JP2001128675A - Monoclonal antibody against endothelin-3 precursor, and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a basic physiological information such as the physiological function of endothelin-3, expression site and a plasma level of the endothelin-3. SOLUTION: The monoclonal antibody having affinity to an precursor of the endothelin-3, a hybridoma cell capable of producing the monoclonal antibody, and a method for the immunoassay of the endothelin-3 precursor, e.g. pig endothelin-3, by a competition method or a sandwich method by using the antibody, are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel and useful antibody having a binding specificity for a precursor of endothelin-3, for example, big endothelin-3. More specifically, the present invention relates to an antibody useful for development of a method for measuring a precursor of endothelin-3, for example, big endothelin-3, based on an antigen-antibody reaction, or for diagnosis and prognosis of a disease involving endothelin-3.

[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 contraction activity and a blood pressure increasing activity. In addition, endothelin-1
From the analysis of the cDNA of the gene, big endothelin-1 consisting of about 40 amino acids was used as an intermediate in the biosynthesis process.
The presence of (endothelin-1 precursor) is also envisioned.
To date, monoclonal antibodies against endothelin-1 and big endothelin-1 have been produced, and a highly sensitive enzyme immunoassay has been developed.
It has become possible to develop a wide range of studies to elucidate the relationship between physiological roles and disease states. Already, by using a monoclonal antibody having a neutralizing activity as a specific antagonist, it was revealed that endothelin-1 had a deep relationship with ischemic disease. Detailed research is being conducted on the relationship with the disease state. On the other hand, chromosome D
From the analysis of NA, new sequences of endothelin-2 and endothelin-3 were found, and it was revealed that endothelin forms a gene family. In particular, endothelin-3 is endothelin-1, endothelin-
As shown in the following example, 6 residues out of 21 amino acid residues (see underline) are different from those of Example 2, and their smooth muscle contraction and blood pressure increasing activity are considerably higher than those of endothelin-1 and endothelin-2. Reported to be weak. Endothelin-1 (pig, human; sometimes referred to as endothelin-α). Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp endothelin-3 (rats, was sometimes referred to as human .γ.) Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp In addition, the analysis of the cDNA of endothelin-3 revealed the precursor structure of endothelin-3 (Japanese Patent Application No. Hei 1-2).
53797), suggesting that endothelin-3 is synthesized using big endothelin-3 having the following sequence as a direct precursor. Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp Ile Asn Thr Pro Glu Gln Thr Val Pro Tyr Gly Leu Ser Asn Tyr Arg Gly Ser Phe Arg X (X = Gly-OH or NH ₂ = Gly-Lys-OH = Gly-Lys-Arg-OH Thus, the results of the study on the structure and pharmacological action show that endothelin-3 forms a receptor system different from endothelin-1 and -2. Strongly suggests that
There is a deep interest in its physiological role.

[0003]

As described above, despite the growing interest in the physiological role of endothelin-3, basic physiological functions such as the expression site of endothelin-3, plasma levels, etc. Information is rarely available. The main cause of this is that no monoclonal antibody specifically recognizing endothelin-3 or endothelin-3 precursor has been produced so far, and endothelin-3 or endothelin-3 precursor is measured specifically and with high sensitivity. Immunological assays have not been developed. These immunological techniques are considered to be one of the most effective means for comprehensively conducting research on endothelin-3, particularly on metabolic pathways, secretory mechanisms, receptor systems, and the association with disease states. The establishment of has been eagerly awaited by various circles.

[0004]

SUMMARY OF THE INVENTION The present inventors have developed an endothelin-3 precursor, such as big endothelin-3.
A monoclonal antibody having binding specificity was prepared, and an immunoassay method capable of detecting big endothelin-3 with high sensitivity and specificity using the antibody was developed. Furthermore, they have discovered that the pharmacological action specific to the antibody, that is, the antibody suppresses contraction of smooth muscle of various animals by endothelin-3. These findings indicate that the antibody can be used as a specific antagonist of endothelin-3, and that the antibody may be a prophylactic or therapeutic agent for various diseases causally or symptomatically related to endothelin-3. It shows that you get. That is, the present invention relates to endothelin-
The present invention relates to a monoclonal antibody capable of binding to the three precursors, a hybridoma cell producing the monoclonal antibody, and an immunoassay for the endothelin-3 precursor, for example, big endothelin-3, which is a competitive or sandwich method using the antibody. .

In the endothelin-3 sandwich method, the antibodies used in the primary reaction and the secondary reaction may be polyclonal antibodies or monoclonal antibodies, respectively, but preferably one of them is endothelin-3.
Is an anti-endothelin-3 monoclonal antibody that does not react with C-terminal of endothelin-3,
An anti-endothelin-3 polyclonal or monoclonal antibody that reacts with the C-terminal of endothelin-3 is used. This latter anti-endothelin-3 polyclonal or monoclonal antibody that reacts with the C-terminal of endothelin-3 is, as can be seen from the above amino acid sequence comparison with endothelin-1 and -2, the C-terminal of endothelin-1 or -2. Including the same antibodies that react with the
This is described in Japanese Patent Application No. 46560/94. Also, in the sandwich method of big endothelin-3, preferably, one of the antibodies used in the primary reaction and the secondary reaction is a monoclonal antibody that reacts with big endothelin-3 but does not react with endothelin-3. A monoclonal antibody that reacts with endothelin-3 is used on the other. Furthermore, a monoclonal antibody that reacts with big endothelin-3 but does not react with endothelin-3 is particularly preferable.
An antibody having a wide specificity with respect to the structure of the C-terminal portion of big endothelin-3, for example, an antibody that reacts to the same extent even when the C-terminal has an Arg-Gly sequence or an Arg-NH2 sequence is used. The preparation of the polyclonal antibody of the present invention generally comprises preparing a complex of the immunizing antigen endothelin-3 or big endothelin-3 with a carrier protein, inoculating the complex with an animal, immunizing the animal, and immunizing the immunized animal with the desired antibody. By collecting the contents and separating and purifying the antibody.

In preparing the monoclonal antibody of the present invention, individuals with high antibody titers are selected from the above immunized animals, and spleen or lymph nodes are collected 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are isolated from bone marrow. By selecting a hybridoma that stably produces a high-titer antibody by fusing with a tumor cell, and obtaining a monoclonal hybridoma. As the immunizing antigen, any of a natural purified product, a synthetic product, and the like can be used, and endothelin-3, big endothelin-3, and a part of big endothelin-3 described above are used. As an immunizing antigen, endothelin-
In some cases, a compound containing the structure of No. 3 or a compound containing a partial structure of endothelin-3 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 synthesizing endothelin-3 by the solid phase method, the solid phase peptide synthesis method of Maryfield [Journal of the American Chemical
It is preferable to use Society (J. Am. Chem. Soc.), 85, 2149 (1963)]. Insoluble resins may be any of those known in the art, such as chloromethylated styrene-divinylbenzene copolymers, phenacylacetic methylated styrene-divinylbenzene copolymers, and the like. Polyamide type resins such as polystyrene type resin and polydimethylacrylamide resin are exemplified. After binding the C-terminal N-protected amino acid to the insoluble resin, the protected amino acids are sequentially coupled from the C-terminal side of endothelin-3 according to a conventional method, and then treated with hydrogen fluoride to form a disulfide bond. Endothelin-3 can be synthesized. As N-protected amino acids, all α-amino groups are protected with Boc groups, the hydroxyl groups of serine and threonine are Bzl groups, and the ω-carboxylic acids of glutamic acid and aspartic acid are OBz
1 group, ε-amino group of lysine is Cl-Z group, 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, tryptophan The indole group is preferably protected with a CHO group. As a means of synthesis by the liquid phase method, for example, “The Peptides (T
he Peptides), Volume 1 (1966), Schroder and Lu
bke, Academic Press, New York, USA or "Peptide Synthesis", Izumiya et al., Maruzen Co., Ltd. (1975)
Methods, such as the azide method, the chloride method,
Acid anhydride method, mixed anhydride method, DCC method, active ester method, method using Woodward reagent K, carbodiimidazole method, oxidation-reduction method, DCC / additive (eg, H
ONB, HOBt, HOSu) method and the like.

[0007] Regarding a protein complex of an immunizing antigen and a 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 antibody against the hapten immunized by coupling to the carrier. If it can be efficiently performed, any one may be coupled at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, and the like may be coupled in a weight ratio of 0.1 to 20, preferably 1 to 5, with respect to one hapten. A method of coupling at a ratio of is used. In addition, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldebit, carbodiimide, maleimide active ester and the like are conveniently used. The condensation product is administered by itself or together with a carrier and a diluent to a site capable of producing an antibody upon administration to a warm-blooded animal, with subcutaneous injection being preferred. 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. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs,
Examples include mice, rats, sheep, goats and chickens. The antibody is collected from the blood, ascites (preferably blood), etc. of the warm-blooded animal immunized by the above method. For example, when the immunogen is endothelin-3 or a partial peptide thereof, the measurement of the anti-endothelin-3 antibody titer in the antiserum is performed, for example, by reacting the labeled endothelin-3 described below with the antiserum and then reacting the antibody with the antibody. This is done by measuring the activity of the bound labeling agent. Antibodies can be separated and purified by immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE)).
, An ultracentrifugation method, a gel filtration method, a specific purification method of collecting only a specific antibody with an antigen-antibody conjugate or an active adsorbent and dissociating the bond to obtain an antibody]. The antibody thus produced contains IgG as a main component and also other immunoglobulins such as IgM and IgA.

On the other hand, in the same manner as in the above-mentioned method for preparing a polyclonal antibody, an individual having an antibody titer is selected from a immunized warm-blooded animal, for example, a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained therein with myeloma cells, hybridomas producing anti-endothelin-3 antibody and anti-endothelin-3 precursor antibody can be prepared. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256 , 495 (1975)]. Polyethylene glycol (P
EG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc.
Particularly, P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells used is about 1: 1 to 20: 1, and PEG (preferably PEG)
1000-PEG6000) at a concentration of about 10-80%,
By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used for screening an anti-endothelin-3 antibody-producing hybridoma or an anti-big endothelin-3 antibody-producing hybridoma. For example, endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 was adsorbed. Solid phase
(E.g., microplate), hybridoma culture supernatant is added, and then horseradish peroxidase (HRP) -labeled anti-immunoglobulin antibody (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used ) Or protein A and detection of anti-endothelin-3 monoclonal antibody bound to the solid phase
An SA (Enzyme-linked immunosorbent assay) method, a hybridoma culture supernatant is added to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and endothelin-3, big endothelin-3, or big endothelin-3 labeled with HRP is added. EIA (Enzyme imine) for detecting the monoclonal antibody bound to the solid phase
munoassay) method. The selection and breeding of hybridomas are usually performed by adding HAT (hypoxanthine, aminopterin, thymidine) to a medium for animal cells containing 10 to 20% fetal bovine serum (eg, RPMI1640). The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

[0009] Separation and purification of the anti-endothelin-3 monoclonal antibody or anti-big endothelin-3 antibody is carried out according to the immunoglobulin separation and purification method as in the above-mentioned separation and purification of the polyclonal antibody. An anti-endothelin-3 polyclonal antibody that reacts with a partial region of endothelin-3 can be prepared by the above method by immunizing a peptide (partial peptide) corresponding to the partial region as a hapten. The anti-endothelin-3 polyclonal antibody prepared as a hapten can also be prepared by affinity chromatography using a column to which a peptide corresponding to a partial region is bound. In addition, an anti-big endothelin-3 polyclonal antibody that reacts with a partial region of big endothelin-3 can be prepared by immunizing a peptide corresponding to the partial region as a hapten by the above method. Can also be prepared from an anti-big endothelin-3 polyclonal antibody prepared by using as a hapten, using affinity chromatography on a column to which a peptide corresponding to a partial region thereof is bound. In addition, a hybridoma that produces a monoclonal antibody that reacts with a partial region of endothelin-3 or big endothelin-3 and a monoclonal antibody that reacts with endothelin-3 or big endothelin-3 but does not react with a partial region thereof are produced. The selection of hybridomas can be performed, for example, by measuring the binding between a peptide corresponding to a partial region thereof and an antibody produced by the hybridoma. Especially endothelin-
3 C-terminal peptide Cys His Leu Asp Ile Ile
For screening of a monoclonal antibody recognizing other than Trp, it is preferable to use a sandwich type EIA using a labeled antibody of the peptide. That is,
The hybridoma culture supernatant was added to the solid phase to which the anti-immunoglobulin antibody or protein A had been adsorbed, endothelin-3 was added, and anti-endothelin-labeled with HRP was further added.
This is an EIA method for measuring enzyme activity on a solid phase after reacting a 3 C-terminal peptide antibody.

[0010] Using the anti-endothelin-3 and anti-big endothelin-3 monoclonal antibodies obtained above, endothelin-3 can be measured or tissue stained. Endothelin-3 and Big Endothelin-3
In general, the competition method described below is used for the measurement method, but the sandwich method described later is preferably used. In the competition method, the anti-endothelin-3 obtained by the present invention is used.
Alternatively, after the anti-big endothelin-3 antibody and the test solution and the labeled endothelin-3 or the labeled big endothelin-3 or its partial peptide are reacted competitively, the ratio of the labeling agent bound to the antibody is measured. Thus, endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 in the test solution is quantified.

The labeling agent for the endothelin-3, the big endothelin-3, or the partial peptide of the big endothelin-3 or the labeling agent for the antibody described below includes radioisotopes, enzymes, fluorescent substances, luminescent substances and the like. Examples of radioisotopes include ¹²⁵ I, ¹³¹ I, ³ H,
^As the above enzyme, ¹⁴ C or the like is preferably a stable enzyme having a large specific activity. Examples of the fluorescent substance include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase.
Fluorescamine, fluorescein isothiocyanate, and the like, and luminescent materials include luminol, luminol derivatives, luciferin, lucigenin, and the like. Further, a biotin-avidin system can be used for binding an antibody, endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 to a labeling agent. In the measurement of the activity of the labeling agent, for example, in the case of measurement of endothelin-3, labeled endothelin-3 bound to an antibody and free labeled endothelin-3 are separated (hereinafter, abbreviated as B / F separation). However, when an enzyme is used as a labeling agent, an antibody against an anti-endothelin-3 antibody insolubilized in a reagent for this or an active adsorbent such as insolubilized protein A is advantageously used. For example, using an anti-IgG antibody (corresponding to an antibody against anti-endothelin-3 antibody) as a solid phase,
The labeled endothelin-3 can be bound to an anti-IgG antibody on a solid phase via the above-mentioned antibody reactive with the antibody, and the labeling agent on the solid phase can be measured.
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, an antibody against anti-endothelin-3, sodium sulfate, dextran charcoal powder, polyethylene glycol, etc., which is not insolubilized other than the above reagents for B / F separation. 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, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. As the carrier, agarose, dextran, insoluble polysaccharides such as cellulose,
Examples include synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.

In the competition method, for example, endothelin-
In the case of the measurement of 3, the anti-endothelin-3 antibody, the test solution, the labeled endothelin-3, and the reagent for separating B / F can be reacted in any order. The reaction may be carried out simultaneously, but at least the labeled endothelin-3 may be reacted with the test solution and the anti-endothelin-
3 It is preferable that the compound is added to the reaction system simultaneously with or after the reaction with the 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, insolubilized anti-endothelin-3 or big endothelin-3 is used.
After contacting (reacting) the test solution with the antibody (primary reaction) and further reacting a labeled anti-endothelin-3 or big endothelin-3 antibody (secondary reaction), the activity of the labeling agent on the insolubilized carrier is determined. By measuring, the amount of endothelin-3 or big endothelin-3 in the test solution can be quantified. 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 an anti-endothelin-3 antibody or an anti-big endothelin-3 antibody used in the secondary reaction, the anti-endothelin-3 antibody or the anti-big endothelin-3 antibody used in the primary reaction is an endothelin-3 or a big endothelin-3 antibody. Antibodies having different sites for binding to the antibody are preferably used. For example, endothelin-3
When the antibody used in the primary reaction has the ability to bind to the C-terminus of endothelin-3, the secondary reaction preferably involves an antibody that binds to a moiety other than the C-terminus (eg, N-terminus). When an endothelin-3 antibody is used and the antibody used in the primary reaction has an ability to bind to the N-terminal of endothelin-3, in the secondary reaction, preferably other than the N-terminal (eg,
An anti-endothelin-3 antibody that binds to the (C-terminal) is used. The antibody used for the primary reaction and the secondary reaction may be a polyclonal antibody or a monoclonal antibody, respectively. For example, in the case of measuring endothelin-3, preferably one of them reacts with endothelin-3,
An anti-endothelin-3 monoclonal antibody which does not react with the C-terminus of endothelin-3, and the other anti-endothelin-3 polyclonal or monoclonal antibody which reacts with the C-terminus of endothelin-3 is used. In the immunoassay for endothelin-3 by the sandwich method, particularly preferred is the C-terminal peptide of endothelin-3, that is, Cys-His-Leu-Asp-Ile-Ile-T.
An anti-endothelin-3 polyclonal antibody that reacts with rp and an anti-endothelin-3 monoclonal antibody that reacts with endothelin-3 but does not react with the C-terminal peptide of endothelin-3 are used. In the sandwich immunoassay, the antibody for solid phase and the antibody for labeling may be of any class or subclass, and if antibody activity is retained, Fc 'or Fc region is removed from them. (ab ') ₂ fractions,
The Fab ′ fraction or the Fab 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 kind, and two or more kinds of antibodies are used for the purpose of improving measurement sensitivity and the like. May be used. In addition, the immunoassay using the antibody obtained in the present invention can be used for diagnosis and prognosis management of a disease involving endothelin-3.

As test samples, plasma, serum, urine, cerebrospinal fluid, body fluids such as ascites, pleural effusion and amniotic fluid, sputum, stool and the like can be used. These samples can be used as they are, or can be diluted or extracted with various buffers and then concentrated, and used as immunoassay samples. As a solvent used for diluting or extracting a sample, any buffer or organic solvent may be used, preferably an immunoassay buffer, 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 bound with anti-endothelin-3 antibody, eluted under appropriate elution conditions, and then reduced under reduced pressure or normal pressure. It may be concentrated under a stream of air. As the carrier for concentration, a C2, C8 or C18 cartridge of a carrier for reversed phase chromatography is particularly preferably used. The concentrate is dissolved in an immunoassay buffer and used as a sample for immunoassay. Further, the anti-endothelin-3 and anti-big endothelin-3 antibodies obtained in the present invention can be used for immunohistochemical staining of endothelin-3 and big endothelin-3. The method includes, for example, a direct method using a labeled anti-endothelin-3 or a labeled big endothelin-3 antibody, an indirect method using an anti-endothelin-3 antibody or a big endothelin-3 antibody and a labeled antibody of the antibody. Etc. can be followed.

Further, among the anti-endothelin-3 antibodies obtained by the present invention, an antibody capable of neutralizing the vasoconstrictive ability of endothelin-3 can be used as a specific antagonist of endothelin-3. As a method of screening an antibody that specifically suppresses the action of endothelin-3 from among anti-endothelin-3 antibodies, any method for detecting the pharmacological action of endothelin-3 can be used. In vitro measurement system using various vascular smooth muscle contraction activities as an index, such as pig, rat, rabbit, guinea pig, dog, human, etc. A biological measurement system may be used. Antibodies that specifically suppress the action of endothelin-3 obtained are IgG, Ig
A and IgM may be of any class, or may be Fab 'or Fab fractions from which Fc' or Fc region has been removed, or polymers thereof. A chimeric antibody obtained by fusing a variable gene portion of a monoclonal antibody capable of specifically suppressing the action of endothelin-3 with a human immunoglobulin constant gene portion and expressing it as a recombinant can also be used.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention. Hybridoma cell A producing mouse monoclonal antibody AET-30a used in Examples described later
ET-30 was granted to the Fermentation Research Institute (IFO) under the accession number IF
O 50193 has been deposited on July 7, 1999. In addition, the hybridoma was obtained from the Ministry of International Trade and Industry of the Ministry of Industry and Technology's Institute of Industrial Science and Technology (FRI) under the accession number FERMBP-25.
No. 23 has been deposited on July 20, 1999 based on the Budapest Treaty and stored at the FRI. Mouse monoclonal antibody bET-31 used in Examples described later
The hybridoma cell bET-31 producing a has been deposited with the Fermentation Research Institute (IFO) under the accession number IFO 50247 from May 24, 1990. The hybridoma was manufactured by the Research Institute of Microorganisms, Ministry of International Trade and Industry
(FRI), deposited under the Budapest Treaty on June 12, 1990 under the accession number FERM BP-2949.
Archived at RI. The hybridoma cell bET-23 that produces the mouse monoclonal antibody bET-23a used in the Examples described later was obtained from the Fermentation Research Institute (I
FO) and a deposit number IFO 50246 from May 24, 1990. The hybridoma has been deposited with the Ministry of International Trade and Industry at the Research Institute of Microbial Industry (FRI) under the Accession No. FERM BP-2948 from June 12, 1990 under the Budapest Treaty, and stored at the FRI.

I. Preparation of enzyme-labeled anti-endothelin-3 C-terminal peptide antibody I-1. Synthesis of peptide i) Synthesis of H-Cys-His-Leu-Asp-Ile-Ile-Trp-OH (C end of endothelin-3) Boc-Ile-Trp-OBzl: 84 g of H-Trp-OBzl-pTsOH DMF100
dissolved in TEA, neutralized with 25 ml of TEA, and then Boc-Ile-OH.1 /
Prepared from 48.9 g of 2 H ₂ O, 40.12 g of HONB, 46.20 g of DCC
Boc-Ile-ONB was added and stirred overnight. The solvent was distilled off under reduced pressure, and 400 ml of diethyl ether and 20 ml of ethyl acetate were added to the residue.
and dissolved in 0.5N-HCl, 5% -NaHCO ₃
It was washed with water, washed with water and dried over anhydrous MgSO ₄ . The solvent was distilled off, petroleum ether was added to the residue, and the crystals were collected by filtration. Yield 83.1 g (90.9%) [α] _D ¹⁸ -19.7 ° (c = 0.99 MeOH) Elemental analysis: Calculated as C ₂₉ H ₃₇ N ₃ O ₅ Calculated: C, 68.62; H, 7.35; N, 8.28 Found: C, 68.69; H, 7.35; N, 8.16 Boc-Ile-Ile-Trp-OBzl: 53.3 g of Boc-Ile-Trp-OBzl was dissolved in 120 ml of dioxane, and 160 ml of 7.1 N-HCl-dioxane was added under ice cooling. And stirred for 90 minutes. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was collected by filtration. This was dissolved in a mixed solvent of 150 ml of acetonitrile and 250 ml of DMF, neutralized with 14.6 ml of TEA under ice cooling, and the precipitated TEA.HCl was removed by filtration. Boc-Ile-ONB 45.
33 g was added and stirred overnight. The solvent is distilled off and the residue is CHCl
₃ Dissolve in 1 liter and add 0.5N-HCl, 5% -NaHC
Washed with O ₃ , water and dried over anhydrous MgSO ₄ . The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was collected as crystals, washed with acetonitrile and diethyl ether, and dried. Yield 54.95 g (84.3%) [α] _D ¹⁸ -12.0 ° (c = 0.98, CHCl ₃ ) Elemental analysis: Calculated as C ₃₅ H ₄₈ N ₄ O ₆ : C, 67.72; H, 7.79; N, 9.03 Found: C, 67.72; H, 7.91; N, 8.87 Boc-Asp- (OBzl) -Ile-Ile-Trp-OBzl: Boc-Ile-Ile-Trp-O
49.7 g of Bzl was dissolved in 250 ml of TFA containing 10% -1,2-ethanedithiol under ice-cooling, and allowed to stand at room temperature for 15 minutes. The solvent was distilled off, and 4N-HCl-dioxane 2 was added to the residue.
After adding 0 ml and stirring well, diethyl ether was added and the precipitate was collected by filtration. This was dissolved in DMF (250 ml), neutralized with TEA (11.1 ml) under ice cooling, and the precipitated TEA.HCl was removed by filtration. Boc-Asp- (OBzl) -OH 3 in the filtrate
Boc-Asp- prepared from 1.0 g, HONB 20.6 g, DCC 23.8 g
(OBzl) -ONB was added and stirred overnight. The solvent was distilled off under reduced pressure,
The residue was dissolved by adding 800 ml of ethyl acetate, washed with 10% aqueous citric acid, 5% -NaHCO ₃ and water, and dried over anhydrous MgSO _4.
And dried. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was collected as crystals by filtration and recrystallized from acetonitrile. Yield 54.0 g (81.7%) [α] _D ¹⁸ -13.7 ° (c = 1.04, CHCl ₃ ) Elemental analysis: Calculated as C ₄₆ H ₅₉ N ₅ O ₉ : C, 66.89; H, 7.20; N, 8.48 Found: C, 66.69; H, 7.23; N, 8.31 Boc-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl: Boc-Asp (OBzl)-
48.0 g of Ile-Ile-Trp-OBzl was dissolved in 210 ml of TFA containing 10% -1,2-ethanedithiol under ice-cooling, and the solution was dissolved at room temperature in 15 ml.
Let stand for minutes. The solvent was distilled off, and 4N-HCl-
After 14.5 ml of dioxane was added and mixed well, diethyl ether was added, and the precipitate was collected by filtration. This is DMF 3
After dissolving in 100 ml and neutralizing with 42 ml of TEA under ice-cooling, the precipitated TEA-HCl was removed by filtration and the filtrate was Boc-Leu-ONB 27.
3 g was added and stirred overnight. The solvent was distilled off under reduced pressure, and the residue was dissolved by adding 600 ml of CHCl ₃ to obtain a 10% aqueous citric acid solution.
% NaHCO _3, and dried over wash anhydrous MgSO ₄ in water. Petroleum ether was added to the residue, and the precipitate was collected by filtration and recrystallized from aqueous acetonitrile. Yield 53.1 g (97.5
%) [Α] _D ¹⁸ -20.6 ° (c = 1.02, CHCl ₃ ) Elemental analysis: Calculated as C ₅₂ H ₇₀ N ₆ O ₁₀ .1 / 2H ₂ O: C, 65.87; H, 7.55; N, 8.86 Found: C, 66.08; H, 7.58; N, 8.78 Boc-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl: Boc-Leu-As
46.5 g of p (OBzl) -Ile-Ile-Trp-OBzl was added to 10% under ice cooling.
Dissolved in 220 ml of 1,2-ethanedithiol-containing TFA,
It was left at room temperature for 15 minutes. The solvent was distilled off under reduced pressure, and diethyl ether was added to the residue, followed by filtration. This was dissolved in DMF (25 ml), TEA (20 ml) was added, and the mixture was stirred well, and then diethyl ether (500 ml) was added, and the precipitate was collected as a powder by filtration. This was dissolved in 200 ml of DMF, and Boc-His (Tos) -OH 2
Boc-H prepared from 2.3g, HONB 11.7g, DCC 13.5g
is (Tos) -ONB was added and stirred overnight. The solvent was distilled off, acetonitrile was added to the residue, and the powder was collected by filtration. yield
45.9 g (86.2%) [α] _D ¹⁸ -16.6 ° (c = 1.0, DMF) Elemental analysis: Calculated as C ₅₈ H ₇₇ N ₉ O ₁₁ .2.5H ₂ O: C, 62.13; H, 7.37; N, 11.24 Found: C, 62.25; H, 7.02; N, 11.03 Boc-Cys (MeBzl) -His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBz
l: 17.2 g of Boc-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl under ice-cooling, TFA88 containing 10% -1,2-ethanedithiol.
The solution was dissolved in ml and left at room temperature for 15 minutes. The solvent was distilled off under reduced pressure, ether was added to the residue, and the resulting powder was collected by filtration.
Dissolved in 25 ml of MF. After adding 25 ml of TEA thereto and stirring well, 500 ml of ether was added and the precipitate was collected as a powder by filtration. This was dissolved in 40 ml of DMF, and Boc-Cys (Me
Boc-Cys (MeBzl) -ONB prepared from 5.72 g of Bzl) -OH, 3.48 g of HONB, and 4.00 g of DCC was added and stirred overnight. The solvent was distilled off, acetonitrile was added to the residue, and the precipitate was collected by filtration as a powder, washed in hot acetonitrile, cooled to room temperature, and filtered. Yield 15.0 g (72.9%) [α] _D ¹⁸ -23.3 ° (c = 1.02, DMF) Elemental analysis: Calculated as C ₆₉ H ₉₀ N ₁₀ O ₁₂ S.3H ₂ O: C, 61.96; H, 7.23 N, 10.47; S, 2.40 found: C, 62.24; H, 6.90; N, 10.49; S, 2.53 H-Cys-His-Leu-Asp-Ile-Ile-Trp-OH: Boc-Cys (MeBzl) -
100 mg of His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl was added to 10 ml of 10% -1,2-ethanedithiol-containing TFA under ice-cooling.
And left at room temperature for 15 minutes, after which the solvent was distilled off under reduced pressure.
Ether was added to the residue, and the mixture was collected by filtration as a powder, dried and then mixed with 0.5 ml of m-cresol and 5 ml of HF at 0 ° C.,
Time processed. HF was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered as a powder and washed with water.
This was dissolved in 60% acetic acid, and applied to a Sephadex LH-20 (2.5 × 90 cm) column packed with the same solvent, and 160 ml to 180 ml.
The ml fractions were collected and lyophilized. Yield 23mg (32.8%)

Ii) H-Arg-His-Leu-Asp-Ile-Ile-Trp-OH
Synthesis of Boc-His-Leu-Asp (OBzl) -Ile-Ile-Trp-OBzl (538 mg) under ice-cooling, 20% -1,2-ethanediotyl Contained TFA 5ml
After standing at room temperature for 15 minutes, 95 mg of pTsOH.H ₂ O was added and dissolved, and the solvent was distilled off under reduced pressure. Residue in DMF
Dissolve in 5 ml, and add 10% -TEA-containing DMF 0.75 under ice-cooling.
ml was added for neutralization. Boc-Arg (NO ₂ ) -OH 60mg, HON
50 mg of B and 50 mg of WSCD.HCl were added, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure, water was added to the residue, and the precipitate was collected by filtration. This was dried, suspended in 10 ml of hot acetonitrile, cooled to room temperature, and the precipitate was collected by filtration. Yield 0.5 g (78.3%) Of these, 102 mg was treated with HF together with 0.2 ml of anisole and 0.2 ml of 1,2-ethanedithiol for 60 minutes at 0 ° C.
F was distilled off under reduced pressure. The residue was washed with diethyl ether, dissolved in 6 ml of 50% acetic acid, applied to a column of Sephadex G-25 (2.5 × 90 cm) filled with the same solvent, developed, and collected from 100 to 137 ml. Dried. Yield 2
0 mg (26.3%)

I-2 Preparation of Immunogen The polypeptide Cys His L obtained in the above I-1 (i)
A condensate of eu Asp Ile Ile Trp and bovine serum albumin (hereinafter abbreviated as BSA) was prepared by the maleimide crosslinking method described below and used as an immunogen. That is, 1.4 ml of BSA 20 mg
Was dissolved in 0.1 M phosphate buffer, pH 7.0, and N- (γ
-Maleimidobutylyloxy) succinimide (hereinafter GMB)
This was mixed with 100 μl of a DMF solution containing 2.6 mg, and reacted at room temperature for 40 minutes. After the reaction, fractionation was performed on a Sephadex G-25 column previously equilibrated with 0.1 M phosphate buffer, pH 6.5, containing 2.5 mM EDTA. Next, 1.2 ml of the eluted fraction containing 5.4 mg of BSA into which a maleimide group was introduced, and an aqueous solution containing 90% dimethyl sulfoxide.
Cys H dissolved or dispersed in 1.2 ml
Is Leu Asp Ile Ile Trp 1.8 mg was reacted at 4 ° C. for 3 days. After the reaction, the solution was dialyzed against physiological saline at 4 ° C. for 2 days.

I-3 Immunity 450 μl of physiological saline containing 600 μg of the immunogen obtained in I-2 above
Add 550 μl complete Freund's adjuvant (Freund comple
te adjuvant) was added thereto and mixed well to prepare an emulsion, which was inoculated subcutaneously into about 20 rabbits. Six weeks later, an incomplete Freund's adjuvant was used to prepare an emulsion in the same manner as above and inoculated subcutaneously into rabbits. This operation was repeated four times every other month thereafter. Seven days after the booster immunization, a part of the blood was collected from the rabbit to obtain an antiserum by a conventional method.

I-4 Preparation of Affinity Solid Phase Polypeptide Arg His Leu Asp Ile Ile Trp was directly converted to C
It was bound to NBr-activated Sepharose 4B to obtain an affinity solid phase. That is, Arg His Leu Asp Ile Ile Trp 1.
5 mg was dissolved in 10 ml of 0.1 M sodium bicarbonate containing 0.5 M salt, and 1 g of CNBr-activated Sepharose 4B
And 3 hours at room temperature. Next, unreacted active groups are reduced to 0.1
After treatment with M Tris-HCl buffer, pH 8, PBS
And stored at 4 ° C.

I-5 Purification of Anti-Endothelin-3 C-terminal Peptide Antibody Using Affinity Solid Phase From 16 ml of rabbit antiserum described in I-3 above, the antibody was partially purified by ammonium sulfate precipitation. That is, PB was added to 10 ml of antiserum.
S10 ml was added and a further 16.5 ml of saturated ammonium sulfate were added with slow stirring (final 45%). After leaving for 30 minutes, 1
After centrifugation at 2,000 × g for 20 minutes, the precipitate was dissolved in 10 ml of PBS. Next, saturated ammonium sulfate was similarly added to a final 30% saturation, followed by centrifugation. The precipitate is washed with 0.15M saline containing 0.15M.
01M borate buffer, pH 8 (hereinafter abbreviated as BBS), 10m
After dissolving in 1 L, the mixture was dialyzed against 0.01 M phosphate buffer (pH 8) containing 0.01 M salt (buffer B) at 4 ° C. for 2 days.
After the antibody fraction was dialyzed against BBS, it was applied to a column (10 mmφ × 40 mm) packed with the affinity solid phase described in I-4 above. After washing thoroughly with BBS, the specific antibody was eluted with 0.1 M acetic acid buffer containing 0.5 M salt, pH 4.5, and further eluted.
0.05 M glycine-hydrochloric acid buffer containing 0.1 M salt, pH 2.0
Eluted. After the eluate was neutralized, it was dialyzed against BBS. The specific antibody was observed in the fraction eluted at pH 2, and 18 mg of the specific antibody was obtained from the fraction.

I-6 Horseradish peroxidase (H
RP) Preparation of labeled anti-endothelin-3 C-terminal peptide antibody Affinity purified anti-Cys His Leu described in I-5 above
Ishikawa et al. Method from Asp Ile Ile Trp antibody [Journal
Of Applied Biochemistry (J. Appl. Bio
Chem .., 6: 56-63 (1984)]. That is, pepsin (Sigma, twice crystal) was added to 6.4 mg of the specific antibody dissolved in 0.1 M acetic acid buffer solution, pH 4.5.
After adding 60 μg and reacting at 37 ° C for 16 hours, FPLC using Superose 12 column equilibrated with BBS
(Pharmacia) to purify the F (ab ') 2 fraction. The fraction was dialyzed against a 0.1 M acetic acid buffer solution, pH 5, and the final 20
mM β-mercaptoethylamine was added, and the mixture was left at 37 ° C. for 90 minutes. The reaction solution was separated by FPLC using a Superose 12 column equilibrated with 0.1 M phosphate buffer containing 2.5 mM EDTA and pH 6.0 to obtain a Fab ′ fraction. On the other hand, 5 mg of horseradish peroxidase was added to 0.9 ml of 0.1 M
It was dissolved in a phosphate buffer solution, pH 7, and 1.05 mg of GMBS dissolved in 50 μl of DMF was added, followed by a reaction at room temperature for 40 minutes. The reaction solution was applied to a Sephadex G-25 column (eluent 0.1
M phosphate buffer, pH 6.8), 3.5 mg of the obtained maleimidated peroxidase and 0.8 mL of the above Fab 'fraction
g and mix with Collodion Pack (MS Instruments)
After concentrating to 0.3 ml, it was left at 4 ° C. for 16 hours.
The reaction solution was applied to an Ultrogel AcA44 column (10 mmφ × 40 mm) using a 0.1 M phosphate buffer, pH 6.5 as an eluent.
The ab'-peroxidase complex fraction was purified.

II. Preparation of monoclonal anti-endothelin-3 antibody II-1 Preparation of immunogen Endothelin-3 and bovine thyroglobulin (TG) were condensed by a maleimide crosslinking method described below to obtain an immunogen. That is, 265 nmole of endothelin-3 (purchased from Peptide Institute) was used in 450 μl of 0.1 M phosphate buffer, pH 7.0.
0 (containing 10% DMF) and GMBS.
It was mixed with 50 μl of a DMF solution containing 6 μmole and reacted at room temperature for 30 minutes. On the other hand, 20 mg (40 nmole) of TG was dissolved in 1.4 ml of 0.02 M phosphate buffer containing 0.15 M salt, pH 6.8, and N-succinimidyl-3- (2-pyridyldithio) propionate (hereinafter abbreviated as SPDP) 2.5 mg (8.0
After mixing 100 μl of DMF solution containing
The reaction was performed for 0 minutes. After the reaction, dithiothreitol 12.4mg
(80 μmole) in 0.1 M acetate buffer, pH 4.5, 0.5
After the reaction was carried out at room temperature for 20 minutes, fractionation was carried out on a Sephadex G-25 column.
12 mg (24 nmole) were obtained. Next, 190 nmole of maleimide group-introduced endothelin-3 and 5.9 nmole of SH group-introduced TG were used.
Was mixed and reacted at 4 ° C. for 2 days, and then 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 above with an adjuvant. Thereafter, booster immunizations were performed two to three times every three weeks.

II-3 Preparation of HRP-labeled endothelin-3 Endothelin-3 80 nmole was dissolved in 450 μl of 0.1 M phosphate buffer, pH 7.0, and 295 μg of GMBS (2.0 μmol
The mixture was mixed with 50 μl of a DMF solution containing e) and reacted at room temperature for 30 minutes. After the reaction, fractionation was carried out on a Sephadex G-15 column, and the maleimide group-introduced polypeptide 60 n
got a mole. On the other hand, HRP 10 mg (250 nmole)
It was dissolved in 1.4 ml of 0.02 M phosphate buffer containing sodium chloride, pH 6.8, mixed with 100 μl of a DMF solution containing 1.17 mg (3.75 μmole) of SPDP, and allowed to react at room temperature for 40 minutes. After the reaction, 0.1 ml containing 12.4 mg (80 μmole) of dithiothreitol
After adding 0.5 ml of M acetate buffer, pH 4.5, and reacting at room temperature for 20 minutes, fractionation was performed on a Sephadex G-25 column to obtain 6 mg (150 nmole) of the enzyme into which the SH group was introduced. Next, maleimide group-introduced endothelin-3 50 nmol
e and SH-introduced peroxidase 20 nmole,
The reaction was performed at 4 ° C. for 16 hours. After the reaction, Ultrogel AcA
Fractionation was performed using a 44 (LKB-Pharmacia) column to obtain peroxidase-labeled endothelin-3.

II-4 Cell Fusion Mice showing a relatively high antibody titer were immunized intravenously with 240 μg of the immunogen dissolved in 0.25 ml of physiological saline intravenously. 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 (P3U
1) [Current Topics in Microbiology and Immunology, 81 , 1 (1978)]. Cell fusion was performed according to the original method [Nature, 256 , 495 (1975)]. That is, the spleen cells and P3U1 were each washed three times with serum-free MEM, and the spleen cells and P3U1 were washed three times.
Mix the ratio of U1 number so that it becomes 5: 1.
The cells were sedimented by centrifugation for 15 minutes. After sufficiently removing the supernatant, the precipitate was loosened gently, and 0.3 ml of 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite) was added. After fusion, MEM was added to the cells at a rate of 2 ml per minute, for a total of 12 ml.
After adding 1 MEM, the supernatant was removed by centrifugation at 600 rpm for 15 minutes. The cell sediment was purified using G containing 10% fetal bovine serum.
P3U1 was suspended in IT Medium (Wako Pure Chemical) (GIT-10FCS) at 2 × 10 ⁶ per ml,
1 ml per well in a 4-hole multi-disc (manufactured by Limbro)
Seeded into 120 wells. After seeding, the cells were cultured at 37 ° C. in a 5% carbon dioxide furan apparatus. Twenty-four hours later, a GIT-10FCS medium containing HAT (hypoxanthine 1 × 10 ⁻⁴ M, aminobriteline 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻³ M) (H
HAT selection culture was started by adding 1 ml per well of AT medium). The HAT 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 9-14 days after cell fusion, and when the culture broth turned yellow (about 1 × 10 ⁶ cells / ml), the supernatant was collected,
The antibody titer was measured by the EIA method described below.

II-5 Screening of hybridoma The antibody titer in the culture supernatant of the hybridoma was measured by the following two methods. In each case, an anti-mouse immunoglobulin antibody-bound microplate was used. The plate was first prepared with an anti-mouse immunoglobulin antibody (IgG fraction,
0.1 M carbonate buffer containing 20 μg / ml
100 μl of the H9.6 solution was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. Next, plate
After washing with BS, PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products) to block excess binding sites in the wells
Was dispensed in 300 μl aliquots and treated at least at 4 ° C. for 24 hours. i) EIA method using HRP-labeled endothelin-3 Buffer E (10% block ace, 2 mg / ml BS) was added to the anti-mouse immunoglobulin antibody-bound microplate.
A, 0.4 M NaCl, 2 mM EDTA and 0.1% Na
50 μl of a 0.02 M phosphate buffer containing N ₃ (pH 7.0) and 50 μl of the hybridoma culture supernatant were added, and reacted at room temperature for 4 hours. After the reaction, the plate was washed with PBS, and 100 μl of the HRP-labeled endothelin-3 prepared in II-3 described above [100% diluted with 0.02 M phosphate buffer containing 1% BSA, pH 7 (buffer A)] was added thereto. The reaction was carried out at 16 ° C for 16 hours. After the reaction, the plate was washed with PBS, and then 100 μl of a 0.1 M citrate buffer containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide, pH 5.5 was dispensed at 100 μl to measure the enzyme activity on the solid phase. The reaction was performed for 10 minutes. After stopping the reaction by adding 100 μl of 4N sulfuric acid, the absorbance at 492 nm was measured using a plate reader (MTP-32, manufactured by Corona). In this manner, the supernatants of all 120 wells in which hybridoma growth was observed were examined. 5 and No. 5 Strong antibody titers were detected in 93 wells. ii) EIA method using HRP-labeled endothelin-3 C-terminal peptide antibody 50 μl of buffer E, 50 μl of hybridoma culture supernatant, and 50 μl of buffer E solution containing 6 ng / ml endothelin-3 were added to the anti-mouse immunoglobulin antibody-bound microplate. The reaction was carried out at room temperature for 4 hours. After washing the plate with PBS, the HR prepared in I-6 above was used.
P-labeled endothelin-3 C-terminal peptide antibody [buffer C (1% BSA, 0.4 M NaCl, 2 mM EDTA
Diluted 300 times with 0.02M phosphate buffer (pH 7.2) containing
0 μl was added and reacted at 4 ° C. for 16 hours. Next, after the plate was washed with PBS, the enzymatic activity on the solid phase was determined using II-
It was measured by the method described in 5 (i). In this way,
When the supernatant of all 120 wells in which hybridoma growth was observed was examined, Strong antibody titers were detected in 93 wells.

II-6 Cloning No. showing positive antibody activity. 5 and No. 5 Each hybridoma in 93 wells 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). At the time of dispensing, thymocytes of BALB / C mice were added as feeder cells 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. 41 in 93 hybridomas
Seven of the clones produced antibodies. Among these clones, clone A obtained from No. 93-18
ET-30 and its monoclonal antibody AET-
Focusing on 30a, the following experiment was performed.

II-7 Preparation of Large Amount of Monoclonal Antibody Hybridoma A was administered to mice intraperitoneally administered with 0.5 ml of mineral oil or untreated mice (BALB / C).
After intraperitoneal injection of ET-30 1-3 × 10 ⁶ cells / animal,
The antibody-containing ascites was collected 10 to 30 days later.

II-8 Purification of Monoclonal Antibody A monoclonal antibody was purified from the above-prepared ascites II-7 using a protein-A column. That is, 8 ml of ascites was added to an equal volume of a binding buffer (1.5 M containing 3.5 M NaCl and 0.05% NaN _3).
After dilution with glycine (pH 9.0), the mixture is applied to a protein-A-Sepharose (Pharmacia) column which has been equilibrated with a binding buffer in advance, and the specific antibody is eluted with a buffer (0.1 M quencher containing 0.05% NaN _3). Acid buffer, pH 3.
Eluted at 0). By the above operation, 40 mg of the specific antibody was obtained.

II-9 Determination of Class and Subclass of Monoclonal Antibody 0.2 μg / ml of the above purified II-8 monoclonal antibody was added.
100 μl of a 1 M carbonate buffer and a pH 9.6 solution were dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. I above
According to the method described in I-5, an excess binding site in a well is covered with a block ace, and an enzyme-linked immunosorbent assay using an isotype typing kit (Mouse-Typer ^™ Sub-Isotyping Kit, manufactured by Bio-Rad) is performed. The class and subclass were examined by (ELISA). As a result, AET-30a was IgG1, κ
Turned out to belong to the class.

III. Competition method-EIA AE diluted 75-fold with buffer C on the anti-mouse immunoglobulin antibody-bound microplate described in II-5 above
After adding 50 μl of the culture supernatant containing T-30 and 50 μl of the endothelin-1 standard solution or 50 μl of the endothelin-3 standard solution, and reacting at room temperature for 1 hour, the HR described in II-3 above was added.
P-labeled endothelin-3 (100-fold diluted with buffer A)
Was added and reacted at 4 ° C. for 16 hours. After the reaction, the plate was washed with PBS, and the enzyme activity on the solid phase was measured by the method described in II-5 (i). The results are shown in FIG. In the figure,-●-indicates a standard curve of endothelin-3, and-○-indicates a standard curve of endothelin-1. From the results in FIG. 1, AET-30
It was found that a reacted with endothelin-3 but not with endothelin-1.

IV. Sandwich method-EIA Purified monoclonal antibody AET-30a 20 µg / ml
A 0.1 M carbonate buffer, pH 9.6 solution containing 100 μl was dispensed 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. Endothelin-3, endothelin-1, endothelin-2 (human), big endothelin-1 (human), big endothelin-1 (porcine) and big endothelin-3 (22 −42) (Human) standard solution 100
μl was added and reacted at 4 ° C. for 24 hours. After washing with PBS, the HRP-labeled endothelin-3 C-terminal peptide antibody prepared in I-6 above (diluted 300-fold with buffer C)
100 µl was added and the reaction was carried out 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 (i) above. The results are shown in FIG. In the figure,
Is endothelin-3, and-▲-is endothelin-1
,-■-is endothelin-2 (human),-○-is big endothelin-1 (human),-△-is big endothelin-1 (porcine), and-□-is big endothelin-
3 shows a standard curve of 3 (human). From the results in FIG. 2, this assay is specific for endothelin-3,
It was found that ³⁴ × 10 ⁻¹⁷ mol / well can be detected with little reaction with other endothelins (cross-reactivity, 0.1% or less).

V. Preparation of monoclonal anti-big endothelin-3 C-terminal peptide antibody V-1. Synthesis of peptide (1) Big endothelin-3 (big ET-3) (22-42); H-Ile-A
sn-Thr-Pro-Glu-Gln-Thr-Val-Pro-Tyr-Gly-Leu-Ser-Asn
Synthesis of -Tyr-Arg-Gly-Ser-Phe-Arg-Gly-OH Using a commercially available Boc-Gly-OCH2-PAM resin (manufactured by Applied Biosystems), 0.77 g (0.5 nmole) of a peptide synthesizer (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-Arg (Tos), Boc-Phe, Boc-Ser
(Bzl), Boc-Gly, Boc-Tyr (Br-Z), Boc-Asn, Boc-Leu, B
oc-Pro, Boc-Val, Boc-Thr (Bzl), Boc-Gln, Boc-Glu (OBz
l), Boc-Ile in the order of the amino acid sequence of big ET-3 (22-42)
Activated with HOBt / DCC and condensed. DCC or HOBt /
After condensing again with the same amino acid derivative activated by DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 0.88 g of protected big ET-3 (22-42) -OCH2-PAM resin.
0.35 g of this resin was treated with 5 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 0.70 g of p-cresol, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether.
The residue was extracted with 5% 50% aqueous acetic acid. The insoluble material is removed by filtration, and 5
It was washed with 5 ml of 0% acetic acid aqueous solution. Combine the filtrate and washings,
concentrated under reduced pressure, and Sephadex LH-20 (2 × 90 cm)
And eluted with 50% acetic acid. The main fraction is collected, concentrated and dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid.
C-ODS AM120 S-50 resin column (2.6 × 7
cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). The main fractions were combined and lyophilized to give 76 mg of a white powder. Amino acid analysis value Asp 1.97 (2), Thr 1.76 (2), Ser 1.68 (2), Glu 2.04
(2), Gly 3.00 (3), Val 1.00 (1), Pro 1.90 (2), Ile 0.9
5 (1), Leu 0.99 (1), Tyr 0.95 (1), Phe 1.02 (1), Arg
2.27 (2) Mass spectrometry (M + H) ⁺ 2356.16 HPLC elution time 17.01 min Column conditions Column: YMC-ODS (AM-301, S-5 120A) Eluent: Solution A (0.1% -trifluoroacetic acid aqueous solution) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% trifluoroacetic acid) (50 minutes)
: 1.0ml / min

(2) big ET-3 (1-42); H-Cys-Thr-Cys-Phe-T
hr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-Tyr-Cys-His-Leu
-Asp-IleOIle-Trp-Ile-Asn-Thr-Pro-Glu-Gln-Thr-Val-P
ro-Tyr-Gly-Leu-Ser-Asn-Tyr-Arg-Gly-Ser-Phe-Arg-Gly
Synthesis of -OH Using a commercially available Boc-Gly-OCH ₂ -PAM resin (Applied Biosystems) 0.78g (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-Arg (Tos), Boc-Phe, Boc-Ser
(Bzl), Boc-Gly, Boc-Tyr (Br-Z), Boc-Asn, Boc-Leu, B
oc-Pro, Boc-Val, Boc-Thr (Bzl), Boc-Gln, Boc-Glu (OBz
l), Boc-Ile, Boc-Trp (CHO), Boc-Asp (0Bzl), Boc-His (D
NP), Boc-Cys (MeBzl), Boc-Lys (Cl-Z) to big ET-3 (1-42)
Activated and condensed with HOBt / DCC in order according to the amino acid sequence of
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-3 (1-42) -OCH _2- PA
M resin was obtained. This is N, N'-dimethylformamide 20ml
And 2 ml of thiophenol was added thereto. The mixture was gently stirred at room temperature for 2 hours, and then the resin was filtered on a glass filter. The resin was washed with N, N'-dimethylformamide and dichloromethane and dried to obtain 1.00 g of a resin. . 0.39 g of this resin is p-
After treatment with anhydrous hydrogen fluoride (10 ml) at 0 ° C for 60 minutes in the presence of 0.74 g of cresol and 1.0 ml of 1,4-butanedithiol, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether. The residue was extracted with 4 ml of trifluoroacetic acid.
The insoluble material was removed by filtration and washed twice with 2 ml of trifluoroacetic acid. Pour this into 750 ml of 4M-urea aqueous solution, and pH
The mixture was adjusted to 8.25 and stirred overnight while gently blowing air. After confirming that the Ellman test is negative,
The whole solution was applied to a YMC-ODS AM120 S-50 resin column (2.6 × 7 cm), and 0.1% aqueous trifluoroacetic acid and 5%
Elution was performed with a linear gradient between 0% acetonitrile (containing 0.1% trifluoroacetic acid). The main fractions were combined and lyophilized to give 16 mg of a white powder. 20-5 using the same column
Purification was performed again by elution with a linear concentration gradient between 0% to obtain 6.3 mg of the desired product. Amino acid analysis value Asp 3.84 (4), Thr 3.31 (4), Ser 2.02 (2), Glu 3.04
(3), Pro 2.22 (2), Gly 3.31 (3), Cys 1.20 (2), Val 1.7
7 (2), Ile 1.83 (3), Leu 2.00 (2), Tyr 4.64 (5), Phe
1.92 (2), Lys 1.78 (2), His 0.88 (1), Arg 2.12 (2) Mass spectrometry (M + H) ⁺ 4979.48 HPLC elution time 21.00 min Column conditions Column: YMC-ODS (AM-301, S -5 120A) Eluent: Linear concentration gradient elution from solution A to solution B using solution A (0.1% -trifluoroacetic acid aqueous solution) and solution B (0.1% -trifluoroacetic acid-containing acetonitrile) (50 minutes)
: 1.0ml / min

(3) big ET-3 (22-41) -NH ₂ ; H-Ile-Asn-Thr-
Pro-Glu-Gln-Thr-Val-Pro-Tyr-Gly-Leu-Ser-Asn-Tyr-Ar
Synthesis of g-Gly-Ser-Phe-Arg-NH ₂ A peptide synthesizer (Model 430A manufactured by Applied Biosystems) using 0.60 g (0.5 nmole) of commercially available p-methyl BHA resin (manufactured by Applied Biosystems)
And synthesized. Boc-Arg (Tos) is added to resin HOBt / DCC
After the introduction, the Boc group on the resin is reduced to 50% trifluoroacetic acid /
Treatment with methylene chloride liberated the amino group. Boc-Phe, Boc-Ser (Bzl), Boc-Gly, Boc-Ar
g ('Tos), Boc-Tyr (Br-Z), Boc-Asn, Boc-Leu, Boc-Pro,
Boc-Val, Boc-Thr (Bzl), Boc-Gln, Boc-Glu (OBzl), Bo
c-Ile to big ET-3 (22-41) -NH 2 amino acid sequence as HO in order
Activated with Bt / DCC and condensed. DCC or HOBt / DC
After another condensation with the same amino acid derivative activated by C, unreacted amino groups were acetylated with acetic anhydride to obtain 1.96 g of protected big ET-3 (22-41) -BHA resin. This resin
0.23 g in the presence of 0.40 g of p-cresol 5 ml of anhydrous hydrogen fluoride
After treatment at 0 ° C. for 60 minutes, hydrogen fluoride is distilled off under reduced pressure.
After the residue was washed twice with 5 ml of ethyl ether, the residue was
The mixture was extracted with 5 ml of aqueous% -acetic acid. The insolubles were removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washings were combined, concentrated under reduced pressure to 2-3 ml, applied to a column of Sephadex LH-20 (2 × 90 cm) and eluted with 50% acetic acid. The main fraction was collected, concentrated, dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid, and dissolved in YMC-O.
The mixture was applied to a DSAM120 S-50 resin column (2.6 × 7 cm), and eluted with a linear concentration gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions, freeze-dry and white powder
56 mg were obtained. Amino acid analysis value Asp 1.97 (2), Thr 1.88 (2), Ser 1.75 (2), Glu 2.05
(2), Gly 3.00 (3), Val 1.00 (1), Pro 1.93 (2), Ile 0.9
5 (1), Leu 0.98 (1), Tyr 0.95 (1), Phe 1.02 (1), Arg
2.18 (2) Mass spectrometry (M + H) ⁺ 2298.19 HPLC elution time 17.01 min Column conditions Column: YMC-ODS (AM-301, S-5 120A) Eluent: Solution A (0.1% -trifluoroacetic acid aqueous solution) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% trifluoroacetic acid) (50 minutes)
: 1.0ml / min

(4) big ET-3 (1-41) -NH ₂ ; H-Cys-Thr-Cys-
Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-Tyr-Cys-Hi
s-Leu-Asp-Ile-Ile-Trp-Ile-Asn-Thr-Pro-Glu-Gln-Thr-
Val-Pro-Tyr-Gly-Leu-Ser-Asn-Tyr-Arg-Gly-Ser-Phe-Ar
Synthesis of g-NH _{2 Using} 0.60 g (0.5 nmole) of a commercially available p-methyl BHA resin (manufactured by Applied Biosystems), synthesis was performed using a peptide synthesizer (Model 430A manufactured by Applied Biosystems). After introducing Boc-Arg (Tos) into the resin with HOBt / DCC, the Boc group on the resin was treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and then the amino group was replaced with Boc- Phe, Boc-Ser (Bzl), Boc-Gly, Boc-Arg (To
s), Boc-Tyr (Br-Z), Boc-Asn, Boc-Leu, Boc-Pro, Boc-
Val, Boc-Thr (Bzl), Boc-Gln, Boc-Glu (OBzl), Boc-Il
e, Boc-Trp (CHO), Boc-Asp (0Bzl), Boc-His (DNP), Boc-
Cys (MeBzl) and Boc-Lys (Cl-Z) were activated and condensed with HOBt / DCC in the order of the amino acid sequence of big ET-3 (1-41) -NH ₂ . After another condensation with the same amino acid derivative activated with DCC or HOBt / DCC, the unreacted amino group is acetylated with acetic anhydride to convert the protected big-ET-3 (1-41) -BHA resin. Obtained. This was suspended in 20 ml of N, N'-dimethylformamide,
After adding 2 ml of thiophenol and stirring gently at room temperature for 2 hours, the resin was filtered on a glass filter, washed with N, N'-dimethylformamide and dichloromethane, and dried.
1.34 g of resin was obtained. 0.74 g of this resin was added to p-cresol 1.
After treating with 0 ml of 1,4-butanedithiol in the presence of 1.0 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether.
The residue was extracted with 6 ml of trifluoroacetic acid. The insoluble material was removed by filtration and washed twice with 2 ml of trifluoroacetic acid. This is 4M-
The solution was poured into 1000 ml of an aqueous urea solution, adjusted to pH 8.0 under ice cooling, and stirred overnight while blowing air gently. Ellm
After confirming that the test has become negative,
MC-ODS AM120 S-50 resin column (2.6
× 7 cm) and eluted with a linear concentration gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions, freeze-dry and
35 mg of a white powder was obtained. Using the same column, re-chromatographic purification was repeated by elution with a linear concentration gradient between 20-50% to obtain 9.5 mg of the desired product. Amino acid analysis value Asp 3.89 (4), Thr 3.81 (4), Ser 1.98 (2), Glu 3.04
(3), Pro 2.09 (2), Gly 3.30 (3), Cys 1.60 (2), Val 1.7
9 (2), Ile 1.83 (3), Leu 2.00 (2), Tyr 4.72 (5), Phe
1.92 (2), Lys 1.88 (2), His 0.91 (1), Arg 2.20 (2) Mass spectrometry (M + H) ⁺ 4921.31 HPLC elution time 21.00 min Column conditions Column: YMC-ODS (AM-301, S -5 120A) Eluent: Linear concentration gradient elution from solution A to solution B using solution A (0.1% -trifluoroacetic acid aqueous solution) and solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes)
: 1.0ml / min

V-2. Preparation of immunogen Big endothelin-3 C-terminal peptide obtained in V-1 above Ile-Asn-Thr-Pro-Glu-Gln-Thr-Val-Pro-Tyr
-Gly-Leu-Ser-Asn-Tyr-Arg-Gly-Ser-Phe-Arg-Gly and bovine thyroglobulin (TG) were condensed by a maleimide crosslinking method described below to obtain an immunogen. That is, 1.28 μmole of the polypeptide was added to 350 μl of 0.1 M phosphate buffer, pH 7.0.
(Containing 10% DMF), GMBS 25.6μm
It was mixed with 100 μl of a DMF solution containing ole, and reacted at room temperature for 30 minutes. On the other hand, TG 30 mg (60 nmole) contains 0.15 M salt
Dissolve in 1.4 ml of 0.02 M phosphate buffer, pH 6.8,
A DMF solution containing 3.7 mg (12.0 μmole) of PDP was mixed and reacted at room temperature for 40 minutes. After the reaction, 0.1 M acetate buffer containing 18.6 mg (120 μmole) of dithiothreitol, pH 4.
After adding 0.5 and 0.5 ml and reacting at room temperature for 20 minutes, fractionation was carried out using a Sephadex G-25 column to obtain 18 mg (36 nmole) of TG into which the SH group was introduced. Next, 920 nmole of the maleimide group-introduced polypeptide and 29 nmole of TG having the SH group were mixed, reacted at 4 ° C for 2 days, and dialyzed against physiological saline at 4 ° C for 2 days.

V-3. Immunization BALB / c female mice 6 to 8 weeks old were immunized subcutaneously with 15 μg / animal (as big endothelin-3 C-terminal peptide) described in the above V-2 together with an adjuvant. Thereafter, a booster was performed twice every three weeks.

V-4. Preparation of HRP-labeled Big Endothelin-3 C-terminal Peptide
Dissolve in 00 μl of 0.1 M phosphate buffer, pH 7.0, add GMBS
The mixture was mixed with 50 μl of a DMF solution containing 2.78 mg (9.9 μmol) and reacted at room temperature for 60 minutes. After the reaction, Sephadex G
Fractionation was performed using a -25 column to obtain 200 nmole of the polypeptide into which the maleimide group was introduced. On the other hand, HRP 10 mg (250 n
and 6 mg (150 nmole) of the enzyme into which the SH group was introduced by the method described in II-3. Next, maleimide group-introduced big endothelin-3 C-terminal peptide 200 nmole
And SH group-introduced peroxidase 50 nmole,
The reaction was performed at 4 ° C. for 16 hours. After the reaction, Ultrogel AcA44
(LKB-Pharmacia) to obtain a peroxidase-labeled big endothelin-3C-terminal peptide.

V-5. Cell fusion Cell fusion was carried out using a mouse immunized with the big endothelin-3C-terminal peptide described in V-3 according to the method described in II-4 to obtain a hybridoma.

V-6. Screening of hybridoma The antibody titer in the culture supernatant of the hybridoma was measured by the following two methods. That is, a microplate binding to an anti-mouse immunoglobulin antibody or a big endothelin-3C-terminal peptide was used. The plate first contains 20 μg / ml of an anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) or a big endothelin-3 C-terminal peptide.
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, after the plate was washed with PBS, 300 μl of PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products) was dispensed in order to cover the excess binding site of the well, and the plate was treated at least at 4 ° C. for 24 hours. i) EIA method using HRP-labeled big endothelin-3 C-terminal peptide Buffer E (10% Block Ace, 2 mg / ml BS) was added to a microplate bound with an anti-mouse immunoglobulin antibody.
A, containing 0.4 M NaCl, 2 mM EDTA and 0.1% NaN ₃
50 μl of 0.02 M phosphate buffer (pH 7.0) and 50 μl of the hybridoma culture supernatant were added, and reacted at room temperature for 4 hours. After the reaction, the HRP-labeled big endothelin-3 C-terminal peptide prepared in V-4 above [diluted 400-fold with buffer C] 1
After adding 00 μl, the mixture was reacted at 4 ° C. for 16 hours. After reaction PBS
After washing, the enzyme activity on the solid phase was measured by the method described in II-5 (i). As described above, when the supernatant of all 120 wells in which hybridoma growth was observed was examined, strong antibody titers were detected in the No. 44 and No. 77 wells. ii) ELISA method using big endothelin-3 C-terminal peptide-bound microplate ELISA plate using big endothelin-3 C-terminal peptide-bound microplate, buffer E 50 μl, hybridoma supernatant 100
μl was added and reacted at room temperature for 4 hours. After the reaction, the plate was washed with PBS, and 100 µl of an HRP-labeled anti-mouse immunoglobulin antibody [diluted 10000-fold with buffer C] was added.
The reaction was performed for 6 hours. Next, after the plate was washed with PBS, the enzyme activity on the solid phase was measured by the method described in V-6 (i) above. In this way, when the supernatant of all 120 wells in which hybridoma growth was observed was examined, No.
Strong antibody titers were detected in 77 wells.

V-7. Cloning Each of the hybridomas in the No. 44 and No. 77 wells showing positive antibody activity was cloned according to the section II-6. 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 the above V-6 (i). In the No. 77 hybridoma, 7
75 out of 6 clones produced antibodies. Among these clones, clone bET obtained from No. 77-30
-31 and its produced monoclonal antibody bET-31a, N
The following experiment was conducted with attention focused on the clone bET-23 obtained from o.44-52 and the monoclonal antibody bET-23a produced by the clone bET-23.

V-8. Preparation of large amount of monoclonal antibody Hybridoma bET-31 or bET-23 1-3 × 10 ⁶ cells / animal were injected intraperitoneally into mice to which 0.5 ml of mineral oil was intraperitoneally administered or untreated mice (BALB / C) Thereafter, 10 to 14 days later, antibody-containing ascites was collected.

V-9. Purification of monoclonal antibody Monoclonal antibodies bET-31a and bET-23a were purified from the above-prepared ascites V-8 using a protein-A column according to the method of II-8. 30 mg of bET-31a and 50 mg of bET-23a were obtained.

V-10. Determination of Class and Subclass of Monoclonal Antibodies Above V-9 Purified Monoclonal Antibodies bET-31a and bET-2
The class / subclass of 3a was determined according to the method described in II-9. As a result, both bET-31a and bET-23a were Ig
Gl and kappa were found to belong to the class.

VI. Competition method for big endothelin-3-EIA bET diluted 100-fold with buffer C in the anti-mouse immunoglobulin antibody-bound microplate described in V-6 above
50 μl of the culture supernatant containing -31a and bET-23a and the C-terminal peptides of big endothelin-1, endothelin-3, big endothelin-3, and big endothelin-3 (22-
42) Alternatively, 50 μl of a big endothelin-3 C-terminal peptide (22-41) -NH ₂ standard solution was added, and reacted at room temperature for 1 hour. Then, the HRP-labeled big endothelin-3 C-terminal peptide described in V-4 above (buffer) (200-fold dilution with C)
Was added and reacted at 4 ° C. for 16 hours. After the reaction, the plate was washed with PBS, and the enzyme activity on the solid phase was measured by the method described in II-5 (i). The results are shown in FIGS. In the figure,-▲-indicates Big Endothelin-3 and -〇-
Is big endothelin-1 and-●-is endothelin-
3,-■-indicates big endothelin-3 C-terminal peptide (22-42), and-□-indicates big endothelin-3.
Shows a standard curve of C-terminal peptide _{(22-41) -NH 2, (A} ) is b
ET-31a and (B) show the results of the competition method using bET-23a.
From the results in FIG. 3, since both bET-31a and bET-23a do not react with big endothelin-1 and endothelin-3, they are antibodies specific to big endothelin-3, peptide (22-42) and big endothelin-3 C-terminal peptide (22-41) -NH ₂ from to react to the same extent, it was found to have broad specificity for C-terminal site of big endothelin-3.

VII. Sandwich method for big endothelin-3-EIA VII-1. Preparation of HRP-Labeled Monoclonal Antibody bET-31a Fab′-HRP-labeled bET-31a was prepared from bET-31a purified by the method described in V-9 according to the method described in I-6. That is,
Monoclonal antibody b dissolved in 0.1 M acetate buffer, pH 4.5
3.17 μg of pepsin (Sigma, twice crystal) in ET-31a 10.5
Was added, and the mixture was reacted at 37 ° C. for 16 hours. Then, the F (ab ′) ₂ fraction was purified using an Ultrogel AcA44 column equilibrated with BBS. The fraction was dialyzed against 0.1 M acetate buffer, pH 5, and the final 20 mM β-mercaptoethylamine was added.
Left for a minute. The reaction solution was separated on a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer containing 2.5 mM EDTA, pH 6.0, to obtain a Fab ′ fraction. On the other hand, 5 mg of horseradish peroxidase was dissolved in 0.9 ml of 0.1 M phosphate buffer, pH 7, and 1.05 mg of GMBS dissolved in 50 μl of DMF.
Was added and reacted at room temperature for 40 minutes. The reaction solution was separated on a Sephadex G-25 column (eluent: 0.1 M phosphate buffer, pH 6.8), and 3.5 mg of the obtained maleimidated peroxidase was obtained.
And the above Fab 'fraction (1.3 mg),
After concentrating to about 0.3ml with (MS Instruments), 4 ℃
For 16 hours. The reaction solution is an Ultrogel AcA44 column (10 mmφ ×
40 mm) and the Fab'-peroxidase complex fraction was purified.

VII-2. HRP-labeled monoclonal antibody bE
Preparation of T-23a Monoclonal antibody bET-2 according to the method described in VII-1 above
HRP-labeled bET-23a was prepared using 12.7 mg of 3a.

VII-3. 100 μl of a standard solution of Big Endothelin-3, Big Endothelin-1 (Human), Endothelin-3, Endothelin-1 and Endothelin-2 (Human) diluted with Buffer E was added to an AET-30a binding microplate prepared by the sandwich method EIA IV. The reaction was performed at 4 ° C. for 24 hours. After washing with PBS,
HRP-labeled bET-31a prepared in VII-1, VII-2 above
Alternatively, 100 μl of bET-23a (diluted 200-fold with buffer C) was added, and reacted 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 (i). FIG. 4 shows the results when HRP-labeled bET-31a was used, and FIG. 5 shows the results when HRP-labeled bET-23a was used.
Shown in In the figure,-▲-is for Big Endothelin-3, -〇- is for Big Endothelin-1 (human),-●-
Is endothelin-3,-■-is endothelin-1,
-□-indicates the standard curve of endothelin-2. From the results shown in FIG. 4, the measurement method using HRP-labeled bET-31a is specific to big endothelin-3, hardly reacts with other endothelins (cross-reactivity, 0.1% or less), and the measurement sensitivity is high. 6 × 10 ^to 17 mol / well.
Similarly, from the results in FIG. 5, the assay using HRP-labeled bET-23a is specific for big endothelin-3 and hardly reacts with other endothelins (cross-reactivity,
0.1% or less). The measurement sensitivity is 8 × 10
^-16 mol / well.

VIII. Quantification of endothelin-3 in amniotic fluid After amniotic fluid (1 ml) was concentrated and pretreated with a Seppak C-18 cartridge, endothelin-3 was quantified by the sandwich method-EIA described in IV above. The method of pretreatment of amniotic fluid is shown in Scheme 1, and the measurement results are shown in Table 1. From the results in Table 1, 4.2 ± 3.9 pg / ml (mean ± S
D, n = 8) endothelin-3 was found to be present. IX. Detection of endothelin-3 in amniotic fluid by reversed-phase high-performance liquid chromatography (RP-HPLC). After pretreatment of 1.5 ml of amniotic fluid using a Seppak C-18 cartridge described in the above item V, the solution was concentrated under a stream of nitrogen. Next, the concentrate is used for eluent A described later.
After dissolving in 100 μl, it was separated by RP-HPLC.
The separation conditions are shown below. Column: ODS-80TM (4.6 mmφ × 250 mm, manufactured by Tosoh Corporation) Eluent A: 5% acetonitrile containing 0.05% -trifluoroacetic acid (TFA) Eluent B: 60% acetonitrile containing 0.05% TFA Elution method: Eluent The concentration of B was 0-40 during the first 5 minutes.
%, 40-65% in the next 20 minutes, and 65% in the next 5 minutes
Increase linearly to ~ 100%. Flow rate: 1 ml / min Fraction: 0.5 ml / tube The eluted fraction was centrifuged under reduced pressure, concentrated to dryness, dissolved in 250 μl of buffer E, and subjected to the sandwich method-EIA described in IV above. FIG. 6 shows the results. Since the immunoactivity of endothelin-3 in amniotic fluid was detected at the elution position of synthetic endothelin-3, it was confirmed that the sandwich method-EIA detected endothelin-3.

[0053]

Embedded image

[0054]

[Table 1]

X. Quantification of Endothelin-3 in Plasma of Healthy Subjects and Dialysis Patients Human plasma (1 ml) was concentrated and pretreated by the method shown in Scheme 1, and then endothelin-3 was quantified by the sandwich method-EIA described in IV above. 17 healthy people (male, 37.5 ± 4.9 years old) and 24 dialysis patients (male, 46.0 ±
FIG. 7 shows the measurement results of the plasma (7.0 years old). These results indicate that endothelin-3 in plasma can be quantified by this assay and that endothelin-3 in dialysis patients
This assay was shown to be clinically useful because of increased plasma levels of.

XI. RP-HPLC Detection of Endothelin-3 Immunoreactivity in Plasma of Healthy and Dialysis Patients After diluting 20 ml of healthy human plasma or 80 ml of plasma of dialysis patient with the same volume of PBS, AET-30-bound Sepharose 4B
Column (1 g of 1.5 mg of antibody was obtained by the method described in I-4)
Gel coupled to CNBr-activated Sepharose 4B
(Including 5 ml) at a rate of 10 ml / hour. 15 ml column
After washing with 3 ml of PBS, containing 3 ml of 0.05% TFA.
The mixture was eluted with a% acetonitrile solution and concentrated under a stream of nitrogen.
The concentrate was treated with 0.05% TFA and 0.05% CHAPS (3-
[(3-cholamidopropyl) dimethylammonio] 1-propanesulfonate) (3-[(3-cholamidopropyl) d
5% containing imethylammonio] -1-propanesultonate)
After dissolving with an acetonitrile solution, analysis was performed by RP-HPLC under the elution conditions described in IX. FIG. 8 shows the results. Since the immune activity of both the healthy person (A) and the dialysis patient (B) was detected at the elution position of the standard synthetic endothelin-3, the sandwich-EIA detected endothelin-3 in the plasma. It was confirmed that the excellent characteristics were obtained.

XII monoclonal antibody AET-30a
Investigation of the neutralizing activity of porcine A spiral strip of about 2 cm isolated from the porcine left coronary artery
The suspension was suspended in a Magnus tube filled with a Krebs-Henseleit solution (hereinafter referred to as a nutrient solution) under a gas mixture (95% O ₂ + 5% CO ₂ ) ventilation. After leaving at 37 ° C. for 3 hours, the tension generated by the contraction of the vascular smooth muscle was measured with an isometric transducer (Polygraph, manufactured by NEC Saneisha). AE of 20 times mole beforehand as a sample
Endothelin reacted with T-30a at 37 ° C for 20 minutes
When the -3 solution (final endothelin-3 concentration 1 × 10 ^-8 M) is added, the generated tension is 7.0% (n = 5) of the tension induced by the same concentration of endothelin-3. ,
When a solution prepared by reacting the control antibody H272-11 (anti-carcinoembryonic antigen) with endothelin-3 (final 1 × 10 ⁻⁸ M) was added, it was almost the same as endothelin-3 (121%, n =
The tension due to the contraction of 5) was observed. From the above,
AET-30a was found to neutralize endothelin-3 vascular smooth muscle contractile activity.

[0058]

Industrial Applicability The monoclonal antibody against endothelin-3 of the present invention has an extremely high binding ability and can neutralize the endothelin-3 vascular smooth muscle contractile activity. An immunological assay by the sandwich method using the monoclonal antibody and an antibody recognizing the C-terminal portion of endothelin-3 allows endothelin-3 to cross with endothelin-1, endothelin-2 or big endothelin-3 with high sensitivity. It can be quantified without reacting. In addition, the antibody can be used as a potent antagonist of endothelin-3 in various diseases associated with endothelin-3. In addition, the monoclonal antibody against the big endothelin-3 C-terminal peptide of the present invention has a wide specificity with respect to the big endothelin-3 C-terminus, and is therefore advantageous for detecting big endothelin-3 immunoreactivity. And a monoclonal antibody against endothelin-3 by immunoassay using a sandwich method.
It can be quantified without cross-reacting with 2, -3 and big endothelin-1.

[0059]

[Brief description of the drawings]

FIG. 1 shows a standard curve of endothelin-3 and endothelin-1 in a competition method using monoclonal antibody AET-30a-EIA.

FIG. 2: Sandwich method using monoclonal antibody AET-30a and anti-endothelin-3 C-terminal peptide antibody-endothelin-3 (-●-) in EIA,
Endothelin-1 (-▲-), endothelin-2 (-■
-), Human big endothelin-1 (-−１-), porcine big endothelin-1 (-□-), and big endothelin-3 (-△-) standard curves are shown.

Fig. 3 (A) Competition method using monoclonal antibody bET-31a-EIA, (B) Competition method using monoclonal antibody bET-23a-EIA, Big endothelin-3 (-▲-), Big endothelin- 1 (human) (-〇-), endothelin-3 (-●-), big endothelin-3 C-terminal peptide (22-42) (-■-), and big endothelin-3 C-terminal peptide (22-41)- The standard curve of NH ₂ (-□-) is shown.

FIG. 4 shows a sandwich method using monoclonal antibodies AET-30a and monoclonal antibody bET-31a.
The standard curves for-), endothelin-1 (-■-) and endothelin-2 (-□-) are shown.

FIG. 5 shows a sandwich method using monoclonal antibodies AET-30a and monoclonal antibody bET-23a.
The standard curves for-), endothelin-1 (-■-) and endothelin-2 (-□-) are shown.

FIG. 6 shows the results of separation by reverse-phase HPLC of human amniotic fluid after treatment with a Seppak C-18 cartridge and the results of detection by the sandwich method-EIA. Arrows indicate the elution positions of synthetic standard endothelin and big endothelin. Abbreviations: ET-3, endothelin-3; (a) oxide of human big endothelin-1; hbig-ET-1, human big endothelin-1; (b) oxide of endothelin-1; pbig-ET-1, Pig Big Endothelin-
1; ET-1, endothelin-1, and ET-2, endothelin-2.

FIG. 7: Endothelin in plasma of healthy subjects and dialysis patients
The measurement results of three concentrations are shown.

FIG. 8 shows the results of separation of plasma endothelin-3 immunoreactivity of healthy and dialysis patients by reversed-phase HPLC and detection by the sandwich method-EIA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12Q 1/28 C12P 21/08 G01N 33/53 D C12Q 1/28 33/577 B G01N 33 / 53 C12R 1:91) 33/577 (C12P 21/08 // (C12N 15/02 ZNA C12R 1:91) C12R 1:91) C12N 15/00 ZNAC (C12P 21/08 5/00 B C12R 1: 91) C12R 1:91)

Claims (19)

[Claims] 1. A monoclonal antibody having a binding property to endothelin-3 precursor. 2. The monoclonal antibody according to claim 1, wherein the precursor of endothelin-3 has one or more amino acids added to the N-terminal or C-terminal of endothelin-3. 3. The endothelin-3 precursor is a polypeptide having the sequence of formula 1, or big endothelin-3 represented by formula 2, or a part thereof.
Or the monoclonal antibody according to 2. Formula 1 Met Glu Pro Gly Leu Trp Leu Leu Phe Gly Leu Thr Val Thr Ser Ala Ala Gly Phe Val Pro Cys Ser Gln Ser Gly Asp Ala Gly Arg Arg Gly Val Ser Gln Ala Pro Thr Ala Ala Arg Ser Glu Gly Asp Cys Glu Glu Thr Val Ala Gly Pro Gly Glu Glu Thr Val Ala Gly Pro Gly Glu Gly Thr Val Ala Pro Thr Ala Leu Gln Gly Pro Ser Pro Gly Ser Pro Gly Gln Glu Gln Ala Ala Glu Gly Ala Pro Glu His His Arg Ser Arg Arg Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp Ile Asn Thr Pro Glu Gln Thr Val Pro Tyr Gly Leu Ser Asn Tyr Arg Gly Ser Phe Arg Gly Lys Arg Ser Ala Gly Pro Leu Pro Gly Asn Leu Gln Leu Ser His Arg Pro His Leu Arg Cys Ala Cys Val Gly Arg Tyr Asp Lys Ala Cys Leu His Phe Cys Thr Gln Thr Leu Asp Val Ser Arg Gln Val Glu Val Lys Asp Gln Gln Ser Lys Gln Ala Leu Asp Leu His His Pro Lys Leu Met Pro Gly Ser Gly Leu Ala Leu Ala Pro Ser Thr Cys Pro Arg Cys Leu Phe Gln Glu Gly Ala Pro Formula 2 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp Ile As n Thr Pro Glu Gln Thr Val Pro Tyr Gly Leu Ser Asn Tyr Arg Gly Ser Phe Arg X (X = Gly-OH or NH ₂ ) 4. It has a binding property to an endothelin-3 precursor,
And endothelin-3 C-terminal peptide, ie, Cys Hi
The monoclonal antibody according to claim 1, which does not react with the peptide represented by s Leu Asp Ile Ile Trp. 5. It has a binding property to an endothelin-3 precursor,
On the other hand, having the following amino acid sequence, endothelin-1 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp, endothelin-2 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr The monoclonal antibody according to claim 1, which does not react with any of Phe Cys His Leu Asp Ile Ile Trp and mouse endothelin-2 Cys Ser Cys Asn Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp. . 6. An endothelin-3 having a binding property to big endothelin-3 (formula 2) or a partial peptide thereof.
2. The monoclonal antibody according to claim 1, which does not react with. 7. Big endothelin having the sequence of formula 3
The monoclonal antibody according to claim 6, which reacts with a 3C-terminal peptide. Formula 3 Ile Asn Thr Pro Glu Gln Thr Val Pro Tyr Gly Leu Ser Asn Tyr Arg Gly Ser Phe Arg X (X = Gly-OH or NH ₂ ) 8. The mouse monoclonal antibody according to claim 7, which is designated as bET-31a. 9. The mouse monoclonal antibody according to claim 7, which is designated by bET-23a. 10. A hybridoma cell producing a monoclonal antibody having a binding property to endothelin-3 precursor. 11. The method according to claim 10, which is designated by bET-31.
The hybridoma cell as described. 12. The method according to claim 10, which is marked by bET-23.
The hybridoma cell as described. 13. A test solution and a labeled endothelin-3 precursor are reacted with an antibody against the endothelin-3 precursor competitively, and the ratio of the labeled endothelin-3 precursor bound to the antibody is measured. A method for quantifying an endothelin-3 precursor in a test solution, characterized in that: 14. An antibody against endothelin-3 precursor, and a test solution and a labeled big endothelin-3 or a labeled big endothelin-3 C-terminal peptide are allowed to competitively react with each other, and the labeled big endothelin-3 bound to the antibody is reacted. A method for quantifying an endothelin-3 precursor in a test solution, comprising measuring the ratio of endothelin-3 or labeled big endothelin-3 C-terminal peptide. 15. An anti-endothelin-3 insolubilized on a carrier.
After contacting the test solution with monoclonal antibody AET-30a or an antibody against endothelin-3 precursor, labeled endothelin-3 monoclonal antibody AET
Contacting an antibody against -30a or a labeled endothelin-3 precursor, and measuring the activity of the labeling agent on the insolubilized carrier;
3 Precursor quantification method. 16. One of the antibody insolubilized on the carrier and the labeled antibody is AET-30a, and the other is capable of binding to the endothelin-3 precursor,
3. A monoclonal antibody which does not react with No.3.
6. The method for quantifying endothelin-3 precursor according to 5. 17. An antibody against endothelin-3 precursor insolubilized on a carrier, and labeled endothelin-3
The method for quantifying an endothelin-3 precursor according to claim 16, wherein the antibody against the precursor 3 is the monoclonal antibody according to claim 7. 18. An antibody against endothelin-3 precursor insolubilized on a carrier, and labeled endothelin-3
The antibody against the precursor 3 is bET-31a or bET-
The method for quantifying an endothelin-3 precursor according to claim 16, which is 23a. 19. A BET labeled with an endothelin-3 monoclonal antibody AET-30a insolubilized on a carrier.
The method for quantifying endothelin-3 precursor according to claim 18, wherein -31a or bET-23a is used.