immune activity were performed. 1 ACHN cell
The cells were cultured in Eagle Minimum Essential Medium containing 0% fetal bovine serum and a non-essential amino acid solution (available from Dainippon Pharmaceutical) to obtain 4 liters of culture supernatant 2 days after confluence. The supernatant was washed with 1.5 ml gel volume of AwETN4.
0 bound solid phase [3 mg of AwETN40 is added to 1 g of Tres
yl Toyopearl (manufactured by TOSOH) according to a conventional method], washed with 10 ml of PBS, washed with 3 ml of distilled water, and washed with 1 ml of 0.1%
Elution was performed with 60% CH ₃ CN containing TFA. The eluate is Sp
It was concentrated with an eed Vac (manufactured by Sarvant) and analyzed by reverse phase-HPLC. The conditions of reverse phase-HPLC are as follows. The column is TSK, ODS-80TM (4.
6 × 250 mm, manufactured by TOSOH), and elution conditions were as follows: Solution A:
5% CH ₃ CN, 0.5% TFA; solution B: 60% CH ₃ C
N, 0.5% TFA, the proportion of solution B from 0 to 40% in the first 5 minutes, from 40 to 65% in the next 20 minutes,
In the next 5 minutes, increase from 65 to 100%, 1m
Elution was performed at a flow rate of 1 / min. A portion was taken from each fraction (0.5 ml) and measured by the sandwich-EIA described in Example 3. BigET-3 (1-41) N as a standard sample
H ₂ , ET-3, bigET-1, ET-1, bigE
T-2 (1-37) and ET-2 (all manufactured by Peptide Laboratories), and bigET-2 (1-38) (Japanese Patent Application No.
143127), and the elution position was examined. The elution results are shown in FIG. The culture supernatant of ACHN contains bigE
T-2 immunoreactivity was detected, and the elution position revealed that the molecular species was bigET-2 (1-38).

Embodiment 5 FIG. Detection and identification of bigET-2 immunoreactivity in human plasma 30 ml of healthy human plasma was added to an equal volume of PBS containing the following protease inhibitor (0.2 mM PMSF, 2 μM E64,
(2 μg / ml peptitatin, 2 μg / ml leupeptin, 2 mM EDTA, 2000 KIU / ml aprotinin and 0.6 mM phosphoramidon). Next, the bigET-2 immunoreactivity was concentrated on an AwETN40-bound solid phase by the method described in Example 4, and reversed phase-HPL.
Analyzed in C. FIG. 4 shows the results. B in healthy human plasma
IgET-2 immunoreactivity was detected, revealing that the main molecular species was bigET-2 (1-38).

[0038]

[Effect] By using a monoclonal antibody having a binding property to human big endothelin-2 or its C-terminal peptide of the present invention, it does not cross-react with big endothelin-1, big endothelin-3 or ET-2.
Human big endothelin-2 can be measured with extremely high sensitivity. This assay is capable of detecting bigET-2 in cultured cells and human plasma, so that bigET-
It is very useful for elucidating the physiological and pathophysiological roles of 2.

[0039]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Cys Ser Cys Ser Ser Leu Met Asp Lys
Glu Cys Val Tyr Phe Cys His 1510 15 Leu Asp Ile Ile Trp 20.

SEQ ID NO: 2 Sequence length: 21 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20.

SEQ ID NO: 3 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20.

SEQ ID NO: 4 Sequence length: 38 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro Glu His Val Val Pro Tyr Gly 20 25 30 Leu Gly Ser Pro Arg Ser 35.

SEQ ID NO: 5 Sequence length: 37 or 38 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics: Location: 37 Other information: Xaa = Pro or Pro Arg sequence Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro Glu Gln Thr Ala Pro Tyr Gly 20 25 30 Leu Gly Asn Pro Xaa 35.

SEQ ID NO: 6 Sequence length: 41 or 42 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics: Location: 42 Other information: Xaa = Gly-OH or NH ₂ Sequence Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His 1 5 10 15 Leu Asp Ile Ile Trp Ile Asn Thr Pro Glu Gln Thr Val Pro Tyr Gly 20 25 30 Leu Ser Asn Tyr Arg Gly Ser Phe Arg Xaa 35 40.

SEQ ID NO: 7 Sequence length: 16 or 17 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: C-terminal fragment Sequence characteristics: Location: 16 Other information: Xaa = Pro or Pro Arg sequence Val Asn Thr Pro Glu Gln Thr Ala Pro Tyr Gly Leu Gly Asn Pro Xaa 1 5 10 15

SEQ ID NO: 8 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: N-terminal fragment

[Brief description of the drawings]

FIG. 1. Competition method using four kinds of monoclonal anti-bigET-2 (22-37) antibodies-bigET by EIA-
2 (22-37) (-●-), bigET-1 (22-38) (-
−-), bigET-3 (22-42) (-△-), and bigET-3 (22-41) NH ₂ (-□-) are shown.

FIG. 2: BigET-2 (1-37) (− ●) by sandwich method-EIA using an antibody against the N-terminal of bigET-2 and an antibody against the C-terminal obtained by the present invention.
-), BigET-1 (1-38) (-○-), bigE
T-3 (22-41) NH ₂ (-△-) and ET-2 (-
The measurement results of (-) are shown.

FIG. 3. bigET- produced by kidney cancer cell ACHN.
2 shows the results of analysis of immunological activity by reversed-phase HPLC and detection by sandwich-EIA obtained according to the present invention, which revealed that bigET-2 (1-38) was the main molecular species.

FIG. 4 shows the results of analysis of bigET-2 immunoreactivity in healthy human plasma by reversed-phase HPLC and detection by sandwich-EIA obtained by the present invention, and FIG.
ET-2 (1-38) was found to be the main molecular species.

────────────────────────────────────────────────── (5) References European Patent Application Publication 366016 (EP, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/08 C12N 5/16 G01N 33 / 53 G01N 33/577 C12N 15/06

Claims (13)

(57) [Claims] 1. An amino acid sequence having the binding property to human big endothelin-2 or its C-terminal peptide, and having the amino acid sequence of Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp. A monoclonal antibody that does not react with endothelin-2. 2. The monoclonal antibody according to claim 1, which does not bind to human big endothelin-1 and human big endothelin-3. 3. The method according to claim 1, wherein said human big endothelin-2 is of the formula Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu AspIleTropGalThrGrGnProG. Wherein Xaa = Pro or Xaa = ProArg).
The monoclonal antibody as described. 4. A human big endothelin-2 C-terminal peptide having the formula Val Asn Thr Pro Glu Gln Thr Ala Pro Tyr Gly Leu Gly Asn Pro Xaa (where Xaa = Pro or Xaa = Pro Arg). The monoclonal antibody according to claim 1, which has: 5. The monoclonal antibody according to claim 4, which is bET-20a. 6. A hybridoma which produces the monoclonal antibody according to claim 1. 7. The hybridoma according to claim 6, which is bET-20. 8. The method for producing an antibody according to claim 1, wherein the hybridoma according to claim 6 or 7 is cultured. 9. A monoclonal antibody capable of binding to human big endothelin-2 according to claim 1, 2, 3, 4 or 5, a test solution and labeled human big endothelin-2 or labeled human big endothelin-2. 2) reacting with a 2 C-terminal peptide competitively and measuring the ratio of labeled human big endothelin-2 or labeled human big endothelin-2 C-terminal peptide bound to the antibody; Human Big Endothelin-
Method for quantifying 2 or its C-terminal peptide. 10. The method according to claim 1, wherein the insolubilized on the carrier.
Contacting the test solution with the monoclonal antibody according to 4 or 5, followed by contacting an antibody having a binding property with labeled human big endothelin-2, and measuring the activity of the labeling agent on the insolubilized carrier. A method for quantifying human big endothelin-2 in a test solution. 11. The monoclonal antibody according to claim 1, wherein the test solution is contacted with an antibody having a binding property to human big endothelin-2 insolubilized on a carrier and then labeled. And measuring the activity of the labeling agent on the insolubilized carrier. 12. An antibody insoluble on a carrier or a labeled antibody is an antibody having a binding property to human big endothelin-2 C-terminal peptide, and the other is having an binding property to human big endothelin-2. The method for quantifying human big endothelin-2 according to claim 10 or 11, wherein the antibody is an antibody that does not react with the human big endothelin-2 C-terminal peptide. 13. A monoclonal antibody having a binding property to human big endothelin-2 insoluble on a carrier but not reacting with the human big endothelin-2 C-terminal peptide, and a labeled bET-20a. 1
2. The method for quantifying human big endothelin-2 according to 1.