JPH04152894A - Monoclonal antibody against endothelin-3 or endothelin-3 precursor and use thereof - Google Patents

Abstract

NEW MATERIAL:IgG, IgA, IgM having affinity for endothelin-3 or endothelin-3 precursor or Fab or Fab fraction prepared by removing Fc' or Fc range therefrom or a polymer thereof. EXAMPLE:Mouse monoclonal antibody of AET-30a. USE:An antagonist of endothelin-3. PREPARATION:The antibody of the example is obtained by culturing hybridoma cell AET-30.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel antibody that is useful in that it has binding specificity for endothelin-3 and endothelin-3 precursors, such as big endothelin-3. For more details,
The present invention relates to the development of a method for measuring endothelin-3 and endothelin-3 precursors, such as big endothelin-3, based on antigen-antibody reactions, or antibodies useful for diagnosis and prognostic treatment of diseases involving endothelin-3.

[Conventional technology]

Endothelin-1 is a peptide consisting of 21 amino acids found in the culture supernatant of vascular endothelial cells, and has extremely strong and sustained vascular smooth muscle contracting activity and blood pressure increasing activity. Furthermore, analysis of the cDNA of endothelin-1 revealed that approximately 4 amino acids are present as intermediates in its biosynthetic process.
Big endothelin-1 consisting of 0 pieces (endothelin-
1 precursor) is also assumed. So far, monoclonal antibodies against endothelin-1 and big endothelin-1 have been produced, and a highly sensitive enzyme immunoassay method has been developed.
It has become possible to carry out a wide range of research aimed at elucidating the relationship with pathological conditions. It has already been revealed that endothelin-1 has a deep relationship with ischemic diseases by using a monoclonal antibody with neutralizing activity as a specific antagonist, and furthermore, using this measurement method, we have investigated plasma endothelin-level and pathological conditions. Detailed research is underway on the relationship between

On the other hand, analysis of chromosomal DNA revealed that endothelin-
The sequences of endothelin-2 and nindothelin-3 were found, and it was revealed that endothelin forms a gene family. In particular, endothelin-3, endothelin-1,
As shown in the example below, 6 of the 21 amino acid residues (see underlined) differ from endothelin-2, and its smooth muscle contraction and blood pressure increasing activities are also different from endothelin-1.
, has been reported to be considerably weaker than endothelin-2.

Endothelin-1 (pig, human. Sometimes called endothelin-α) Cys Ser Cys Ser Ser Leu M
etAsp Lys Glu Cys Val Tyr
PheCys His Leu Asp I le
I le Trp endothelin-2 (human) Cys Ser Cys Ser Ser Engineering" Positive Le
uAsp Lys Glu Cys Val Tyr
PheCys His Leu Asp I le
Ile Trp Endothelin-3 (rat, human. Sometimes called γ) Cys Pbe Thr
p Lys Glu Cys Val Tyr 1st C
ys His Leu Asp I le I le
From the analysis of Trp and endothelin-3 cDNA,
The precursor structure of endothelin-3 has been revealed (Japanese Patent Application No. 1-253797), and it has been suggested that endothelin-3 is synthesized using big endothelin-3 having the following sequence as a direct precursor.

Cys Thr Cys Phe Thr Tyr L
ysAsp Lys Glu Cys Val Tyr
TyrCys His Leu Asp I
le I Le Trpl 1e As
n Thr Pro Glu Gln Th
rVal Pro Tyr Gly Leu
Ser AsnTyr Arg Gly S
er Phe Arg This strongly suggests that they form a different receptor system from 1 and -2.
There is great interest in its physiological role.

[Problem to be solved by the invention]

As mentioned above, despite increasing interest in the physiological role of endothelin-3, until now little basic physiological information such as the expression site and plasma level of endothelin-3 has been obtained. . The main cause of this has been endothelin-3 or endothelin-3.
A monoclonal antibody that specifically recognizes endothelin-3 precursor has not been produced, and an immunoassay method that specifically and sensitively measures endothelin-3 or endothelin-3 precursor has not been developed. It will be done. These immunological methods are considered to be one of the most effective means for comprehensively conducting research on endothelin-3, particularly regarding metabolic pathways, secretion mechanisms, receptor systems, and relationships with pathological conditions. The establishment of this system was strongly desired from all walks of life.

[Means to solve problems]

The present inventors prepared a monoclonal antibody that has binding specificity for endothelin-3 or an endothelin-3 precursor, for example, big endothelin-3, and used the antibody to enhance endothelin-3 or big endothelin-3. We have developed an immunoassay method that can detect sensitively and specifically. Furthermore, it was discovered that the antibody has a unique pharmacological effect, that is, the antibody suppresses endothelin-3-induced contraction of smooth muscle of various animals. These findings indicate that the antibody can be used as a specific antagonist of endothelin-3, and that the antibody can be used as a prophylactic or therapeutic agent for various diseases that are causally or symptomatically related to endothelin-3. It shows that you can get.

That is, the present invention provides monoclonal antibodies that bind to endothelin-3 or endothelin-3 precursors,
a hybridoma cell producing the monoclonal antibody;
The present invention also relates to an immunoassay method for endothelin-3 and endothelin-3 precursors such as big endothelin-3 by a competitive method or a sandwich method using the antibody.

In the sandwich method for endothelin-3, the antibodies used in the first and second reactions may be polyclonal antibodies or monoclonal antibodies, respectively, but preferably one of them reacts with endothelin-3, but not with endothelin-3. An anti-endothelin-3 monoclonal antibody that does not react with the C-terminus of endothelin-3, and an anti-endothelin-3 polyclonal or monoclonal antibody that reacts with the C-terminus of endothelin-3 on the other hand, is used.

This latter anti-endothelin-3 polyclonal or monoclonal antibody that reacts with the C-terminus of endothelin-3, as seen from the amino acid sequence comparison with endothelin-102 mentioned above,
It contains the same antibody that reacts with the end, and is mentioned in Japanese Patent Application No. 1-46560.

Also, in the sandwich method for big endothelin-3, preferably one of the antibodies used in the first reaction and the second reaction is a monoclonal antibody that reacts with big endothelin-3 but does not react with endothelin-3. A monoclonal antibody, one of which reacts with endothelin-3, is used. Furthermore, among monoclonal antibodies that react with big endothelin-3 but do not react with endothelin-3, particularly preferably antibodies that have broad specificity for the structure of the C-terminal portion of big endothelin-3, such as the C-terminal An antibody that reacts to the same extent whether the sequence is Arg-Gly or Arg-NH is used.

The polyclonal antibody of the present invention is generally prepared by creating a complex between the immunizing antigen endothelin-3 or big endothelin-3 and a carrier protein.

An animal is immunized with this product, and a target antibody-containing substance is collected from the immunized animal, and the antibody is separated and purified.

In preparing the monoclonal antibody of the present invention, an individual with a high antibody titer is selected from the above-mentioned immunized animals, and the lungs or lymph nodes are collected 2 to 5 days after the final immunization.

Monoclonal hybridomas are obtained by fusing the antibody-producing cells contained therein with myeloma cells, selecting hybridomas that stably produce antibodies with high titers.

As the immunizing antigen, any of natural purified preparations, synthetic preparations, etc. can be used, and the aforementioned endothelin-3, big endothelin-3, and a portion of big endothelin-3 are used. Furthermore, a compound containing the structure of endothelin-3 or a compound containing a partial structure of endothelin-3 may be used as the immunizing antigen.

The various peptides used in the present invention can be produced by known conventional methods of peptide synthesis. Either a solid phase synthesis method or a liquid phase synthesis method may be used. For example, when endothelin-3 is synthesized by the in-phase method, Merryfield's solid phase peptide synthesis method [Journal of the American Chemical Society (J, Aa+, Chei, Soc, )] is used.
85, 2149 (1963)] is preferably used.

The insoluble resin may be any of those known in the art, such as chloromethylated styrene-divinylbenzene copolymer, phenacylacetic methylated styrene-divinylbenzene copolymer, etc. Examples include polystyrene type resins and polyamide type resins such as polydimethylacrylamide resins.

After binding the N-protected amino acid at the C-terminus to an insoluble resin, protected amino acids were sequentially combined from the C-terminal side of endothelin-3 according to a conventional method, and then treated with hydrogen fluoride,
Targeted endothelin by forming disulfide bonds
3 can be synthesized. As N-protected amino acids, all α-amino groups are protected by Boc groups, the hydroxyl groups of serine and threonine are protected by Bzl groups, the ω-carboxylic acids of glutamic acid and aspartic acid are protected by ○Bzl groups, and the E-amino groups of lysine are protected by Bzl groups. The group is CQ-Z group, the thiol group of cysteine is Acm group, M e B z 1 group, the hydroxyl group of tyrosine is Br-Z group, the imidazole group of histidine and the guanido group of arginine are Tos group, the indole group of tryptophan is CHO Preferably, it is protected with a group.

As a means of synthesis by liquid phase method, for example, "The Peptides J, Vol. 1 (19
1966), 5chroner and Lubke
Author, Academic Press.

New York, U, S, A, or "Peptide Synthesis", by Azumaya et al., Maruzen Co., Ltd. (1975), such as the azide method, chloride method, Wa anhydride method, mixed anhydride method, DCC method, active ester method, method using Woodward reagent K, carbodiimidazole method, redox method, DCC/additive (e.g., HONB
, HOBt, HO8u) method, etc.

Regarding the protein complex between an immunizing antigen and a carrier protein used to immunize salivary mammals, the type of carrier protein and the mixing ratio of the carrier and hapten are determined so that the antibody is effective against the hapten that has been coupled to the carrier and immunized. If possible, any substance may be coupled in any ratio; for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be coupled to 1 part hapten by weight.
A method is used in which the coupling ratio is 0.1 to 20, preferably 1 to 5.

Further, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldevit, carbodiimide, maleimide active ester, etc. are conveniently used.

The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, either by itself or together with a carrier or diluent, with subcutaneous injection being preferred. In order to increase antibody production ability during administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Administration is usually done once every 2 to 6 weeks.

Examples of warm-blooded animals used include monkeys, rabbits,
Examples include dogs, guinea pigs, mice, rats, sheep, goats, and chickens.

Antibodies can be obtained from blood, ascites (
preferably from blood). For example, when the immunogen is endothelin-3 or its partial peptide, the anti-endothelin-3 antibody titer in the antiserum can be measured by reacting the labeled endothelin-3 described later with the antiserum, and then reacting the antiserum with the antibody. This is done by measuring the activity of the bound labeling agent. Antibodies can be separated and purified using immunoglobulin separation and purification methods [e.g., salting-out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, adsorption/desorption method using an ion exchanger (e.g., DEAE), ultracentrifugation method, gel method. This is carried out according to a specific purification method in which only specific antibodies are collected by filtration, antigen-antibody conjugates, or active adsorbents, and the antibodies are obtained by dissociating the bonds.

The antibodies produced in this manner contain IgG as a main component and also contain other immunoglobulins such as IgM and IgA.

On the other hand, select warm-blooded animals, such as mice, that have been immunized in the same manner as in the preparation method for polyclonal antibodies above, and in which antibody titers have been observed, and collect the lungs or lymph nodes 2 to 5 days after the final immunization. Anti-endothelin-3 antibody and anti-endothelin-3 precursor antibody-producing hybridomas can be prepared by fusing the included antibody-producing cells with myeloma cells. The fusion operation can be performed using known methods, such as the method of Köhler and Milstein [Na
ture), 256.495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include N5-1, P3U1, S P 210, and P3tJ1 is particularly preferably used. The preferred ratio of the number of antibody-producing cells (lung cells) to the number of bone marrow cells used is about 1:1 to 20:1;
(Preferably PEG 1000 to PEG 600
0) is added at a concentration of about 10 to 80%, and 20 to 40%
Cell fusion can be carried out efficiently by incubating at 30-37° C. for 1 to 10 minutes.

Various methods can be used to screen for hybridomas producing anti-endothelin-3 antibodies or hybridomas producing anti-big endothelin-3 antibodies. Add hybridoma culture supernatant to a solid phase (e.g., microplate), then add anti-immunoglobulin antibody labeled with horseradish peroxidase (HRP) (if the cells used for cell fusion are mouse, anti-mouse immunoglobulin antibody) (used) or protein A to detect anti-endothelin-3 monoclonal antibodies bound to the solid phase.
L I S A (Enzyme-1 inked im
n+unosorbent assay) method, hybridoma culture supernatant is added to the same phase adsorbed with anti-immunoglobulin antibody or protein A, and HRP-labeled endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 is added. EI A (Enz
Examples include the yme immunoassay) method.

For selection and breeding of hybridomas, HAT (hypoxanthine, aminopterin, thymidine) is usually added to
Animal cell culture medium containing ~20% tallow serum (e.g. RP
MI 1640). 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.

Separation and purification of anti-endothelin-3 monoclonal antibodies or anti-big endothelin-3 antibodies is carried out in accordance with the method for separation and purification of immunoglobulins, similar to the separation and purification of polyclonal antibodies described above.

An anti-endothelin-3 polyclonal antibody that reacts with a partial region of endothelin-3 can also be prepared by immunizing with a peptide (partial peptide) corresponding to that partial region as a hapten and using the method described above. It can also be prepared from an anti-endothelin-3 polyclonal antibody prepared using it as a hapten, using affinity chromatography using a column bound to a peptide corresponding to one class of the antibody.

Furthermore, an anti-big endothelin-3 polyclonal antibody that reacts with a partial region of big endothelin-3 can also be prepared by immunizing with a peptide corresponding to that region using no)butene and using the method described above. It can also be prepared from an anti-big endothelin-3 polyclonal antibody prepared using Big Endothelin-3 as a hapten, using affinity chromatography using a column coupled with a peptide corresponding to one of the regions.

Also, endothelin-3 or big endothelin-
For example, selection of hybridomas that produce monoclonal antibodies that react with a partial region of 3 and hybridomas that produce monoclonal antibodies that react with endothelin-3 or big endothelin-3 but not with a partial region of This can be carried out by measuring the binding property between the peptide corresponding to the region and the antibody produced by the hybridoma.

Especially endothelin-3C-terminal peptide CysHis Le
To screen for monoclonals that recognize molecules other than u Asp I le I le Trp.

It is preferable to use sandwich-type EIA using a labeled antibody against the peptide. That is, a hybridoma culture supernatant was added to a solid phase adsorbed with an anti-immunoglobulin antibody or protein A, endothelin-3 was added, and an anti-endothelin-3 C-terminal peptide antibody was reacted with 1 m of HRP. This is an EIA method for measuring the above-mentioned stimulant activity.

Using the anti-endothelin-3 and anti-big endothelin-3 monoclonal antibodies obtained above, endothelin-3 measurement, tissue staining, etc. can be performed. The competitive method described below is usually used to measure endothelin-3 and big endothelin-3, but it is preferable to use the sandwich method described below.

In the competitive method, the anti-endothelin obtained in the present invention
3 or competitively react the anti-big endothelin-3 antibody with the test solution and labeled endothelin-3 or labeled big endothelin-3 or its partial peptide, and then measure the proportion of the labeling agent bound to the antibody. By doing this, endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 in the test solution is quantified.

Labeling agents for endothelin-3, big endothelin-3, partial peptides of big endothelin-3, or antibodies described below include radioactive isotopes, enzymes, fluorescent substances, luminescent substances, and the like. Examples of radioactive isotopes include 1zsI, 1311, 3H214
The enzyme is preferably stable and has a large specific activity, such as β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., and the fluorescent substance is preferably fluorescamine. Examples of luminescent substances include luminol, luminol derivatives, luciferin, and lucigenin. Furthermore, a biotin-avidin system can also be used to bind an antibody, endothelin-3, big endothelin-3, or a partial peptide of big endothelin-3 to a labeling agent.

In measuring the activity of the above-mentioned labeling agent, for example, in the case of measuring endothelin-3, labeled endothelin-3 bound to an antibody and free labeled endothelin-3 are separated (hereinafter abbreviated as B/F separation). ), but when an enzyme is used as a labeling agent, an antibody against anti-endothelin-3 antibody insolubilized in a reagent for this purpose 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, labeled endothelin-3 is transferred to the anti-I on the solid phase via the above-mentioned antibody that is reactive with the anti-IgG antibody.
This can be done by binding to gG antibody and measuring the labeling agent on the solid phase. When an enzyme is used as a labeling agent, a conventional colorimetric method or fluorescence method is used to measure the enzyme activity on the insolubilized carrier. When a non-protein substance such as a radioisotope is used as a labeling agent, B/F
In addition to the above-mentioned reagents, reagents such as an anti-endothelin-3 antibody that does not become insolubilized, sodium sulfate, dextran charcoal powder, and polyethylene glycol are used for the separation. In either method, the activity of the labeling agent in the supernatant or precipitate is measured.

For the above insolubilization, physical adsorption may be used,
Alternatively, a method using chemical bonding, which is usually used to insolubilize or immobilize proteins, enzymes, etc., may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicone, and glass.

In the competitive method, for example, in the case of measuring endothelin-3, the anti-endothelin-3 antibody, test solution, labeled endothelin-3, and B/F separation reagent can be reacted in any order. However, at least labeled endothelin-3 may be added to the reaction system at the same time as the reaction between the test solution and the anti-endothelin-3 antibody, or after a delay after the reaction. is preferred.

However, B/F separation reagents such as sodium sulfate, dextran charcoal powder, and polyethylene glycol are mainly used at the end of the reaction system.

On the other hand, in the Sand Inch method, a test solution is brought into contact with (reacted with) insolubilized anti-endothelin-3 or big endothelin-3 antibody (first reaction), and then labeled anti-endothelin-3 or big endothelin-3 antibody is contacted (reacted) with the sample solution. After reacting with big endothelin-3 antibody (secondary reaction), endothelin-3 in the test solution is measured by measuring the activity of the mi agent on the insolubilized carrier.
Alternatively, the amount of big endothelin-3 can be quantified.

The primary reaction and secondary reaction may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be similar to those described above.

The anti-endothelin-3 antibody or anti-big endothelin-3 antibody used in the secondary reaction is different from the anti-endothelin-3 antibody or anti-big endothelin-3 antibody used in the primary reaction. Antibodies having different binding sites from the antibody are preferably used. For example, in the case of measuring endothelin-3, if the antibody used in the first reaction has the ability to bind to the C-terminus of endothelin-3, in the second reaction,
Preferably, an anti-endothelin-3 antibody that binds to a region other than the C-terminus (e.g., N-terminus) is used, and when the antibody used in the primary reaction has the ability to bind to the N-terminus of endothelin-3, In the next reaction, an anti-endothelin-3 antibody that preferably binds to a region other than the N-terminus (eg, the C-terminus) is used.

The antibodies used in the primary reaction and the secondary reaction may be polyclonal antibodies or monoclonal antibodies, respectively, but in the case of measuring endothelin-3, for example, one of them preferably reacts with endothelin-3, but not with endothelin-3. Anti-endothelin that does not react with the C-terminus of 3.
3 monoclonal antibodies, the other being endothelin-
An anti-endothelin-3 polyclonal or monoclonal antibody that reacts with the C-terminus of 3 is used.

In the immunoassay method for endothelin-3 using the sandwich method, particularly preferred is the C-terminal peptide of endothelin-3, that is, Cys-His-Leu-Asp-
An anti-endothelin-3 polyclonal antibody that reacts with 11e-I le -Trp and an anti-endothelin-3 monoclonal antibody that reacts with endothelin-3 but not with the C-terminal peptide of endothelin-3 are used.

In the sandwich method immunoassay, both the solid-phase antibody and the labeling antibody may be of any class or subclass, and if the antibody activity is retained, the Fc' or Fc region may be removed from them. F(
It may be two fractions (ab'), two fractions (Fab'), or two fractions (Fab').

When using a monoclonal antibody in the sandwich method immunoassay, it is not necessary that only one type of antibody be used as the solid phase antibody or labeling antibody.
A mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity.

Furthermore, immunoassay using the antibodies obtained according to the present invention can be used for diagnosis and prognosis management of diseases involving endothelin-3.

Test samples include plasma, serum, urine, brain, cerebrospinal fluid, ascites,
Body fluids such as pleural fluid and amniotic fluid, as well as sputum can be used. These samples can be used as samples for immunoassay as they are, or after being diluted with various buffers or extracted and concentrated. Any buffer or organic solvent may be used as the solvent for diluting or extracting the sample, but preferably immunoassay buffer, water, physiological saline, acetate buffer, acetone, chloroform-methanol, or
These solutions containing surfactants are used.

In addition, the concentration may be performed by directly concentrating the sample under reduced pressure, normal pressure, or nitrogen flow, or after adding the sample to an ion exchange or reversed phase chromatography carrier or an anti-endothelin-3 antibody binding carrier. After elution under appropriate elution conditions, it may be concentrated under reduced pressure or normal pressure under a stream of nitrogen. Particularly preferably, the carrier for concentration is a C2, C8 or C18 cartridge for reverse phase chromatography. The concentrate is dissolved in an immunoassay buffer and used as an immunoassay sample.

Furthermore, the anti-endothelin-3 and anti-big endothelin-3 antibodies obtained in the present invention can be used in immunohistological staining of endothelin-3 and big endothelin-3. The method includes, for example, a direct method using a labeled anti-endothelin-3 or labeled bisoguentothelin-3 antibody, and an indirect method using a labeled anti-endothelin-3 antibody or big endothelin-3 antibody and an antibody against the antibody. It is possible to comply with laws, etc.

Furthermore, among the anti-endothelin-3 antibodies obtained in the present invention, antibodies that can neutralize the vasoconstrictor ability of endothelin-3 can be used as specific antagonists of endothelin-3.

As a method for screening anti-endothelin-3 antibodies for antibodies that specifically suppress the action of endothelin-3, any method that detects the pharmacological action of endothelin-3 can be used. 3. An in vitro measurement system that uses various vascular smooth muscle contractile activity as an indicator, such as pigs, rats, rabbits, guinea pigs, dogs, humans, etc., or the blood pressure increasing activity or blood pressure decreasing activity of endothelin-3 in the above animals as an index. Examples include biological measurement systems.

The obtained antibody that specifically suppresses the action of endothelin-3 may be of any class such as IgG, IgA, or IgM, or may be a Fab' or Fab fraction from which the Fc' or Fc region has been removed, or a polymer thereof. good. Furthermore, it is also possible to use a chimeric antibody obtained by fusing a variable gene region of a monoclonal antibody capable of specifically suppressing the action of endothelin-3 with a human immunoglobulin constant gene region and expressing the antibody as a recombinant.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the present invention should not be limited thereto.

Hybridoma cell AET producing the mouse monoclonal antibody AET-30a used in the Examples below
-30 is the accession number IF to the Institute of Fermentation Foundation (IFO).
○ It has been deposited as No. 50193 since July 7, 1999. In addition, the hybridoma was given accession number FERM B to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
It was deposited as P-2523 on July 20, 1999 based on the Budapest Treaty and is kept at FRI.

Hybridoma cell bET producing the mouse monoclonal antibody bET-31a used in the Examples below
-31 is the accession number IF to the Institute of Fermentation Foundation (IFO).
○ It has been deposited as No. 50247 since May 24, 1990. In addition, the hybridoma was submitted to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with accession number FERM.
June 1990 based on the Budapest Treaty as BP-2949
It has been deposited since April 12th and is kept at FRI.

Hybridoma cell bET producing mouse monoclonal antibody bET-23a used in the Examples below
-23 is the accession number IF to the Fermentation Research Institute (1FO).
○ It has been deposited as No. 50246 since May 24, 1990. In addition, the hybridoma was submitted to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with accession number FERM.
June 1990 based on the Budapest Treaty as BP-2948
It has been deposited since April 12th and is kept at FRI.

■, Preparation of #labeled anti-endothelin-30 end peptide antibody ■-1, Synthesis of peptide i) H-Cys-His-Leu-Asp-11e
-11e-Trp-OH (C-terminus of endothelin-3)
Synthesis of oc-11e-Trp-OBzl: H-Trp-
Dissolve 84g of OBzl-pTsOH in DMFloomQ, neutralize with 25ml of TEA, and then dissolve Boc-11e-0.
111/282048.9g, HONB 40.12
g.

Boc-11e-ONB prepared from 46.20 g of DCC
was added and stirred overnight. The solvent was distilled off under reduced pressure, and the residue was dissolved in 400 mQ of diethyl ether and 20 mQ of ethyl acetate, and dissolved in 0.5N HCQ, 5%-N a HCO3.
It was washed with water, and then dried with anhydrous MgSO4. The solvent was distilled off, petroleum ether was added to the residue, and the crystals were collected by filtration. Yield 83.1g (90.9%) [α] 18-
19.7' (c = 0.99 MeOH) Elemental analysis: C2, H,, N, ○, calculated value: C, 68,
62; H, 7, 35; N, L2g actual value: C, 68
, 69:H, 7, 35:N, 8.16Boa-11e
-11e-Trp-OBzl: Boc-11e-T
rp-OBzl 53゜3g in dioxane 120m
Dissolved in 7.1N-HCQ-dioxane 1 under water cooling.
60mQ was added 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. Add this to 150m of acetonitrile Q and D M F
Dissolved in a mixed solvent of 250mQ and TEA under water cooling.
After neutralization with 14.6 mQ, the precipitated TEA-HCQ was filtered off. 845.33 g of Boa-11e-0 was added to the filtrate and stirred overnight. The solvent was distilled off and the residue was converted into CHCQ.
Dissolved in IQ, 0.5N-HCQ, 5%-NaHC
Washed with O, water and dried with anhydrous MgSO4. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitate was collected as crystals by filtration, washed with acetonitrile and diethyl ether, and dried.

Yield 54.95g (84.3%) (αl 11'-12,0@(c=0.98.CHC
Q,) Elemental analysis: Calculated values as C3sH4I, N40G: C, 67,72; H, 7,79; N, 9.
03 actual measurement value: C, 67,72; H, 7,91; N
, 8.87Boc-Asp-(OBzl)-11e-
11e-Trp-OBzl: Boc-11e-Il
49.7 g of e-Trp-OBzl was diluted to 10% under water cooling.
It was dissolved in 250 mQ of TFA containing -1,2-ethanedithiol and left at room temperature for 15 minutes. The solvent was distilled off, 20 mQ of 4N-HCQ-dioxane was added to the residue, and the mixture was thoroughly stirred, diethyl ether was added, and the precipitate was collected by filtration. This was dissolved in DMF250mQ, and T
E A11.1m TEA precipitates after neutralization with Q
-HCQ was filtered off. Boc-Asp-(OBz
l)-0H31,Og, HONB 20.6g, D
Boc-Asp-(OBz) prepared from CC23.8g
l)-ONB was added and stirred overnight.

The solvent was distilled off under reduced pressure, and 800 mQ of ethyl acetate was added to the residue to dissolve it, followed by 10% citric acid water, 5% NaHCO3,
Washed with water and dried over anhydrous MgSO4. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, the precipitate was collected as crystals by filtration, and recrystallized from acetonitrile.

Yield 54.0g (81.7%) (α)”-13.7° (c = 1.04.C
HCQ,) Elemental analysis: Calculated values as C, GH,, N, ○, C, 66, 89: H, 7, 20: N,
8.48 Actual value: C, 66, 69: H,? , 23
: N, 8.31Boc-Leu-Asp(OBzl
)-11e-11e-Trp-OBzl: Boc-
Asp(OBzl)-11e-11e-Trp-OBz
l 48. Og was dissolved in TFA 210mQ containing 10%-1,2-ethanedithiol under water cooling and left at room temperature for 15 minutes. The solvent was distilled off, and the residue was diluted with 4N-H.
After adding 14.5 m of CQ-dioxane Q and stirring well, diethyl ether was added and the precipitate was collected by filtration. This was dissolved in DMF300mQ and T E A42 under ice cooling.
After neutralization with mQ, precipitated TEA-HCQ was filtered off, 27.3 g of Boc-Leu-ONB was added to the filtrate, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure, and the residue was dissolved in CHCn.
, add 600 m
Dry with O4. Petroleum ether was added to the residue, and the precipitate was collected by filtration and recrystallized from hydrous acetonitrile. yield
53.1% (97.5%) (α) ”-20.6” (
c=1.02. CHCQ,) Elemental analysis: C,2H7
゜NGO.・Calculated value as 1/2H20: C, 6
5,87; H, 7,55; N, 8.86 Actual value:
C, 66,08; H, 7,58: N, 8.78
Boc-His-Leu-Asp(OBzl)-11e
-11e-Trp-OBzl: B.

c-Leu-Asp (OBzl)-11e41e-T
rp-OBzl 46.5g under water cooling at 10%-1,2
-Dissolved in ethanedithiol-containing TFA220mQ and left at room temperature for 15 minutes. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the residue was collected by filtration. DMF25mQ
After adding 20 mQ of TEA and stirring well,
500 mQ of diethyl ether was added and the precipitate was collected as a powder by filtration. Dissolve this in 200ml of DMF and
c-His (T.

5)-0) 122.3g, 1(ONB 11.7g,
Boc-)1is (T
os)-ONB was added and stirred overnight. Distill the solvent,
Acetonitrile was added to the residue, and the powder was collected by filtration.

Yield 45.9g (86.2%) [a) 1s16.6° (c=1.o, DMF) Elemental analysis: CS 8 H7t N qo, 1・2.5H20
Calculated values as: C, 62, 13; H, 7, 37:
N, 11.24 Actual value: C, 62,25; H,
7,02; N, 11.03Boc-Cys(MeB
zl)-His-Leu-Asp(OBzl)-11e
-11e-Trp-OBzl: Boa-His-L
eu-Asp(OBzl)-11e-11e-Trp-
17.2 g of OBzl was dissolved in 88 mQ of TFA containing 10%-1,2-ethanedithiol under water cooling, and the mixture was dissolved at room temperature for 15 minutes.
Leave it for a minute. The solvent was distilled off under reduced pressure, ether was added to the residue, the powder was collected by filtration, and then dissolved in DMF25IIIQ. After adding 25 mQ of TEA to this and stirring well,
Ether 500mQ was added and the precipitate was filtered as a powder. This was dissolved in DMF40mQ, and Boc-Cys
(MeBzl) -OH5,72g, )IONB
Boc prepared from 3.48g, DCC4゜00g
-Cys(MeBzl)-ONB was added and stirred overnight. The solvent was distilled off, acetonitrile was added to the residue, and the precipitate was collected as a powder by filtration, further washed in hot acetonitrile, and after cooling to room temperature, the powder was collected by filtration.

Yield 15.0g (72.9%) [α]Il; 1l-23.3° (c= 1.02.D
MF) Elemental analysis: CG, H9, N,. 0.2S
-Calculated value as 3H20: C, 61, 96;, H,
7,23; N, 10.47; S, 2.40 Actual value: C, 62,24: H, 6,90; N,
10.49; S, 2.53 H-Cys-His-Leu-Asp-11e-11e
-Trp-OH: Boa-Cys(MeBzl)-H
is-Leu-Asp (OBzl) (le-11e-
Trp-OBzlloomg under water cooling, 10%-1,2
- It was dissolved in 1 mM of TFA containing ethanedithiol, and after being left at room temperature for 15 minutes, the solvent was distilled off under reduced pressure. Ether was added to the residue, the powder was collected by filtration, dried, and then treated with 0.5 m m of m-cresol in 5 m Q 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 Cef7 was filled with the same solvent.
Dex LH-20 (2.5 x 90an) (7) A section of 160 m2 to 180 mm was collected and freeze-dried.

Yield 23■ (32.8%) Amino acid analysis value; Asp 1. (1(1), Cys
O, 80(1).

11e 2.13(2), Leu O,99(1
).

His 1.07 (1), Trp O, 67 (1)
.

Average recovery rate 90.5% ii) H-Arg-His-Leu-Asp-11
Synthesis of e-11e-Trp-OH (similar to endothelin-3C end, highly soluble) oa-His-Leu-As
p(OBzl)-11e-11e-Trp-OBzl
538■ was dissolved in TFA 5 mΩ containing 20%-1,2-ethanediothyl under water cooling, and after being left at room temperature for 15 minutes,
After adding and dissolving PTsOH-H2095, the solvent was distilled off under reduced pressure. The residue was dissolved in 5 mQ of DMF, and 0.75 mQ of DMF containing 10%-TEA was added under water cooling for neutralization. To this, Boc-Arg (No□)-0H60
mg, 50 μg of HONB, and 50 μg of WSCD-HCQ were added and stirred overnight. The solvent was distilled off under reduced pressure, water was added to the residue, and the precipitate was collected by filtration.

After drying this, it was suspended in hot acetonitrile 10 mρ,
After cooling to room temperature, the precipitate was collected by filtration.

Yield 0.5g (78.3%) Of this, 102■ is anisole 0.2m Q, 102-
The mixture was treated with HF together with 0.2 mM ethanedithiol for 60 minutes at ○C, and the HF was distilled off under reduced pressure. After washing the residue with diethyl ether, it was dissolved in 6 m Q of 50% acetic acid and washed with Sephadex G-25 (2,5 x 9
0C11) (Developed with 71 columns, 100-13
A 7mQ section was collected and lyophilized.

Yield 20■ (26.3%) Amino acid analysis value: Asp O, 98 (1), Ile
1.95(2).

Leu 1. Of+), His□, 95(1).

Arg O, 98 (1), Trp 0.72 (1).

Average recovery rate 89.7% 1-2 Preparation of immunogen Polypeptide Cys H obtained in I-1(i) above
A condensate of is Lau 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, B5A20a+g was dissolved in 1.4mM of 0.1M phosphate buffer, pH 7.0, and 100μQ of a DMF solution containing 2.611Ig of N-(γ-maleimidobutyryloxy)succinimide (hereinafter abbreviated as GMBS) was added. The mixture was mixed and reacted at room temperature for 40 minutes. After the reaction, the mixture was cleared and fractionated using a Sephadex G-25 column equilibrated with 0.1 M phosphorus a buffer, pH 6.5, containing 2.5 mM EDTA. Next, B5A with maleimide group introduced
1.2 mQ of the elution fraction containing 3.4■ and the polypeptide Cys H□5Leu Asp dissolved or dispersed in 1.2 mM of an aqueous solution containing 90% dimethyl sulfoxide.
IIeIle Trp 1.8m< was reacted at 4° C. for 3 days. After the reaction, the mixture was dialyzed against physiological saline at 4°C for 2 days.

1-3 Immunology above? 550 μQ of complete Freund's adjuvant (550 μQ) was added to 450 μQ of physiological saline containing 1 μg of immunogen 60 (1 μg) obtained in Example 2.
), mix well to create an emulsion, and apply it under the rabbit's skin for about 2 minutes.
Inoculated at 0 locations. Six weeks later, an emulsion was prepared in the same manner using incomplete Freund's adjuvant and inoculated subcutaneously into rabbits. From then on, repeat this operation 4 times every other month.
Seven days after the booster immunization, blood was partially collected from the rabbit and antiserum was obtained using a conventional method.

Preparation of I-4 Affinity Solid Phase Polypeptide Arg His Leu Asp Il
e Ile Trp was directly coupled to CNBr-activated Sepharose 4B as an affinity solid phase.

That is, Arg His Leu Asp Ile I
Dissolve 1.5 mg of le Trp in 0.1 M sodium bicarbonate containing 10 mA of 0.5 M common salt, and add 1 g of
of CNB r-activated Sepharose 4B at room temperature for 3 hours. Next, the unreacted active groups were treated with 0.1M)-Lis-HCl buffer (pH 8), and then dispersed in PBS.
Stored at 4°C.

Purification of anti-endothelin-3 C-terminal peptide antibody using I-5 affinity solid phase Antibody was partially purified from the 16 mΩ rabbit antiserum described in 1-3 above by ammonium sulfate salting out method. That is, antiserum 10 m
Add P B S 10m to Q and add 16.5m
Saturated ammonium sulfate of Q was gradually added with stirring (final 45%
). After leaving it for 30 minutes, 20 at 12,0OOX g
The mixture was centrifuged for a minute, and the precipitate was dissolved in PB610mQ.

Next, saturated ammonium sulfate was added in the same manner to a final saturation of 30%, followed by centrifugation. The precipitate was mixed with 0.0
．． OIM borate buffer, PH8 (hereinafter abbreviated as BBS),
0.0 containing 0.01M salt after dissolving in 10mQ
For 1M phosphate buffer pH 8 (Buffer B).

Dialysis was performed at 4°C for 2 days. After the antibody fraction was dialyzed against BBS, it was applied to a column (10 naφ x 40 m) packed with affinity homophase described in 1-4 above. After thorough washing with BBS, the specific antibody was dissolved in 011M acetate buffer containing 0.5M NaCl, P)I 4.5, and then 0.05M glycine-HCl buffer containing 0.1M NaCl, pH 2. ,0
It was eluted.

The eluate was neutralized and then dialyzed against BBS.

Specific antibodies were found in both fractions eluted at pH 2, and 18 μg of specific antibodies were obtained from both fractions.

I-6 Preparation of horseradish peroxidase (HRP) labeled anti-endothelin-30 end peptide antibody Affinity purified anti-Cys His described in 1-5 above
From the Leu Asp Ile Ile Trp antibody, Ishikawa et al.'s method [Journal of Applied Biochemistry (J, Appl, Biochem), 6:
56-63 (1984))
P jlA11! The body was created.

That is, 160 μg of pepsin (Sigma, twice crystallized) was added to 6.4+ng of the specific antibody dissolved in 0.1M acetate buffer, pH 4.5, and the mixture was reacted at 37° C. for 16 hours.

Both F(ab') fractions were purified by FPLC (manufactured by Pharmacia) using a Superose 12 column equilibrated with BBS. Both aliquots were dialyzed against 0.1M acetate buffer, pH 5, and then a final 20mM of β-mercaptoethylamine was added and left at 37°C for 90 minutes. 2.5m of reaction solution
0.1M phosphate buffer containing MEDTA, PH6.0
FPL using a Superose 12 column equilibrated with
The Fab' fraction was obtained.

On the other hand, horseradish peroxidase 5mg is 0.911
Dissolved in Q's 0.1M phosphate buffer, pH 7, 50μ
51.05 mg of GMB dissolved in Q's DMF
was added and reacted at room temperature for 40 minutes.

The reaction solution was transferred to a Sephadex G-25 column (eluent 0.1
M phosphate buffer, pH 6, 8), 3.5 µm of maleimidated peroxidase obtained and 0.8 µm of the above Fab' fraction were mixed, and the mixture was separated with collodion bag (Munis Kiki Co., Ltd.) to approximately 0.0 µm. After concentrating to .3 mQ, 1
It was left for 6 hours. The reaction solution was applied to an Ultrogel AcA44 column (10 mw++φX40 mm) using 0.1 M phosphate buffer, pH 6.5, as an eluent to purify the Fab'-peroxidase complex fraction.

(2) Preparation of monoclonal anti-endothelin-3 antibody 11-1 Preparation of immunogen Endothelin-3 and bovine thyroglobulin (TG) were condensed by the maleimide crosslinking method described below to prepare an immunogen.

Namely, endothelin-3 (purchased from Peptide Institute)
265 nmole in 450 μM 0.1 M phosphate buffer.

Dissolve in pH 7.0 (containing 10% DMF).

It was mixed with 50 μQ of a DMF solution containing 6 μn+ole of GMBS 6 and reacted at room temperature for 30 minutes.

On the other hand, 0.15 mg (40 nmole) of TG
0.02M phosphate buffer containing M NaCl, pH 6,8
,1,4m Q, N-succinimidyl-3-
(2-pyridyldithio)propionate (hereinafter 5PDP
(abbreviated as) DM containing 2.5■ (8.0 μmole)
After mixing 100 μQ of F solution, the mixture was allowed to react at room temperature for 40 minutes. After the reaction, dithiothreitol 12.4μ (80μ
0.1M acetate buffer containing mole), pH 4, 5, 0
, 5m Q were added, and the mixture was allowed to react at room temperature for 20 minutes, followed by fractionation using a Sephadex G-25 column to obtain TG12 (24 nmole) into which an SH group had been introduced.

Next, maleimide group-introduced endothelin-3190 nmol
e and 5.9 n mole of SH group-introduced T G were mixed.

After reacting at 4℃ for 2 days, the temperature was increased to 4℃ against physiological saline.
, dialyzed for 2 days.

If-2 immunization 6-8 week old BALB/C11i! ! Mice were subcutaneously immunized with 100 μg/mouse of the immunogen described in ①-1 above together with an adjuvant. Thereafter, booster immunizations were performed 2 to 3 times every 3 weeks.

Preparation of n-3HRP41m endothelin-3 Dissolve 380 nmole of endothelin in 450 μQ of 0.1 M phosphate buffer, pH 7.0, and add 295 μg of GMBS.
(2,0p mole) of DMF solution 50μ12
and reacted at room temperature for 30 minutes. After the reaction, fractionation was performed using a Sephadex G-15 column to obtain a polypeptide 60 nInole into which a maleimide group had been introduced.

On the other hand, HRP long (250nl!ole) was added to 0.02M phosphate buffer containing 0.15M salt, pH 6.
,8,1,4m12 and S P D P 1.
DMF solution containing 17 mg (3,75 μmole) 10
After mixing 0 μQ, the mixture was allowed to react at room temperature for 40 minutes. After the reaction, 12.4 mg of dithiothreitol (80 p mole
) containing 0.1M acetate buffer, pH 4,5,0,5m
After reacting at room temperature for 20 minutes, fractionation was carried out using a Sephadex G-25 column to obtain 6 µm (150 nmole) of the enzyme into which an SH group had been introduced.

Next, maleimide group-introduced endothelin-350n mo
le and SR group-introduced peroxidase 20 n mole
were mixed and reacted at 4°C for 16 hours. After the reaction, fractionation was performed using an Ultrogel AcA44 (LKB-Pharmacia) column, and peroxidase-reduced endothelin-3
I got it.

11-4 Cell Fusion Mice that showed relatively high antibody titers were given a final immunization by intravenously inoculating 240 μg of immunogen dissolved in 0.25 mQ of physiological saline. Three days after the final immunization, the lungs were removed from the mice, compressed with a stainless steel mesh, filtered, and suspended in Eagle's Minimum Essential Medium (MEM) to obtain a lung cell suspension. BALB/C as cells used for cell fusion
Mouse-derived myeloma cells P3-X 63. Ag8.
Using Ul (P2H4) [Current Tobacco In Microbiology and Immunology, 81.
1 (1978)). Cell fusion is described in the original note [Nature,
256°495 (1975)). That is, lung cells and P2H4 were each washed three times with serum-free MEM, and the ratio of lung cells and P3H1 numbers was adjusted to 5.
:1, and centrifuged at 800 rpm for 15 minutes to precipitate the cells. After thoroughly removing the supernatant,
Lightly loosen the precipitate and add 45% polyethylene glycol (P
EG) 6000 (manufactured by Kotsuholite) at 0°3+n
Q was added and allowed to stand for 7 minutes in a 37°C hot water bath to perform fusion. After fusion, add MEM to the cells at a rate of 2 mQ/min.
After adding a total of 12 mQ of MEM, the mixture was centrifuged at 600 rpm for 15 minutes and the supernatant was removed. This cell precipitate was mixed with GIT medium (Wako Pure Chemical Industries, Ltd.) containing 10% beef tallow serum (GI
P2H4 was suspended in 2×10' particles per 1 mg of P2H4 (T-10Fcs).

1 m in 24-hole multi-dish (manufactured by Linpro)
Q was seeded in 120 wells. After seeding, hold the cells at 37°
The cells were cultured at 5% carbon dioxide in a furan vessel. HA after 24 hours
T (hypoxanthine I x 10'''' 4M, aminobritelin 4 x 10'''' 7M, thymidine 1.6 x 10-3
GIT-10FC5 medium (HAT medium) containing M)
By adding 1 mg per well, HAT
Selective culture was started. HAT selective culture is performed at the start of culture 3.
After 6.9 days, 1rnQ of the old solution was discarded, and the incubation was continued by adding 1rr+Q of HAT medium. Hybridoma proliferation was observed 9 to 14 days after cell fusion, when the culture medium turned yellow (approximately 1X10' cells/mQ).
The supernatant was collected, and the antibody titer was measured by the EIA method described below.

Screening of n-5 Hybridoma Antibody titers in hybridoma culture supernatants were measured by the following two methods. Anti-mouse immunoglobulin antibody-bound microplates were used in both methods. The plate was first coated with anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel).
O, 1M carbonate buffer containing 20μg/lQ, pH 9.6
The solution was dispensed into 96-well microplates in 100 μQ portions and left at 4° C. for 24 hours. Next, the plate is PB
After washing with S, PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) was added to block excess binding sites in the wells.
was dispensed into 300 μΩ portions and treated at 4° C. for at least 24 hours.

1) EIA method using HRP 41 fragile endothelin-3 Buffer E (10% Block Ace, 2 mg/mQ
BsA, 0.4M N a CQ, 2mM EDTA
50 μQ of 0.02 M phosphate buffer (pH 7.0) containing 0.1% NaN and 50 μΩ of hybridoma culture supernatant were added, and the mixture was allowed to react at room temperature for 4 hours. After the reaction,
After washing with PBS, the HRP prepared in 11-3 above
s recognition endothelin-3 [0.02M containing 1% BSA
IJ acid buffer, pH 7 (Buff 77-A).

0-fold dilution] 100 μQ was added and reacted at 4° C. for 16 hours. After the reaction and washing with PBS, 0.2% orthophenylenediamine and 0.2% orthophenylenediamine were added to measure the enzyme activity on the same phase.
0.1M citrate buffer containing 0.2% hydrogen peroxide, pH
5, 5 was dispensed into 100 μΩ portions and allowed to react at room temperature for 10 minutes. After adding 100 μΩ of 4N sulfuric acid to stop the reaction, the absorption at 492 nm was measured using a plate reader (MTP-3).
2, manufactured by Corona Corporation).

When the supernatants of all 120 wells in which hybridoma growth was observed were examined in this way, no.

Strong antibody titers were detected in wells No. 5 and No. 93.

n) EIA method using HRP-labeled endothelin-3 C-terminal peptide antibody Anti-mouse immunoglobulin antibody binding 50μQ of buffer E and 50μ of hybridoma culture supernatant were added to the microplate.
Q, and 6 ng/m Q of endothelin-3
50 μQ of buffer E solution containing was added, and the mixture was allowed to react at room temperature for 4 hours. After washing the plate with PBS, proceed as above 1.
-6 HRP-labeled endothelin-30 end peptide antibody [Buffer C (1% BSA, 0.4M N
a 100 μQ diluted 300 times with CQ, 0.02 M phosphate buffer containing 2 mM EDTA, pH 7,2) was added, and the mixture was allowed to react at 4° C. for 16 hours. Next, plate the plate in PBS
After washing with
Measured by the method described.

When the supernatants of all 120 wells in which hybridoma growth was observed in this manner were examined, no.

Strong antibody titers were detected in 93 wells.

11-6 Cloning No. 5 and No. 5 showed positive antibody activity.

Each hybridoma in 93 wells was subjected to cloning by limiting dilution. That is, 1.5 hybridomas/
The cells were suspended in RPM11640-20FC8 to a concentration of 0.2 mQ per well and dispensed into a 96-well microplate (manufactured by Nunc). During dispensing, BALB/C mouse thymocytes were added as feeder cells at 5×10′ cells per well. After about a week, cell proliferation was observed, and the antibody titer in the supernatant was examined by the EIA method described in 11-5 above.
Eight out of 41 clones of the o.5 hybridoma and 7 out of 41 clones of the no.93 hybridoma produced antibodies.

Among these clones, we focused on clone AET-30 obtained from No. 93-18 and the monoclonal antibody AET-30a produced thereby, and conducted the following experiments.

n-7 Preparation of a large amount of monoclonal antibody Hybridoma AET-301~3X10' cells/mouse were intraperitoneally administered to mice intraperitoneally administered with 0.5 m Q of mineral oil or to untreated mice (B A L'B/'C). Antibody-containing ascites fluid was collected 10 to 30 days after the intravenous injection.

(1) Purification of monoclonal antibody-8 Monoclonal antibody was purified from the ascites prepared above from U-7 using a protein-A column.

That is, 8 mQ of ascites was mixed with an equal volume of binding buffer (3,5M NaC
Q, 1.5M glycine with 0.05% NaN3, pH
The specific antibody was diluted with elution buffer (0.05% N
0.1 M citrate buffer, pH 3, containing a N )
0) was eluted. Through the above operations, 40 μ specific antibodies were obtained.

11-9 Determination of class/subclass of monoclonal antibody A 0.1 M carbonate buffer, pH 9.6 solution containing 1 μg/mQ of the above n-8 purified monoclonal antibody was dispensed into a 96-well microplate in 100 μα portions, and incubated at 4°C for 24 hours. I left it for a while. Following the method described in n-5 above, block the excess binding sites in the wells with BlockAce, and then use an isotype typing kit (Mouse-TyperT).
Classes and subclasses were investigated by enzyme linked immunosorbent assay (ELISA) using MSub-Isotyping Kit (manufactured by Biorat). As a result, AET-30a was IgG1. was found to belong to the class.

■, Competitive method - EIA Add 50 μQ of AET-30-containing culture supernatant diluted 75 times with buffer C to the anti-mouse immunoglobulin antibody-bound microplate described in 1-5 above, and 50 μQ of endothelin-1 standard solution or endothelin-3. After adding 50 μQ of the standard solution and reacting at room temperature for 1 hour, the above l
HRP-labeled endothelin-3 described in 1-3 (100-fold diluted with buffer A) was added and reacted at 4°C for 16 hours. After the reaction, after washing with PBS, the enzyme activity on the same phase was removed.
It was measured by the method described in 1-5(i). The results are shown in Figure 1. In the figure, -.- indicates the standard curve of endothelin-3, and -0- indicates the standard curve of endothelin-1.

From the results shown in Figure 1, AET-30a is endothelin-3
It was found that the enzyme reacted with the endothelin-1 and did not react with the endothelin-1.

■, Sandwich method - EIA Purified monoclonal antibody AET-30a 20
0.1 M carbonate buffer containing ug/m Q, pH 9
, 6 solution was dispensed into a 96-well microplate in an amount of 100μQ and left at 4°C for 24 hours. Excess binding sites in the wells were inactivated by adding 300 μQ of Block Ace (manufactured by Snow Brand Milk Products Co., Ltd., sold by Dainippon Pharmaceutical Co., Ltd.) diluted 4 times with PBS.

Add endothelin-3, endothelin-10, endothelin-2 (human), big endothelin-1 (human), big endothelin-1 (pig), and big endothelin-3 (22- 42) 100 μQ of (human) standard solution was added and reacted at 4° C. for 24 hours. After washing with PBS,
HRP-labeled endothelin-3C prepared in 1-6 above
End peptide antibody (300-fold diluted with buffer C) 10
0 μQ 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 ①-5(i) above. The results are shown in Figure 2. In the figure,
-・- is endothelin-3, -mu is endothelin-1, -■- is endothelin-2 (human), -0
- indicates the standard curve of big endothelin-1 (human), -8- indicates big endothelin-1 (pig), and -low indicates big endothelin-3 (human).

From the results shown in Figure 2, this measurement method is specific for endothelin-3, and it is possible to detect endothelin-34 x 10-17 mol/well with almost no reaction with other endothelins (cross-reactivity, 0.1 % or less), it was found that it was detectable.

■ Preparation of monoclonal anti-big endothelin-30 end peptide antibody ■ Synthesis of -16 peptide (1) Big endothelin-3 (big ET-3) (
22-42);H-11e-Asn-Thr-Pro-
G 1u-Gin-Thr-Va 1-Pro-Tyr
-G 1y-Leu-5er-A sn-Tyr-A
rg-G 1y-5e r-Phe-A rg-G 1
Synthesis of y-OH Commercially available Roe-Gly-OCH2-PAM resin (manufactured by Abride Biosystems) 0.77 g (0.5
using a peptide synthesizer (applied
Synthesis was performed using Biosystems Model 430A).

After treating the Boa group on the resin with 50% trifluoroacetic acid/methylene chloride to liberate the amino group, the amino group is treated with Boa-Arg(Tos), Boc-Phe,
Boc-5er (Bzl), Boa-Gly, Bo
c-Tyr(Br-Z), Boc-Asn, Boc
-Leu, Boc-Pro, Boc-Val, B
oc-Thr(Bzl), Boc-Gin.

Boc-Glu (OBzl), Boc-11e bi
g H in order according to the amino acid sequence of ET-3 (22-42)
It was activated and condensed with OBt/DCC.

After further condensation with the same amino acid derivative activated with DCC or HOBt/DCC, unreacted amino groups were acetylated with acetic anhydride to form the protected big ET.
-3(22-42)-0CH2-PAM resin 0.88
I got g.

After treating 0.35 g of this resin with 5 ml of anhydrous hydrogen fluoride in the presence of 0.70 g of p-cresol at 0°C for 60 minutes,
Hydrogen fluoride was distilled off under reduced pressure, and the residue was dissolved in 5 ml of ethyl ether.
After washing twice with , the residue was extracted with 50% aqueous acetic acid. Insoluble materials were removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing liquid were combined, concentrated under reduced pressure to 2 to 3 ml, and applied to a Sephadex LH-20 (2x90an) column.
Elute with 50% acetic acid. The main fractions were collected and concentrated to 0.1
% trifluoroacetic acid water, YMC-O
DS AM120 5-50 resin column (2,6X7
c++), 0.1% trifluoroacetic acid water and 50%
Elution was performed with a linear gradient between acetonitrile (containing 0.1% trifluoroacetic acid).

The main fractions were combined and lyophilized to obtain 76 ml of white powder.

Amino acid analysis value Asp 1.97 (2), Thr 1.76 (2),
Set 1.68 (2), Glu 2゜04 (2)
, Gly 3,00 (3), Val 1.00 (1
), Pro 1.90(2).

11e 0.95(1), Leu O,99(1),
Tyr O, 95 (1), Phe 1002 (1)
, Arg 2,27(2) by mass spectrometry (M +
H) 2356.16 HPLC elution time 17.0
1 minute column condition Column: YMC-ODS (AM-301, S-51
2OA) Eluent: Solution A (0.1% trifluoroacetic acid water) Solution B (acetonitrile containing 0.1% trifluoroacetic acid) was used in a linear 1-degree gradient elution ratio (50 minutes) from solution A to solution B. ) Flow rate: 1.0 ml/min (2) big ET-3 (1-42); H-Cys
-Thr-Cys-Phe-Thr-Tyr-Lys-
Asp-Lys-Glu-Cys-Val-Tyr
-Tyr-Cys-Hi 5-Leu -A sp-I
leo I le-Trp-I 1e-Asn-Th
r-Pro-G 1u-G ln-Thr-Val
-Pro-Tyr-Gly-Leu-5er-As
n-Tyr-Arg-Gly-5er-Phe-Arg
Synthesis of -Gly-OH Commercially available Boc-Gly-OCH2-PAM resin (manufactured by Applied Biosystems) 0.78 g (0.5
using a peptide synthesizer (applied
1 was synthesized using Biosystems Ura Model 430A).

After treating the Boa group on the resin with 50% trifluoroacetic acid/methylene chloride to liberate the amino group, the amino group is treated with Boa-Arg(Tos), Boc-Phe,
Boc-5er (Bzl), Boc-Gly, Bo
c-Tyr(Br-Z), Boc-Asn, Boa
-Leu, Boc-Pro, Boc-Val, B
oc-Thr(Bzl), Boc-Gin, Boc-
Glu(OBzl), Boa-11e, Boc-T
rp(CHO), Boc-Asp(OBzl), Bo
c-His (DNP), Boc-Cys (MeBzl
), Boc-Lys (C1-Z) to big ET-3 (
1-42)) 10Bt/DCC
was activated and condensed. Furthermore, DCC or HOBt/
After condensation again with the same amino acid derivative activated with DCC, unreacted amine groups were acetylated with acetic anhydride to form the protected big-ET-3(1-42)-0CH,-P.
A resin was obtained. This was suspended in 20 ml of N,N'-dimethylformamide, 2 ml of thiophenol was added, and the mixture was gently stirred at room temperature for 2 hours. The resin was then filtered onto a glass filter and washed with N,N'-dimethylformamide and dichloromethane. After drying, 1.00 g of resin was obtained.

0.39 g of this resin, 0.74 g of p-cresol, 10
After treating with anhydrous hydrogen fluoride 101111 in the presence of 1.0 ml of 4-butanedithiol at ○℃ for 60 minutes, the hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether, and the residue was dissolved in trifluoroacetic acid. Extracted with 4 ml. Insoluble materials were removed by filtration and washed twice with 2 ml of trifluoroacetic acid. This was poured into 750 ml of 4-Tourea aqueous solution, the pH was adjusted to 8.25 while cooling with water, and the mixture was stirred overnight while gently blowing air. After confirming that the El1man test is negative, transfer the entire solution to YMC-ODS AM12
Attach to O5-50 resin column (2,6X7a11) and
．． 1% trifluoroacetic acid water and 50% acetonitrile (0
, 1% trifluoroacetic acid). The main fractions were combined and lyophilized to give a white powder 1
I got 6■. Using the same column, rechromatography was carried out using a linear gradient elution between 20% and 50% to obtain the target product 6.
,3■ were obtained.

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 1.77 (2), Ile 1.83 (3),
Leu 2.00 (2), Tyr 4°64 (5)
, Phe 1.92 (2), Lys 1.78 (2
), His O, 88(1).

Arg 2.12(2) (M + H) 4979.48HP by mass spectrometry
LC elution time 21.00 minutes Column conditions Column: YMC-ODS (AM-301, S-51
2OA) Eluent: Linear concentration gradient elution from solution A to solution B (50 minutes) flow rate using solution A (0.1% trifluoroacetic acid water) solution B (acetonitrile containing 0.1% trifluoroacetic acid) : 1. Oml/min (3) big ET-3 (22-41)-NO3:
H-Ile-Asn-Thr-Pro-Glu-Gln
-Thr-Val-Pro-Tyr-Gly-Leu-
5er-Asn-Tyr-Arg-Gly-5er-P
Synthesis of he-Arg-NO3 Commercially available p-methyl BOA resin (manufactured by Applied Biosystems) 0.60 g (0.5 n mole)
Synthesis was performed using a peptide synthesizer (Model 430A manufactured by Applied Biosystems).

After introducing Boc-Arg (Tos) into the resin by HOBt/DCC, the 80c group on the resin was added with 50% trifluoroacetic acid w! /methylene chloride to liberate the amino group.

Boc-Phe, Boa-5er (
Bzl), Boc-Gly, Boc-Arg('T
os), Boc-Tyr (Br-Z), Boa-A
sn, Boc-Leu.

Boc-Pro, Boc-Val, BoC-Thr
(Bzl), Boa-Gin, Boa-Glu(O
Bzl), Boa-11e to Mg ET-3 (22-
41)-I08t/D (in order according to the amino acid sequence of NO3)
It was activated with CC and condensed.

After further condensation with the same amino acid derivative activated with DCC or HOBt/[)CC, unreacted amino groups were acetylated with acetic anhydride to form the protected bigE
T-3(22-41)-Bl (1.96 g of A resin was obtained.

After treating 0.23 g of this resin with 5 ml of anhydrous hydrogen fluoride in the presence of 0.40 g of p-cresol at 0°C for 60 minutes, the hydrogen fluoride was distilled off under reduced pressure, and the residue was dissolved in 5 ml of ethyl ether.
After washing twice with l, the residue was extracted with 5 ml of 50% aqueous acetic acid. Insoluble materials were removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing liquid were combined and concentrated under reduced pressure to 2 to 3 ml.
It was applied to a column of Sephadex LH-20 (2x90ω) and eluted with 50% acetic acid. Collect and concentrate the main fractions to 0.
．． Dissolve in 100ml of 1% trifluoroacetic acid water, YMC
-ODS AM12O5-50 resin column (2,6x
7) and eluted with a linear concentration gradient between 1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid).

The main fractions were combined and lyophilized to obtain 56 lbs of white powder.

Amino acid sequence Asp 1.97 (2), Thr 1.88 (2
), Ser 1.75(2), Glu 2゜0
5(2), Gly 3.00(3), Vat 1.
00(1), Pro 1.93(2).

11e 0.95(1), Leu O,9[1),
Tyr O, 95(1), Phel.

02(1), Arg 2.18(2) by mass spectrometry (M + H etc. 2298.19 HPLC elution time
17.01 minute column conditions Column: YMC-〇D S (AM-301, S-5
12OA) Eluent: Linear concentration gradient elution from solution A to solution B (50 minutes) flow rate using solution A (0.1% trifluoroacetic acid water) solution B (acetonitrile containing 0.1% trifluoroacetic acid) : 1. o+++1/min (4) big ET-3(1-41)-NO3;H
-Cys-Thr-Cys-Phe-Thr-Tyr-
Lys-Asp-Lys-Glu-Cys-Va 1-
Tyr-Tyr-Cys-His-Leu-A sp-
Ile-11e-Trp-I1e-Asn-Thr
-Pro-G 1u-Gln-Thr-Va 1-P
ro-Tyr-G ly-Leu-5er-Asn-T
yr-Arg-Gly-5er-Phe-Arg-NO
Commercially available p-methyl BHA resin (manufactured by Applied Biosystems) 0.60 g (0.5 nma
ie) and a peptide synthesizer (Model 430A manufactured by Applied Biosystems).

Boa-Arg (Tos) on the resin) lOBt/D
After introduction with CC, the Boa group on the resin is treated with 50% trifluoroacetic acid/methylene chloride to liberate the amino group, and then this amino group is treated with Boa-Phe, Boa-5.
er(Bzl), Boc-Gly, B.

c-Arg (Tos), Boc-Tyr (Br-Z)
, Boc-Asn, Boa-Leu.

Boc-Pro, Boc-Val, Boc-Thr
(Bzl), Boc-Gin, B.

c-Glu(OBzl), Boc-11e, Boc
-Trp (CHO), Boc-Asp (OBzl),
Boa-His (DNP), Boa-Cys (Me
Bzl), Boa-Lys (C1-Z) as big E
H in order according to the amino acid sequence of T-3(1-41)-NO3
It was activated and condensed with OBt/DCC. After further condensation with the same amino acid derivative activated with DCC or HOBt/DCC, unreacted amino groups were acetylated with acetic anhydride, resulting in protected big-ET-3(1-41).
-BHA resin was obtained. This was suspended in 20 ml of N,N'-dimethylformamide, and 2 ml of thiophenol was added.
After gently stirring at room temperature for 2 hours, the resin was filtered onto a glass filter, washed with N,N'-dimethylformamide and dichloromethane, and dried to obtain 1.34 g of resin.

0.74 g of this resin was mixed with 1.0 g of p-cresol, 10
After treatment with 10 Ill of anhydrous hydrogen fluoride in the coexistence of 1.0+ Il of 4-butanedithiol at 0°C for 60 minutes, hydrogen fluoride was distilled off under reduced pressure, the residue was washed twice with 5 ml of ethyl ether, and the residue was dissolved in trifluoroacetic acid. Extracted with 6 liters. Insoluble materials were removed by filtration and washed twice with 2 ml of trifluoroacetic acid. This was poured into 1,000 parts of 4-Tourea aqueous solution, the pH was adjusted to 8.0 while cooling with water, and the mixture was stirred overnight while gently blowing air. After confirming that the El1man test is negative, transfer the entire solution to YMC-ODS AM12
Attach to O5-50 resin collar A (2,6x70m), 0
．． 1% trifluoroacetic acid water and 50% acetonitrile (0
, 1% trifluoroacetic acid). The main fractions were combined and lyophilized to produce a white powder 3
I got 5 ■. Re-chromatographic purification was repeated using the same column and eluting with a linear concentration gradient between 20% and 50%, yielding 9.5μ of the desired product.

Amino acid analysis value Asp 3.89 (4), Thr 3.81 (4),
Ser 1.9g (2), Glu 3゜04 (3)
, Pro 2.09 (2), Gly 3.30 (3
), Cys 1.60(2).

Val 1.79 (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) by mass spectrometry (M 10H) 4921.31HPL
C elution time 21.00 minutes Column conditions Column: YMC-ODS (AM-301, S-51
2OA) Eluent: Linear concentration gradient elution from solution A to solution B (50 minutes) flow rate using solution A (0.1% trifluoroacetic acid water) solution B (acetonitrile containing 0.1% trifluoroacetic acid) : 1. oml/min ■-2, Preparation of immunogen Big endothelin-3 C-terminal peptide obtained in above V-1 l1e-Asn-Thr-Pro-Glu-G
in-Thr-Va 1-Pro-Tyr-G 1 y
-Leu-5e r-Asn -Tyr-A rg-G
ly-Ser-Phe-Arg-Gly and bovine thyroglobulin (TG) were condensed by the maleimide crosslinking method described below and used as an immunogen.

That is, 350 μm of 1.28 μmole of polypeptide
0°1M phosphate buffer, pH 7.0 (10% D
G M B 525.6 μm
Mix with 100 μm of DMF solution containing ole and incubate at room temperature for 3
It was allowed to react for 0 minutes.

On the other hand, TG'30mg (60n mole) is 0.1
0.02M phosphate buffer containing 5M salt, pH 6.8,
Dissolve in 1.4 ml, S P D P 3.7 gg
After mixing a DMF solution containing (12,0tt mole), the mixture was reacted at room temperature for 40 minutes. After reaction.

Dithiothreitol 18.6mg (120p mole
) containing 0.1M acetate buffer, pH 4, 5, 0, 5m
1 and reacted at room temperature for 20 minutes, then Sephadex G
-25 column to perform fractionation, and T0 with SH group introduced.
18B (36 n mole) was obtained 6 Next, 920 n s of maleimide group-introduced polypeptide.

le and SH group-introduced TG29 n @ole were mixed and allowed to react at 4°C for 2 days.
Dialysis was performed at ℃ for 2 days.

■-3, Immunization BALB/c R mice aged 6 to 8 weeks were subcutaneously immunized with 15 μg/mouse of the immunogen described in V-2 above (as big endothelin-30 end peptide) together with an adjuvant. Thereafter, booster immunizations were performed twice every 3 weeks.

V-4, Preparation of HRP-labeled big endoserine-30-end peptide 3300 mol of big endoserine-30-end peptide
e was dissolved in 500 μm of 0.1 M phosphate buffer, pH 7.0, and 52.78 g of GMB (9.9 p mol
The mixture was mixed with 50 μl of a DMF solution containing e) and allowed to react at room temperature for 60 minutes.

After the reaction, fractionation was performed using a Sephadex G-25 column to obtain 200 n mo of polypeptide into which a maleimide group had been introduced.
I got le.

On the other hand, HRP 10+sg (250n mole)
Using this, enzyme 6@g (150 n mole) into which an SH group was introduced was obtained by the method described in ①-3.

Next, 200 n moles of maleimide group-introduced big endothelin-3 C-terminal peptide and 50 n moles of SH group-introduced peroxidase were mixed and reacted at 4°C for 16 hours. After the reaction, Ultrogel AcA44 (LK
B-Pharmacia) column to obtain a peroxidase-labeled big endothelin-36 end peptide.

(1)-5, Cell fusion Using mice immunized with the big endothelin-30 end peptide described in V-3, cell fusion was carried out by the method described in 11-4 to obtain hybridomas.

■-6. Screening of hybridomas Antibody titers in hybridoma culture grooves were measured by the following two methods. That is, an anti-mouse immunoglobulin antibody or a big endothelin-30 end peptide-bound microplate was used.

The plate was first coated with anti-mouse immunoglobulin antibody (I
gG fraction, Kabbell Co. 1 or big endoserine-3
A 0.1M carbonate buffer, pH 9.6 solution containing 20 μg/ml of C-terminal peptide was added to a 96-well microplate.
00 μl was dispensed and left at 4° C. for 24 hours.

Next, the plate was washed with PBS, and 300 μl of PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) was dispensed in order to block excess binding sites in the wells, and treated at 4° C. for at least 24 hours.

1) EIA method using HRP-labeled big endothelin-30-terminal peptide Buffer E (10% Block Ace, 2 m/ml
0.02M phosphate buffer containing BSA, 0.4M NaCl,2*eMEDTA and 0.1% NaN, p
H7,0) 50 μl and hybridoma culture supernatant 50
Add μm.

After 0 reaction at room temperature for 4 hours, HRPII! produced in the above V-4! ! Add 100 μl of converted big endothelin-3C-terminal peptide [400-fold dilution with buffer C],
The reaction was carried out at 4°C for 16 hours. After the reaction and washing with PBS, the enzyme activity on the solid phase was measured by the method described in Tl-5(i).

In this way, we examined the supernatants of all 120 wells in which hybridoma growth was observed, and found that Nn44 and Nn44
A strong antibody titer was detected in the n77 well.

■) ELISA method using big endothelin-3 C-terminal peptide binding microplate big endothelin-3
Add buffer E5 to the C-terminal peptide binding microplate.
0 μm, add 100 μl of hybridoma supernatant, and leave at room temperature 4.
After 0 hours of reaction, after washing with PBS, HRP
&? 100 μl of an anti-mouse immunoglobulin antibody [diluted 10,000 times with buffer C] was added, and the mixture was allowed to react at 4° C. for 16 hours. Next, after washing the plate with PBS.

The enzyme activity on the solid phase was measured by the method described in V-6(i) above.

When the supernatants of all 120 wells in which hybridoma growth was observed in this manner were examined, a strong antibody titer was detected in the Nα77 well.

(2)-7, Cloning Each hybridoma in wells Nα44 and Nα77, which showed positive antibody activity, was cloned according to the section IF-6. After about a week, the cells begin to proliferate, and the antibody titer in the supernatant is determined by
When investigated using the IA method.

&44 hybridoma, 2 out of 76 clones, and Nα77 hybridoma, 7 out of 76 clones.
Five clones were producing antibodies.

Among these clones, we focused on clone bET-31 obtained from &77-30 and the monoclonal antibody bET-31a produced by it, and clone bET-23 obtained from &44-52 and the monoclonal antibody bET-23a produced by it. We conducted the following experiments.

■-8. Preparation of large amounts of monoclonal antibodies Hybridoma bET-3 was administered to mice intraperitoneally administered with 0.5 ml of mineral oil or to untreated mice (BALB/C).
Antibody-containing ascites fluid was collected 10-14 days after intraperitoneal injection of 1 or bET-231~3X10 cells/mouse.

■-9. Purification of monoclonal antibody V-8r! Monoclonal antibodies bET-31a and bET-2 were detected using a protein-A column from ascites prepared from 4
3a was purified according to the method of ll-8. bET-31
A specific antibody with a of 30 μb and ET-23a of 50 μ was obtained.

Determination of the class and subclass of V-10 and monoclonal antibodies The class and subclass of the V-9 purified monoclonal antibodies bET-31a and bET-23a were determined according to the method described in 11-9. As a result, it was found that both bET-31a and bET-23a belong to the IgG1 class.

(2) Competitive method for big endothelin-3 - EIA Add 50 mL of culture medium containing bET-31a and bET-23a diluted 100 times with buffer C to the anti-mouse immunoglobulin antibody-bound microplate described in v-6 above.
μl, and big endothelin-1, endothelin-
3, Big End Serin-3, Big End Serin-3
After adding 50 μm of 0-end peptide (22-42) or big endothelin-3C-end peptide (22-41)-NH2 standard solution and reacting at room temperature for 1 hour, the HRP-labeled big endothelin-3C described in V-4 above was added. Add end peptide (200-fold diluted with buffer C) and incubate at 4°C.
The reaction was carried out for 16 hours. After the reaction, the solid phase was washed with PBS, and the enzyme activity on the solid phase was measured by the method described in 11-5(i). The results are shown in FIGS. 3(A) and 3(B). In the figure, -mu- is big endoselin-3, -〇- is big endoselin-1, -・- is endothelin-3, -
m- is big endothelin-36 terminal peptide (22-4
2), and -low indicates the standard curve of big endothelin-30 end peptide (22-41)-Nl(,
A) shows the results of the competition method using bET-31a and (B) shows the results of the competition method using bET-23a.

From the results in Figure 3, bET-31a and bET-23
Since both a does not react with big endothelin-1 and endothelin-3, big endothelin-3
It is an antibody specific for big endothelin-
Since it reacted to the same extent as the 30-terminal peptide (22-42) and the big endothelin-30-terminal peptide (22-41)-NH2, it was found that it had broad specificity for the C-terminal region of big endothelin-3.

■Sandwich method for big-end serine-3
EIA ■-1, HRPIIIlt monoclonal antibody bET
- Preparation of bET-31a purified by the method described in V-9 above -
Fab'-HRP 41 antibody was produced according to the method described in 6.

That is, 3.17 μg of pepsin (Sigma, twice crystallized) was added to 10.5 μg of monoclonal antibody bET-31a dissolved in 0.1 acetic acid buffer, p14.5, and
After reacting at ℃ for 16 hours, the F(ab') fraction was purified using an Ultrogel AcA44 column equilibrated with BBS. After the fraction was dialyzed against 0.1M acetate buffer, pH 5, a final 20 cod β-mercaptoethylamine was added, and the mixture was left at 37°C for 90 minutes. The reaction solution was 2.5 mN
0.1M phosphate buffer containing EDT A, pH 6
, 0 to obtain a Fab' fraction.

Meanwhile, horseradish peroxidase 5■ was dissolved in 0.9 ml of 0.1 M phosphate buffer, p)17, and 50 μm
81.05 ml of GMB dissolved in DMF was added and reacted at room temperature for 40 minutes.

The reaction solution was transferred to a Sephadex G=25 column (eluent 0.1
M phosphate buffer, pH 6, 8), and the resulting maleimidated peroxidase (3.5) and the above Fab' fraction (1.3aIg) were mixed and separated using a collodion bag (Munis Kiki Co., Ltd.) at approximately 0.0%. After concentrating to 3 ml, heat at 4°C.
It was left for 16 hours. Kurtrogel AcA44 using 0.1M phosphate buffer, pF16.5 as the eluent for the reaction solution
The Fab'-peroxidase complex fraction was purified by applying it to a column (10 ml φ x 40 m).

■-2. HRP-labeled monoclonal antibody bET-23
Preparation of monoclonal antibody bET according to the method described in ①-1 above.
HRPIJ-conjugated bET using -23a12.7+ag
-23a was produced.

■-3. Big endothelin-3, big endothelin-1 (human), endothelin-3, endothelin-1 and endothelin-2 (human) standard solutions 10 diluted with buffer E were added to the AET-30a binding microplate prepared by sandwich method EIA ①.
0 μl was added and reacted at 4° C. for 24 hours. After washing with PBS, add HRP-labeled embrittled T-31a or bET-23a (Buffer C
(200-fold dilution) was added thereto, and the mixture was 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 U-5(i) above. Figure 4 shows the results when using HRP-labeled embrittled T-31a.
FIG. 5 shows the results when RP-labeled embrittled T-23a was used. In the figure, -mu- is big endoserin-3, and -〇- is big endoserine-1 (human), -・-
is for endothelin-3, -1- is for endothelin-1,
-Rho indicates the standard curve of endothelin-2.

From the results shown in Figure 4, the measurement method using HRP41-identified bET-31a is specific to big endothelin-3;
It hardly reacts with other endothelins (cross-reactivity,
(0.1% or less), and the measurement sensitivity was found to be 6XIO''17 mol/well.

Similarly, from the results shown in Figure 5, HRP-labeled embrittled T-238
It was found that the measurement method using the method is specific for big endothelin-3 and hardly reacts with other endothelins (cross-reactivity, 0.1% or less). In addition, the measurement sensitivity was 8 x 10'''' mole/well.

(2) Determination of endothelin-3 in amniotic fluid After amniotic fluid (1 mQ) was concentrated and pretreated using a Motsupak C-18 cartridge, endothelin-3 was determined by the sandwich method-EIA described in (1) 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±3.9p was found in the amniotic fluid at the time of birth.
It was found that endothelin-3 of g/m Q (mean±SD, n=8,) was present.

■, Detection of endothelin-3 in amniotic fluid by reversed phase high performance liquid chromatography (RP-HPLC) Table 1, No.
, 3 amniotic fluid L5m 12 was added to Motsupu Pack C described in ■ above.
After pretreatment using a -18 cartridge, the mixture was concentrated under a nitrogen stream. Next, the concentrate was mixed with eluent A10, which will be described later.
After dissolving in 0 μQ, it was separated by RP-HPLC. The separation conditions are shown below.

Column: OD S-, 80TM (4,6nnφX
250m+++.

Tosoh Corp. Solution A: 0.05%-IJ)) Ii 5% acetonitrile containing myovine vinegar (TFA) Solution B: 60% acetonitrile containing 0.05% TFA Elution method: Eluent The concentration of B was increased from 0 to 4 during the first 5 minutes.
0%, then 40-65% in the next 20 minutes, then 5% in the next 20 minutes.
Increase linearly from 65 to 100% in minutes.

Flow rate: 1 mQZ fraction Fraction: 0.5 mQ/lube After centrifuging the eluted fraction under reduced pressure and concentrating it to dryness, 250 μQ
The sample was dissolved in Buffer E and subjected to the sandwich method-EIA described in (1) above. The results are shown in Figure 6. Since the immunoreactivity of endothelin-3 in amniotic fluid was detected at the elution position of synthetic endothelin-3, the sandwich method-EI
It was confirmed that A detected endothelin-3.

Scheme 1 Extraction method of endothelin in amniotic fluid Pretreatment of Seppak C-18 cartridge - 5 mQ of 86% ethanol containing 4% acetic acid - 5 mQ of methanol - 5 mQ of distilled water - 5 mQ of 4% acetic acid Using 3 mQ of 4% acetic acid, Amniotic fluid (1,0-5,0m
Loading on acidified cartridge of Q) Washing with 10 mΩ distilled water Elution with 4 mQ of 86% ethanol containing 4% acetic acid Concentration under nitrogen gas stream at 35°C Reconstitution with 0.25 mfl of buffer E ↓ Sandwich EIA 1) Rosmalen F et al., (1
987) Clinic Himiriki Acta 331-340 Table 1 Quantification of endothelin-3 in amniotic fluid 1 4, 3 2 <0. 5 3 13.3 4 2, 6 5 3, 3 6 4, 3 7 2, 2 8 3, 2 mean ±SD 4.2±3.9SD:
5 standard deviation (standard deviation) X, endothelin-3 in plasma of healthy subjects and dialysis patients
After human plasma (1i) was concentrated and pretreated by the method shown in Scheme 1, endothelin-3 was quantified by the sandwich method-EIA described in (1) above. Figure 7 shows the measurement results of plasma from 17 healthy subjects (male, 37.5±4.9 years old) and 24 dialysis patients (male, 46.0±7.0 years old).

These results demonstrate that endothelin-3 in plasma can be quantified using this measurement method, and that the plasma level of endothelin-3 increases in dialysis patients, making this measurement method clinically useful. revealed.

■Detection of endothelin-3 immunoreactivity in plasma of healthy subjects and dialysis patients by RP-HPLC Healthy subjects' plasma, 20 m
1. Alternatively, after diluting dialysis patient plasma 80 ml with the same amount of PBS, conjugate 1.5 μ of antibody to 1 g of CNBr-activated Sepharose 4B using the AET-30-coupled Sepharose 4B column (method described in I-4). gel 1
．． 5+sl) at a rate of 10a+1/hour. After washing the column with 1511 PBS, it was eluted with 3 ml of 60% acetonitrile solution containing 0.05% TFA, and concentrated under a nitrogen stream. Concentrate with 0.05% TFA
and 0.05% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate) (3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate)
yl) dimethylaIIn+onio] -1
-propanesultonate) and then analyzed by RP-HPLC under the elution conditions described in (1).

The results are shown in FIG. Healthy subjects (A) and dialysis patients (B)
) Since any immunoreactivity was detected at the elution position of standard synthetic endothelin-3, the sandwich-EI
It was confirmed that A detected endothelin-3 in plasma, and its excellent properties became clear.

Tomo: Examination of the neutralizing activity of monoclonal antibody AET-30a Approximately 2 spiral strips excised from the left coronary artery of a pig were
It was suspended in a Magnus tube filled with Taleb-Henseleit solution (hereinafter abbreviated as nutrient solution) while aerating with a mixed gas (95% 02 + 5% CO2). After leaving it at 37℃ for 3 hours,
The tension generated by the contraction of vascular smooth muscle is measured using an isometric transducer (polygraph, manufactured by NEC Sanei Co., Ltd.).
It was measured by

As a sample, 20 times the mole of AET-30 was prepared in advance.
Even when adding an endothelin-3 solution (final endothelin-3 concentration lXl0-'M) reacted with a for 20 minutes at 37°C, the tension generated is 7.0 of the tension induced by the same concentration of endothelin-3. % (n: 5), whereas a solution in which control antibody H272-11 (anti-carcinoembryonic antigen) and endothelin-3 were reacted (final 1 x 10''''M
) is added to the same level as endothelin-3 (12
The tension due to contraction of 1%, n=5) was measured in WA. From the above, it was revealed that AET-30a neutralizes the vascular smooth muscle contractile activity of endothelin-3.

〔Effect of the invention〕

The monoclonal antibody against endothelin-3 of the present invention has extremely high binding ability and can neutralize the vascular smooth muscle contractile activity of endothelin-3. An immunoassay method using the sandwich method using the monoclonal antibody and an antibody that recognizes the C-terminus of endothelin-3 allows endothelin-3 to be detected with high sensitivity and endothelin-1 to be detected with high sensitivity.
Alternatively, it can be quantified without cross-reacting with endothelin-2 or big endothelin-3. The antibody can also be used as a potent antagonist of endothelin-3 in various diseases associated with endothelin-3.

In addition, 1 monoclonal antibody against big endothelin-30 end peptide of the present invention is a monoclonal antibody against big endothelin-3
Because it has a wide range of specificity for the C-terminus, it is advantageous for detecting big endothelin-3 immunoreactivity, and an immunoassay method using the sandwich method using the monoclonal antibody and a monoclonal antibody against endothelin-3, Big End Serin-3 with high sensitivity.

It can be quantified without cross-reacting with endothelin-1, -2, -3, and big endothelin-1.

[Brief explanation of the drawing]

FIG. 1 shows a standard curve for endothelin-3 and endothelin-1 in competitive EIA using monoclonal antibody AET-30a. Figure 2 shows endothelin-3 (-・-), endothelin-1 (-mu-), endothelin-2 (-■-
), standard curves for human big endothelin-1 (-〇-), porcine big endothelin-1 (-low), and big endothelin-3 (-Δ-) are shown. Figure 3 (A) is competitive method-EIA using monoclonal antibody bET-31a, (B) is monoclonal antibody bE
Competitive method using T-23a - EIA, big endoserine-3 (-mu-), big endoserine- in each
1 (human) (-〇-), endothelin-3 (-・-),
Big endothelin-3C-terminal peptide (22-42)
(-■-) and the standard curve of big endothelin-3C-terminal peptide (22-41)-NH2 (-rho). FIG. 4 shows big endothelin-3 (-mu-) big endothelin-1 (human) (-〇-) in sandwich method-EIA using monoclonal antibody AET-30a and monoclonal antibody bET-31a. Endothelin-3 (-・-), endothelin-1 (-1
-) and endothelin-2 (-rho). Figure 5 shows big endothelin-3 (-mu-), big endothelin-1 (human) (-〇-), endothelin-3 (-・-), endothelin-1 (-■-), and endothelin-2 (-) are shown. Figure 6 shows the separation of human amniotic fluid by reverse phase HPLC after treatment with a Seppac C-18 cartridge. The arrows indicate the elution positions of synthetic standard endothelin and big endothelin, showing the detection results by the above-mentioned Sand Inch method-EIA. Abbreviations: ET-3, endothelin-3; (a) oxide of human big endothelin-1; hbig-ET-1, human big endothelin-1: (b) oxide of endothelin-1;
p b i g-ET-1, porcine big endothelin-1; ET-1, endothelin-1, and ET-2,
Endothelin-2゜Figure 7 shows the measurement results of endothelin-3 concentration in the plasma of healthy subjects and dialysis patients.
The figure shows the results of separation of endothelin-3 immunoreactivity in plasma from healthy individuals and dialysis patients by reverse phase HPLC and detection by the above-mentioned Sandwich method-EIA.

Claims (26)

[Claims] (1) A monoclonal antibody capable of binding to endothelin-3 or endothelin-3 precursor. (2) The monoclonal antibody according to claim 1, wherein endothelin-3 has the following amino acid sequence. (3) The precursor of endothelin-3 is endothelin-3
The monoclonal antibody according to claim 1, which has one or more amino acids added to the N-terminus or C-terminus. (4) The monoclonal antibody according to claim 1 or 3, wherein the precursor of endothelin-3 is a polypeptide having the sequence of formula 1, or big endothelin-3 represented by formula 2, or a portion thereof. Formula 1 [There is a gene sequence] Formula 2 [There is a gene sequence] [There is a gene sequence] (X=Gly-OH or NH_2) (5) The monoclonal antibody according to claim 1, which has a binding property to an endothelin-3 precursor and does not react with an endothelin-3 C-terminal peptide, that is, a peptide represented by [there is a gene sequence]. (6) The monoclonal antibody according to claim 1, which has a binding property to endothelin-3 and does not react with the endothelin-3 C-terminal peptide, that is, the peptide represented by [there is a gene sequence]. (7) has binding properties with endothelin-3 precursor, while
The monoclonal antibody according to claim 1, which has the following amino acid sequence and does not react with either endothelin-1 [gene sequence is available] endothelin-2 [gene sequence is available] or mouse endothelin-2 [gene sequence is available] . (8) Endothelin-1 [gene sequence available], mouse endothelin-2 [gene sequence available], and mouse endothelin-2 [gene sequence available] The monoclonal antibody according to claim 1, which does not react with any of the following. (9) The monoclonal antibody according to claim 1, which has binding property to big endothelin-3 (Formula 2) or a partial peptide thereof and does not react with endothelin-3. (10) Big endoserine-3C having the sequence of formula 3
The monoclonal antibody according to claim 9, which reacts with the terminal peptide. Formula 3 [There is a gene sequence] (X=Gly-OH or NH_2) (11) The mouse monoclonal antibody according to claim 8, which is labeled with AET-30a. (12) The mouse monoclonal antibody according to claim 10, which is labeled with bET-31a. (13) The mouse monoclonal antibody according to claim 10, which is labeled with bET-23a. (14) Hybridoma cells that produce monoclonal antibodies that bind to endothelin-3 or endothelin-3 precursors. (15) The hybridoma cell according to claim 14, which is labeled with AET-30. (16) The hybridoma cell according to claim 14, which is labeled with bET-31. (17) The hybridoma cell according to claim 14, which is labeled with bET-23. (18) The antibody against endothelin-3 or endothelin-3 precursor is competitively reacted with the test solution and labeled endothelin-3 or labeled endothelin-3 precursor, and the labeled endothelin-3 bound to the antibody is 3
Alternatively, a method for quantifying endothelin-3 or endothelin-3 precursor in a test liquid, which comprises measuring the proportion of labeled endothelin-3 precursor. (19) An antibody against endothelin-3 precursor is competitively reacted with a test solution and labeled big endothelin-3 or labeled big endothelin-3 C-terminal peptide, and labeled big endothelin-3 bound to the antibody is reacted competitively.
Alternatively, a method for quantifying endothelin-3 precursor in a test liquid, which comprises measuring the proportion of labeled big endothelin-3 C-terminal peptide. (20) After bringing the test solution into contact with the antibody against endothelin-3 or endothelin-3 precursor insolubilized on the carrier, the labeled antibody against endothelin-3 or endothelin-3 precursor is brought into contact with the insolubilized carrier. A method for quantifying endothelin-3 or endothelin-3 precursor in a test solution, which comprises measuring the activity of the above labeling agent. (21) One of the antibody against endothelin-3 insolubilized on the carrier and the labeled antibody against endothelin-3 is a polyclonal antibody or monoclonal antibody that has binding property to endothelin-3 C-terminal peptide, and the other is a polyclonal antibody or monoclonal antibody that has binding property to endothelin-3 C-terminal peptide. 21. The method for quantifying endothelin-3 according to claim 20, which is a monoclonal antibody that has binding properties to but does not react with the C-terminal peptide of endothelin-3. (22) One of the antibody against endothelin-3 or endothelin-3 precursor insolubilized on the carrier and the labeled antibody against endothelin-3 or endothelin-3 precursor is a monoclonal antibody that has binding property to endothelin-3. Yes, and the other is endothelin-3
21. The method for quantifying endothelin-3 precursor according to claim 20, which is a monoclonal antibody that has binding properties to the precursor but does not react with endothelin-3. (23) One of the antibody against endothelin-3 or endothelin-3 precursor insolubilized on the carrier and the labeled antibody against endothelin-3 or endothelin-3 precursor is the monoclonal antibody according to claim 8, and the other The method for quantifying endothelin-3 precursor according to claim 22, wherein is the monoclonal antibody according to claim 10. (24) Anti-endothelin-3 monoclonal antibody AET-30a insolubilized on a carrier and labeled anti-endothelin-3 C-terminal peptide (CysHisLeuAspIl)
22. The method for quantifying endothelin-3 according to claim 21, which uses the eIleTrp) antibody. (25) One of the antibodies against endothelin-3 or endothelin-3 precursor insolubilized on the carrier and the labeled antibody against endothelin-3 or endothelin-3 precursor is AET-30a, and the other is b
Claim 22 which is ET-31a or bET-23a.
The described method for quantifying endothelin-3 precursor. (26) bET-3 labeled with anti-endothelin-3 monoclonal antibody AET-30a insolubilized on a carrier
26. The method for quantifying endothelin-3 precursor according to claim 25, using 1a or bET-23a.