JP4314365B2 - Specific antibody against endothelin-2 / VIC, production method and use thereof - Google Patents

Description

  The present invention relates to a novel antibody having binding specificity to endothelin-2 and / or VIC, its production method, endothelin-2 and / or VIC quantitative reagent using the antibody, carrier for identification amount, and endothelin using these -2 and / or VIC quantification method. In particular, it can be used for the development of clinical test drugs or pathophysiology research in medicine and veterinary livestock.

Along with the endothelium-dependent vasodilator response, endothelium-dependent vasoconstrictor responses to various stimuli have been reported. Angiotensin is obtained from endothelial cells (Circulation Research, 60,4221 (1987)), and then has a strong vascular smooth muscle contraction effect from mammalian vascular endothelial cell culture supernatant, A peptide consisting of 21 amino acids (endothelin-1) was discovered.
On the other hand, the present inventors discovered a vascular / intestinal smooth muscle contractile peptide VIC from analysis of the mouse genome, and showed for the first time that endothelin has three types of peptides (endothelin gene family) (Patent Literature). 1 and non-patent document 1). In humans, it has been reported that endothelin-2 is a corresponding peptide (see Patent Document 2 and Non-Patent Document 2).

These amino acid sequences are shown below.
Mouse VIC
CysSerCysAsnSerTrpLeuAspLysGluCysValTyrPheCysHisLeuAspIleIleTrp
Human ET-2
CysSerCysSerSerTrpLeuAspLysGluCysValTyrPheCysHisLeuAspIleIleTrp
Furthermore, as a precursor of endothelin-2 or VIC, a peptide comprising about 40 amino acids (hereinafter referred to as big-endothelin-2 and big-VIC, respectively) and a peptide comprising about 200 amino acids (hereinafter endothelin-2 precursor). Body / VIC precursor). In this specification, endothelin-2 / VIC, big-endothelin-2 / big-VIC and endothelin-2 precursor / VIC precursor are collectively referred to as endothelin-2 / VIC.
Examples of big-endothelin-2 / big-VIC include mouse and rat big-VIC having the amino acid sequences shown by the following amino acid sequences (a) and (b), and human big having the amino acid sequence shown by amino acid sequence (c). -Examples include endothelin-2.
(A) Mouse Big VIC
CysSerCysAsnSerTrpLeuAspLysGluCysValTyrPheCysHisLeuAspIleIleTrpValAsnThrAlaG lyGlnThrAlaProTyrGlyLeuGlyAsnProProArgArgArgArgArg
(B) Rat Big VIC
CysSerCysAsnSerTrpLeuAspLysGluCysValTyrPheCysHisLeuAspIleIleTrpValAsnThrAlaGlyGlnThrAlaProTyrGlyLeuGlyAsnProProGlnArgArgArgArg
(C) Human Big ET-2
CysSerCysSerSerTrpLeuAspLysGluCysValTyrPheCysHisLeuAspIleIleTrpValAsnThrProGluGlnThrAlaProTyrGlyLeuGlyAsnProProArgArgArgArgArg
Examples of the endothelin-2 precursor / VIC precursor include mouse and rat VIC precursor having the amino acid sequence 1-203, human endothelin-2 precursor having the amino acid sequence 1-212, and the like.

Japanese Patent No. 2807471 Japanese Patent No. 2795346 Saida K, Mitsui Y, Ishida N. A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family.Molecular cloning, expression, and biological activity.J Biol Chem 1989; 264: 14613-14616 Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes.Proc Natl Acad Sci U S A 1989; 86: 28633-2867.

  The endothelin-2 / VIC may be causally or symptomatically related to various diseases such as essential hypertension, heart failure, intestinal disease, etc. from the viewpoint of its potent vascular / intestinal smooth muscle contractile action. It is considered. In order to elucidate these, it is necessary to conduct comprehensive research on endothelin-2 / VIC, especially on metabolic pathways, secretion mechanisms, receptors, etc. Immunological techniques such as immunoassay are considered to be one of the most effective means for that purpose, and production of antibodies specific to endothelin-2 / VIC is regarded as extremely important. However, until now, endothelin-2 / VIC is very similar in amino acid sequence to other endothelin analogs, especially endothelin-1 (2/3 of 21 amino acids are different in sequence) and specific. Antibody could not be obtained.

  Therefore, only a method based on gene expression has been known as a method for detecting endothelin-2 / VIC. For future research, it is essential to develop a more rapid, simple and sensitive detection method using a specific antibody. The problem of the present invention is to produce an antibody specific to endothelin-2 / VIC, It is intended to enable detection of endothelin-2 and / or VIC simply and with high sensitivity.

As a result of intensive studies, the present inventors have studied various antigens or immunization methods against animals, and succeeded in obtaining polyclonal antibodies and monoclonal antibodies having binding specificity to endothelin-2 and / or VIC.
That is, the present invention relates to the following (1) to (5) .

（２） キャリアタンパク質を結合せしめた、エンドセリン−２及びエンドセリン−２の部分ペプチド、又はキャリアタンパク質を結合せしめた、VIC及びVICの部分ペプチドを動物に免疫して、免疫細胞を採取し、該免疫細胞と骨髄腫細胞とを融合させ、得られる融合細胞の中から、エンドセリン−２及びVICに対して特異的に反応する抗体を産生する細胞を選別し、選別された融合細胞を培養して、上記（１）に記載モノクローナル抗体を得る方法であって、エンドセリン−２の部分ペプチドが、以下ａ）のアミノ酸配列からなる環状ペプチドであり、ＶＩＣの部分ペプチドが以下ｂ）のアミノ酸配列からなる環状ペプチドであることを特徴とする、上記（１）に記載のモノクローナル抗体の製造方法。

（３） 以下ａ）又はｂ）のアミノ酸配列からなる環状ペプチドを有する、上記（１）に記載のエンドセリン−２及びVICに対する特異的抗体の製造用ハプテン。

（４） 上記（１）に記載の抗体あるいはこれらの標識化抗体を主成分として含有する、エンドセリン−２及び／又はVICの定量試薬。
（５） 担体上に上記（１）に記載の抗体を結合して不溶化せしめたことを特徴とする、エンドセリン−２及び／又はVICの定量用担体。 (1) Any of endothelin-1 and endothelin-3 does not recognize, a monoclonal antibody having specific reactivity to endothelin-2 and VIC, cyclic peptide consisting of the following a) or b) amino acid sequence The above-mentioned antibody, characterized by recognizing

(2) Immunizing animals with endothelin-2 and endothelin-2 partial peptides bound with carrier protein, or VIC and VIC partial peptides bound with carrier protein, and collecting immune cells, Cells and myeloma cells are fused, and from the resulting fused cells, cells that produce antibodies that specifically react with endothelin-2 and VIC are selected, and the selected fused cells are cultured, a method of obtaining a description monoclonal antibody in the above (1), partial peptides of the endothelin-2 is a cyclic peptide comprising the amino acid sequence: a) an annular partial peptide of the VIC comprises the amino acid sequence: b) characterized in that it is a peptide, the method of producing a monoclonal antibody according to the above (1).

(3) A hapten for producing a specific antibody against endothelin-2 and VIC according to (1) above , which has a cyclic peptide consisting of the amino acid sequence of a) or b) below.

(4) A quantitative reagent for endothelin-2 and / or VIC containing the antibody according to (1) above or a labeled antibody as a main component.
(5) A carrier for quantification of endothelin-2 and / or VIC, wherein the antibody according to (1) above is bound on a carrier and insolubilized.

The polyclonal antibody and the monoclonal antibody of the present invention have a high binding ability to endothelin-2 or VIC, and are extremely useful in that endothelin-1 is not recognized, which has been difficult to separate. Therefore, the above-mentioned antibody of the present invention can be advantageously used for the detection and purification of endothelin-2 and / or VIC and its partial peptides by immunoassay. In particular, endothelin-2 and VIC are obtained by immunoassay by sandwich method using an antibody having binding ability to a partial region of endothelin-2 or VIC and an antibody having binding ability to a region different from the partial region. / Or VIC can be quantified with extremely high sensitivity.

  Hereinafter, the present invention will be described in more detail. In this specification, when amino acids, peptides, protecting groups, active groups, and the like are indicated by abbreviations, these are abbreviations by IUPAC-IUB Commission on Biological Nomenclature or commonly used in the field. Based on the abbreviations, examples are given below. In addition, when there are optical isomers with respect to amino acids and the like, L form is shown unless otherwise specified.

(Abbreviation)
Trp: Tryptophan
Ile: Isoleucine
Leu: Leucine
Asp: Aspartic acid
Pro: Proline
Thr: Threonine
Gly: Glycine
His: Histidine
Cys: cysteine
Arg: Arginine
Ser: Serine
Met: methionine
Asn: Asparagine
Lys
Glu: Glutamic acid
Gln: Glutamine
Val: Valin
Tyr: tyrosine
Phe: Phenylalanine
Boc: t-butoxycarbonyl
Tos
OBzl: benzyl ester
MeBzl: 4-methylbenzyl
Bzl: benzyl
Cl-Z: 2-chlorobenzyloxycarbonyl
Br-Z: 2-bromobenzyloxycarbonyl
Acm: Acetamidomethyl
TFA: trifluoroacetic acid
TEA: Triethylamine
PAM: 4- (oxymethyl) phenylacetamidomethyl
pTs ・ OH: p-Toluenesulfonic acid
HONB: N-hydroxy-5-norbornene-2,3-dicarboximide
ONB: N-hydroxy-5-norbornene-2,3-dicarboximide ester
DMF: N, N'-dimethylformamide
DCC: N, N'-cyclohexylcarbodiimide
WSCD · HCl: 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride

  The monoclonal antibody or polyclonal antibody of the present invention is an endothelin analog, in particular, endothelin-3, and also endothelin-1 can be effectively distinguished (= not bound). Therefore, by using the antibody of the present invention, an immunoassay system for endothelin-2 / VIC is established, and histochemical knowledge can be obtained. In addition, endothelin-2 / VIC can be isolated and purified by immunoadsorption chromatography using the antibody. These facts are very useful for the physiological significance of endothelin-2 / VIC, the explanation of its relation to various diseases, and the research and development of these diagnostic and therapeutic agents.

  Preparation of the polyclonal antibody of the present invention was extremely difficult. Because, generally, a complex of an immune antigen endothelin-2 / VIC and a carrier protein is formed, and this is inoculated into an animal to immunize, and an anti-endothelin-2 / VIC antibody-containing material is collected from the immunized animal, When this is separated and purified, it becomes an antibody that also recognizes endothelin-1. Therefore, specific immunizing antigen ET-2 (3-11) / VIC (3-11) Claims 9 and 10 are synthesized, and immunized with them, and endothelin-1 is not recognized. An antibody that specifically binds only to was obtained. In preparing the monoclonal antibody of the present invention, an individual with high antibody titer is selected from the above immunized animals, the spleen or lymph nodes are collected 3-5 days after the final immunization, and the antibody producing cells contained therein are myeloma cells. By selecting a hybridoma that stably produces a high-titer antibody and obtaining a monoclonal hybridoma.

As an immunizing antigen, in addition to ET-2 (3-11) / VIC (3-11), a peptide containing it corresponds. For example, endothelin-2 / VIC, big-endothelin-2 / big-VIC, endothelin-2 precursor / VIC precursor and parts thereof are included therein. However, as a practical matter, a peptide longer than ET-2 (3-11) / VIC (3-11) increases the common part with ET-1, and the specificity is drastically reduced. Various peptides used in the present invention can be produced by known conventional means of peptide synthesis. Either a solid phase synthesis method or a liquid phase synthesis method may be used.
When ET-2 (3-11) / VIC (3-11) and peptides containing it are synthesized by the solid phase method, Maryfield's solid phase peptide synthesis method [J.Am.Chem. Soc.), 85, 2149 (1963)]. Any insoluble resin known in the art may be used, such as a chloromethylated styrene-divinylbenzene copolymer, a phenacylacetic methylated styrene-divinylbenzene copolymer, etc. Polyamide type resins such as polystyrene type resin and polydimethylacrylamide resin can be mentioned.

  After binding the N-protected amino acid at the C-terminal to the insoluble resin, the protected amino acids are sequentially bonded from the C-terminal side of ET-2 (3-11) / VIC (3-11) and the peptide containing the same according to a conventional method Then, after treatment with hydrogen fluoride, a disulfide bond is formed, and the target ET-2 (3-11) / VIC (3-11) and a peptide containing the same can be synthesized. As N-protected amino acids, all α-amino groups are protected with a Boc group, the hydroxyl groups of serine and threonine are Bzl groups, ω-carboxylic acids of glutamic acid and aspartic acid are OBzl groups, and the ε-amino group of lysine is Cl. -Z group, cysteine thiol group is preferably Acm group, MeBzl group, tyrosine hydroxyl group is Br-Z group, histidine imidazole group and arginine guanide group are preferably protected by Tos group, and tryptophan indole group is preferably protected by CHO group. .

  As a means of synthesis by the liquid phase method, for example, “The Peptides”, Volume 1 (1966), by Schroder and Lubke, Academic Press, New York, USA or “peptide synthesis”, by Izumiya et al., The methods described in Maruzen Co., Ltd. (1975), such as the azide method, chloride method, acid anhydride method, mixed anhydride method, DCC method, active ester method, method using Woodward reagent K, carbodiimidazole method, Examples thereof include a redox method and a DCC / additive (eg, HONB, HOBt, HOSu) method.

Regarding ET-2 (3-11) / VIC (3-11) used to immunize mammals and protein complexes of peptides containing them and carrier proteins, the types of carrier proteins and carriers and haptens (in this case) As long as the antibody can be efficiently produced against the hapten immunized by coupling to a carrier, any ratio can be coupled, for example, bovine serum albumin or bovine serum A method is used in which thyroglobulin, hemocyanin, etc. are coupled at a weight ratio of 0.1-20, preferably 1-5, to one hapten.
In addition, various condensing agents can be used for coupling the hapten and the carrier, but glutaraldehyde, carbodiimide, maleimide active ester, and the like are conveniently used.

The condensation product is administered to a warm-blooded animal by itself, together with a carrier or diluent, at a site where antibody production is possible by administration, and subcutaneous injection is particularly preferable. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2-6 weeks, about 3-6 times in total.
Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens.
The antibody is collected from blood, ascites (preferably blood) of a warm-blooded animal immunized by the above method. The anti-endothelin-2 / VIC antibody titer in the antiserum is measured by, for example, reacting the labeled endothelin-2 / VIC described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The Separation and purification of antibodies include immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation, gel A specific purification method in which only a specific antibody is collected by a filtration method, an antigen-antibody conjugate or an active adsorbent, and the binding is dissociated to obtain an antibody].

The antibody thus prepared contains IgG as a main component and includes other immunoglobulins such as IgM and IgA. In addition, it specifically binds to endothelin-2 / VIC and does not bind to endothelin-1.
On the other hand, a warm-blooded animal immunized in the same manner as in the above polyclonal antibody preparation, for example, an individual with an antibody titer selected from a mouse, and a spleen or lymph node is collected 2-5 days after the final immunization. Anti-endothelin-2 / VIC antibody-producing hybridomas can be prepared by fusing the antibody-producing cells contained with myeloma cells. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is particularly preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) used to the number of bone marrow cells is about 1: 1-20: 1, PEG (preferably PEG1000-PEG6000) is added at a concentration of about 10-80%, Cell fusion can be carried out efficiently by incubating at 20-40 ° C, preferably 30-37 ° C for 1-10 minutes.
Various methods can be used for screening anti-endothelin-2 / VIC antibody-producing hybridomas. For example, hybridoma culture supernatant is added to a solid phase (eg, microplate) to which endothelin-2 / VIC is adsorbed, and then An anti-immunoglobulin antibody labeled with horseradish peroxidase (HRP) (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A is added, and anti-endothelin-2 / bound to a solid phase. Anti-endothelin bound to solid phase by adding hybridoma culture supernatant to EIA method detecting VIC monoclonal antibody, solid phase adsorbed with anti-immunoglobulin antibody or protein A, adding endothelin-2 / VIC labeled with HRP EIA method that detects -2 / VIC monoclonal antibody. Selection and breeding of anti-endothelin-2 / VIC monoclonal antibody is usually performed in animal cell culture medium (eg RPMI1640) containing 10-20% fetal calf serum with addition of HAT (hypoxanthine, aminopterin, thymidine) .

The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-described measurement of the anti-endothelin-2 / VIC antibody titer in the antiserum.
The separation and purification of the anti-endothelin-2 / VIC monoclonal antibody is performed according to the method for separating and purifying immunoglobulin as in the case of the separation and purification of the polyclonal antibody described above.
Using the anti-endothelin-2 / VIC polyclonal antibody obtained above, an antiserum containing the antibody or an anti-endothelin-2 / VIC monoclonal antibody, according to a normal immunoassay method or the like, Tissue staining and the like can be performed. For the immunoassay of endothelin-2 / VIC, the competitive method or sandwich method described below is preferably used.

In the competition method, the anti-endothelin-2 / VIC antibody obtained in the present invention is reacted competitively with the test solution and labeled endothelin-2 / VIC, and then labeled endothelin-2 bound to the antibody. By measuring the ratio of / VIC, endothelin-2 / VIC in the test solution is quantified.
Examples of the endothelin-2 / VIC labeling agent or the antibody labeling agent described below include radioisotopes, enzymes, fluorescent substances, and luminescent substances. Examples of the radioisotope include 125I, 131I, 3H, and 14C, and the enzyme preferably has a stable and high specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, apple Examples of acid dehydrogenase include fluorescent substances such as fluorescamine and fluorescein isothiocyanate, and examples of luminescent substances include luminol, luminol derivatives, luciferin, and lucigenin. Furthermore, a biotin-avidin system can also be used for the binding of the antibody or endothelin-2 / VIC and the labeling agent.

  In measuring the activity of the above-mentioned labeling agent, it is necessary to separate labeled endothelin-2 / VIC bound to the antibody from free labeled endothelin-2 / VIC (hereinafter abbreviated as B / F separation). However, when an enzyme is used as the labeling agent, an active adsorbent such as an antibody against anti-endothelin-2 / VIC antibody insolubilized in the reagent for this purpose or protein A insolubilized is advantageously used. For example, an anti-IgG antibody using an anti-IgG antibody (corresponding to an antibody against anti-endothelin-2 / VIC antibody) as a solid phase, and labeled endothelin-2 / VIC in the solid phase via the above-mentioned antibody reactive with this And the labeling agent on the solid phase is measured. When an enzyme is used as the labeling agent, the usual colorimetric method or fluorescence method is used for measuring the enzyme activity on the insolubilized carrier. When non-protein substances such as radioisotopes are used as the labeling agent, antibodies against anti-endothelin-2 / VIC that do not insolubilize other than the above reagents for B / F separation, sodium sulfate, dextran charcoal, polyethylene glycol, etc. These reagents are used. In any method, the activity of the labeling agent in the supernatant or the sediment is measured.

  In the insolubilization, physical adsorption may be used, or a method using a chemical bond that is usually used to insolubilize and immobilize proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the competition method, the anti-endothelin-2 / VIC antibody, the test solution, the labeled endothelin-2 / VIC, and the B / F separation reagent can be reacted in any order, and all or Some of them may be reacted at the same time, but at least labeled endothelin-2 / VIC should be added to the reaction system simultaneously with the reaction between the test solution and anti-endothelin-2 / VIC antibody or after the reaction solution. Is preferred. However, B / F separation reagents such as sodium sulfate, dextran charcoal, and polyethylene glycol are mainly used at the end of the reaction system.

  In the sandwich method, the test solution is contacted (reacted) with the insolubilized anti-endothelin-2 / VIC antibody (primary reaction), and further reacted with the labeled anti-endothelin-2 / VIC antibody (secondary reaction). The amount of endothelin-2 / VIC in the test solution can be quantified by measuring the activity of the labeling agent on the insolubilized carrier. The primary reaction and the secondary reaction may be performed at the same time or may be performed at different times. The labeling agent and the insolubilization method can be the same as those described above.

  As the anti-endothelin-2 / VIC antibody used in the secondary reaction, an antibody having a different site binding to the antibody of endothelin-2 / VIC from the anti-endothelin-2 / VIC antibody used in the primary reaction is preferably used. It is done. For example, when the antibody used in the primary reaction has the ability to bind to the C-terminal part of endothelin-2 / VIC, in the secondary reaction, anti-endothelin- preferably binds to other than the C-terminal part (eg, N-terminal part). When a 2 / VIC antibody is used and the antibody used in the primary reaction has a binding ability to the N-terminal part of endothelin-2 / VIC, the secondary reaction is preferably other than the N-terminal (eg, C-terminal). Anti-endothelin-2 / VIC antibody that binds to (part). The antibody used for the primary reaction and the secondary reaction may be a polyclonal antibody or a monoclonal antibody, respectively. Preferably, one of them reacts with endothelin-2 / VIC, but the C-terminal part of endothelin-2 / VIC. An anti-endothelin-2 / VIC monoclonal antibody that does not react with the anti-endothelin-2 / VIC, and the other anti-endothelin-2 / VIC polyclonal or monoclonal antibody that reacts with the C-terminal of endothelin-2 / VIC is used.

Particularly preferably, in the immunoassay of endothelin-2 / VIC by sandwich method, anti-endothelin-reactive with the C-terminal peptide of endothelin-2 / VIC, ie, Cys-His-Leu-Asp-Ile-Ile-Trp 2 / VIC polyclonal antibody and anti-endothelin-2 / VIC monoclonal antibody which reacts with endothelin-2 / VIC but does not react with the C-terminal peptide of endothelin-2 / VIC are used.
The same applies to the immunoassay for big-endothelin-2 / big-VIC by the sandwich method.

  Furthermore, the immunoassay using the antibody obtained in the present invention can be used for diagnosis and prognosis management of heart disease, intestinal disease or renal disease. As test samples, body fluids such as plasma, serum, urine, cerebrospinal fluid, ascites, pleural effusion, amniotic fluid, sputum, feces, and the like can be used. These samples can be used as immunoassay samples as they are or after dilution or extraction with various buffer solutions and concentration. Any buffer or organic solvent may be used as a solvent used for sample dilution or extraction, but preferably an immunoassay buffer, water, physiological saline, acetate buffer, acetone, chloroform-methanol, or These solutions containing a surfactant are used. Concentration may be performed by directly concentrating the sample under reduced pressure, normal pressure, or nitrogen flow, or adding the sample to an ion exchange or reverse phase chromatography carrier or an anti-endothelin-2 / VIC antibody binding carrier. Then, after elution with an appropriate eluent, the solution may be concentrated under reduced pressure, normal pressure, or nitrogen stream.

As the concentration carrier, an ODS cartridge as a carrier for reverse phase chromatography is particularly preferably used. The concentrate is dissolved in an immunoassay buffer and used as a sample for immunoassay.
Furthermore, the anti-endothelin-2 / VIC antibody obtained in the present invention can also be used for immunohistochemical staining of endothelin-2 / VIC. The method follows, for example, a direct method using a labeled anti-endothelin-2 / VIC antibody, an indirect method using an anti-endothelin-2 / VIC antibody and a labeled antibody against the anti-endothelin-2 / VIC antibody, etc. be able to.

Furthermore, among the anti-endothelin-2 / VIC antibodies obtained in the present invention, an antibody capable of neutralizing endothelin-2 / VIC's ability to contract blood vessels and intestinal tract can be used as a specific antagonist of endothelin-2 / VIC. .
In addition to being used for immunohistochemical staining, the antibody of the present invention can be used as a diagnostic kit as an ELISA or RIA kit. In the present invention, a sequence that can emphasize an amino acid different from endothelin-1 was selected as an antigen, and in addition to this, VIC (3-11) and VIC were immunized alternately, so that amino acids common to both were used. Have the effect of boosting each other, resulting in highly specific antibodies that recognize the differences. Specificity was measured by ELISA (with RIA). This can be understood from the example shown in the figure. The actual immunostaining is shown in FIGS.

  The present invention will be described in more detail with reference to the following examples, but the present invention should not be limited thereto.

Example (1) Production of Synthetic Peptide i) Synthesis of VIC (3-11) and Peptides Containing It Vic-synthesizer using commercially available Boc-Trp (CHO) -PAM resin (Applied Biosystems) (Applied Biosystems, Model 430A) was used and synthesized by a conventional method. In the condensation method, the Boc group on the resin is treated with 50% trifluoroacetic acid in methylene chloride to release a terminal amino group, and this free amino group is converted into Boc-Ile, Boc-Asp (OBzl), Boc-Leu, Boc-His (Tos), Boc-Cys (Acm), Boc-Tyr (Br-z), Boc-Val, Boc-Phe, Boc-Glu (OBzl), Boc-Lys (Cl-Z), Boc-Met , Boc-Ser (Bzl) was repeatedly condensed in the presence of DCC according to the amino acid sequence of VIC and a part thereof (including VIC (3-11)) from the C-terminal side.

Part of the protected VIC (3-11) and VIC-containing resin thus obtained was swollen with 1 ml of anisole and 1 ml of 1,2-ethanediotil, and treated with 10 ml of hydrogen fluoride at 0 ° C. for 60 minutes. Excess hydrogen fluoride was distilled off under reduced pressure. The residue was washed with 5 ml of ethyl acetate, extracted into 50% aqueous acetic acid, applied to a dextran gel (Sephadex LH-20) column (2 × 90 cm), and the main fraction eluted with the same solvent was collected and frozen. Drying gave a white powder. A part of this was dissolved in 20% 80% acetic acid water, mercuric trifluoroacetate was added, stirred at room temperature for 60 minutes, diluted with the same solvent, the precipitate was filtered off through hydrogen sulfide gas, Lyophilized. This was dissolved in dilute acetic acid, adjusted to pH 8 with ammonium bicarbonate, subjected to air oxidation for 6 hours, adjusted to pH 3 with acetic acid, and then lyophilized. This was applied to a Sephadex LH-20 column (2 × 90 cm) packed with 30% -acetic acid, and the main fraction was collected, and further HPLC (column: YMC, solvent: 0.1% -trifluoroacetic acid water and 0.1% -The product was fractionated with a linear concentration gradient elution of acetonitrile containing trifluoroacetic acid to obtain the desired product.

Measurement condition column: Nucleosyl 50DS-H (4.6 mmφ × 250 mm) manufactured by Chemco
Eluent: Liquid A (0.1% -trifluoroacetic acid water)
Liquid B (0.1%-trifluoroacetic acid-50% water-containing acetonitrile)
Elution of linear concentration gradient from solution A to solution B (20 minutes)
Flow rate: 1.0ml / min Measurement condition column: Toyo Soda DEAE-2SW (4.6mmφ × 250mm)
Eluent: Liquid A (10 mM Tris / hydrochloric acid pH 7.5)
B liquid (1M NaCl-containing A liquid)
Elution from solution A to solution B using linear gradient elution (40 minutes)
Flow rate: 1.0ml / min

(2) Production of immunogen (i) Immunogen (I)
VIC (3-11) and a peptide containing the same [obtained in (1) above] and KLH were bound using glutaraldehyde. That is, 4.5 mg of the polypeptide and 13.5 mg of TG were dissolved in 4.5 ml of 0.2 M phosphate buffer pH 7.0, glutaraldehyde having a final concentration of 0.2% was added, and the mixture was reacted at room temperature for 3 hours. After the reaction, it was dialyzed against physiological saline at 4 ° C. for 2 days.
The amino acid sequence and structure of the synthesized hapten VIC (3-11) and hapten VIC (3-11) -KLH used as an immunogen are shown in FIG. 1 together with VIC, ET-2, ET-1, and ET-3. Show.

(Ii) Immunization Add 550 μl of Freund complete adjuvant to 450 μl of physiological saline containing 400 μg of the immunogen (I) obtained in (2) above and mix well to prepare an emulsion. I was inoculated in one place. Six weeks later, an emulsion was prepared in the same manner using incomplete Freund's adjuvant and inoculated subcutaneously into rabbits. This procedure is performed 3 times every other month [immunogen (I)] 4 times [immunogen (II)] or 4 times [immunogen (III)], and 7 days after the booster, blood is partially collected from the rabbit. Antiserum was obtained by a conventional method. Similarly, 6-8 week old BALB / C female mice were immunized subcutaneously with an immunogen (I) 70 μg / mouse together with an adjuvant. Thereafter, booster immunization was performed 2-3 times every 3 weeks, and partial blood was collected 7 days after immunization to obtain antiserum.

(4) Preparation of enzyme-labeled antigen i) Preparation of peroxidase-labeled endothelin-2 / VIC Endothelin-2 / VIC 210 nmole was dissolved in 450 μl of 0.1 M phosphate buffer, pH 7.0, and GMBS 295 μg (1.05 μmole) The mixture was mixed with 50 μl of DMF solution containing and reacted at room temperature for 30 minutes. After the reaction, fractionation was performed using a Sephadex G-15 column to obtain 100 nmole of a polypeptide having a maleimide group introduced therein. On the other hand, horseradish peroxidase 10 mg (250 nmole) was dissolved in 0.02 M phosphate buffer solution, pH 6.8, 1.4 ml containing 0.15 M sodium chloride, mixed with 100 μl of DMF solution containing 1.17 mg (3.75 μmole) SPDP, and room temperature. The reaction was allowed for 40 minutes. After the reaction, 0.1M acetic acid buffer solution containing 12.4 mg (80 μmole) of dithiothreitol, pH 4.5, 0.5 ml was added and reacted at room temperature for 20 minutes, followed by fractionation with a Sephadex G-25 column. 6 mg (150 nmole) of the introduced enzyme was obtained. Next, maleimide group-introduced endothelin-2 / VICI 50 nmole and SH group-introduced peroxidase 10 nmole were mixed and reacted at 4 ° C. for 16 hours. After the reaction, fractionation was performed using a Ultrogel AcA44 (manufactured by LKB-Pharmacia) column to obtain peroxidase-labeled endothelin-2 / VIC.

(5) Measurement of antibody titer The antibody titer in rabbit antiserum against the immunogen (I) was measured by the following method. That is, 100 μl each of 0.1 M carbonate buffer solution and pH 9.6 solution containing 20 μg / ml of anti-rabbit IgG antibody (IgG fraction, manufactured by Kappel) was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. After washing the plate with phosphate buffered saline (PBS), 300 μl of 1% BSA-containing PBS was dispensed in order to block excess binding sites in the wells, and treated at 4 ° C. for 24 hours. Rabbit antiserum diluted in buffer A (0.02M phosphate buffer, 1% BSA, pH 7.0) (6 chickens, No.1a-No.6a) and the above-mentioned peroxidase label on the plate prepared as described above Endothelin-2 / VIC (I) (diluted 100-fold with buffer A) (50 μl) was added, and the mixture was reacted at 4 ° C. for 16 hours. After the reaction, wash well with PBS, and dispense 100 μl of 0.1 M citrate buffer, pH 5.5 containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide to measure enzyme activity on the solid phase. For 10 minutes. After adding 100 μl of 4N sulfuric acid and stopping the reaction, the absorbance at 492 nm was measured with a plate reader (MTP-32, manufactured by Corona) to determine the activity of the antibody.

  The antibody titer in the mouse antiserum against the immunogen (I) was measured by the following method. That is, 100 μl of 0.1 M carbonate buffer and ph9.6 solution containing 20 μg / ml of anti-mouse IgG antibody (IgG fraction, H chain, L chain specific Cappel) was dispensed into a 96-well microplate. It was left at 24 ° C. for 24 hours. After the plate was washed with PBS, 300 μl of 1% BSA-containing PBS was dispensed in order to block excess binding sites in the wells and treated at least at 4 ° C. for 24 hours. 50 μl of mouse antiserum diluted with buffer A (8 mice, No.1a-No.8a) and the above-mentioned peroxidase-labeled endothelin-2 / VIC (I) (100 times with buffer A) were prepared on the plate prepared as described above. Dilution) 50 μl was added and reacted at 4 ° C. for 16 hours. After the reaction, the plate was thoroughly washed with PBS, and the enzyme activity on the solid phase was measured by the method described above. Further, anti-endothelin-2 / VIC antibody-producing hybridomas were selected by measuring the antibody titer in the hybridoma culture supernatant by the same method.

(6) Preparation of affinity solid phase (i) Affinity solid phase (I)
A condensate of the polypeptide Cys His Leu Asp Ile Ile Trp and human serum albumin (hereinafter abbreviated as HSA) was bound to CNBr activated Sepharose 4B (Pharmacia) to obtain an affinity solid phase (I). That is, according to the method described in (2) above, 20 mg of HSA and 2.1 mg of GMBS were reacted at room temperature for 60 minutes, and then fractionated on a Sephadex G-25 column. Next, 1 ml of the elution fraction containing 5 mg of HSA introduced with a maleimide group and 1 mg of the polypeptide Cys His Leu Asp Ile Ile Trp dissolved in 1 ml of an aqueous solution containing 90% dimethyl sulfoxide are reacted at 4 ° C. for 3 days. I let you. After the reaction, the mixture was dialyzed against 0.1M sodium hydrogen carbonate containing 0.5M sodium chloride, and then reacted with CNBr-activated Sepharose 4B1g for 3 hours at room temperature. Next, unreacted active groups were treated with 0.1 M Tris-HCl buffer, pH 8, washed, dispersed in PBS containing 1% BSA, and stored at 4 ° C.

(7) Purification of anti-endothelin-2 / VIC polyclonal antibody i) Purification by anion exchange chromatography Rabbit anti-endothelin-2 / VIC antiserum was salted out and fractionated by DEAE-cellulose column chromatography. Anti-endothelin-2 / VIC antibody was purified to IgG fraction. That is, 10 ml of PBS was added to 10 ml of rabbit anti-endothelin-2 / VIC antiserum No. 1a, and further 16.5 ml of saturated ammonium sulfate was added while gradually stirring (final 45%). After standing for 30 minutes, 20 ml at 12,000 × g. Centrifugation was performed for 30 minutes, and the precipitate was dissolved in 10 ml of PBS. Next, similarly, saturated ammonium sulfate was added to a final saturation of 30%, followed by centrifugation. The precipitate was dissolved in 0.01 M borate buffer containing 0.15 M salt, pH 8 (hereinafter abbreviated as BBS), 10 ml, and then dissolved in 0.01 M phosphate buffer pH 8 (buffer B) containing 0.01 M salt. Dialysis was performed at ℃ for 2 days. Next, the antibody fraction after salting-out and dialysis was added to a DEAE-cellulose column (Whatman, DE-52, 20 mmφ × 100 mm) previously equilibrated with buffer B. After washing the column with buffer B, the antibody was eluted using a continuous ionic strength gradient of buffer B-buffer C (0.01 M phosphate buffer containing 0.35 M NaCl, pH 8). The antibody titer in the eluted fraction was measured according to the method described in (5) above.

  Rabbit immunoglobulin was detected in the same manner as described in (5) using antibodies specific for each immunoglobulin on the solid phase. That is, an anti-rabbit IgGFc component or anti-rabbit IgM (Cappel, goat antibody, IgG fraction) was immobilized on a microplate according to the method described in (5). Next, 50 μl of each fraction of DEAE-cellulose eluted anti-endothelin-2 / VIC antibody diluted 102-107 times with buffer A and 50 μl of peroxidase labeled endothelin-2 / VIC (diluted 100 times with buffer A) were added, The reaction was performed at 4 ° C. for 16 hours. After washing after the reaction, the enzyme activity on the solid phase was measured. As a result, the strongest reaction was observed when the anti-rabbit IgGFc component was used as a solid phase, but a specific reaction was also observed when anti-rabbit IgM was used as a solid phase.

ii) Purification by affinity solid phase Antibody was partially purified from rabbit antiserum against immunogen (I) by ammonium sulfate salting-out. The antibody fraction was dialyzed against BBS and then applied to a column (10 mmφ × 40 mm) packed with the affinity solid phase (I). After thoroughly washing with BBS, the specific antibody was eluted with 0.1 M acetate buffer containing 0.5 M sodium chloride, pH 4.5, and further eluted with 0.05 M glycine-hydrochloric acid buffer containing 0.1 M sodium chloride, pH 2.0. As a result of measuring the antibody titer in the eluted fraction by the E1A method described in (5) above, a strong antibody titer was observed only in the fraction eluted at pH 2, and a specific antibody was obtained from this fraction.

(8) Cell fusion Final immunization was performed by inoculating a mouse showing a relatively high antibody titer intravenously with 240 µg of immunogen (I) dissolved in 0.25 ml of physiological saline. The spleen was removed from the mouse 3 days after the final immunization, pressed with a stainless steel mesh, filtered, and suspended in Eagles, Minimum, Etsensial Medium (MEM) to obtain a spleen cell suspension. BALB / C mouse-derived myeloma cells P3-x63.Ag8.U1 (P3U1) were used as cells used for cell fusion [Current Topics in Microbiology and Immunology, 81, 1 (1978)]. Cell fusion was performed according to the original method [Nature, 256, 495 (1975)]. That is, spleen cells and P3U1 were each washed 3 times with MEM without serum, mixed so that the ratio of spleen cells and P3U1 number was 5: 1, and centrifuged at 800 rpm for 15 minutes to precipitate the cells. It was. After sufficiently removing the supernatant, the precipitate was lightly loosened, 0.3 ml of 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite) was added, and the mixture was allowed to stand in a 37 ° C. hot water bath for 7 minutes for fusion. After fusion, MEM was added to the cells at a rate of 2 ml / min, and a total of 12 ml of MEM was added, followed by centrifugation 15 times for 600 minutes to remove the supernatant.

  This cell precipitate was suspended in RPMI1640 medium (RPMI1640-10FCS) containing 10% fetal bovine serum so that P3U1 would be 2 × 10 6 per ml, and 1 ml per well in a 24-well multi-disc (Limbro) 120 Wells were seeded. After seeding, the cells were cultured at 37 ° C. in a 5% carbon dioxide furan vessel. After 24 hours, add 1 ml per well of RPMI1640-10FCS medium (HAT medium) containing HAT (hypoxanthine 1 × 10-4M, aminobruterin 4 × 10-7M, thymidine 1.6 × 10-3M) HTA selective culture was started. The HAT selective culture was continued by discarding 1 ml of the old solution 3, 6 and 9 days after the start of the culture and then adding 1 ml of HAT medium. Hybridoma growth was observed 9-14 days after cell fusion, and when the culture broth turned yellow (about 1 × 10 6 cells / ml), the supernatant was collected, and the EIA method described in (5) above was used. The antibody titer was measured. Thus, when the supernatant of all 120 wells in which hybridoma growth was observed was examined, strong antibody activity was observed.

(9) Cloning Each hybridoma in a well showing a positive antibody activity was subjected to cloning by limiting dilution. That is, the cells were suspended in RPMI 1640-20FCS so that the number of hybridomas was 1.5 / ml, and 0.2 ml per well was dispensed into a 96-well microplate (manufactured by Nunk). At the time of dispensing, BALB / C mouse thymocytes were added at 5 × 10 5 cells per well as feeder cells. After about 1 week, cell proliferation was observed, and the antibody titer in the supernatant was examined by the EIA method. As a result, antibody activity was observed. Paying attention to these clones and the monoclonal antibodies produced by them, the following experiments were conducted.

(10) Preparation of a large amount of monoclonal antibody After intraperitoneal injection of hybridoma AwETN40 1-3 × 10 6 cells / mouse into mice administered 0.5 ml of mineral oil intraperitoneally or untreated mice (BALB / C), 10 − After 30 days, antibody-containing ascites was collected.

(11) Purification of monoclonal antibody Ascites fluid described in (10) above was salted out according to the method described in (7) above, and then fractionated by DEAE-cellulose column chromatography to purify the monoclonal antibody. That is, 7 ml of PBS was added to 7 ml of ascites, and 11.5 ml of saturated ammonium sulfate (final 45%) was added while gradually stirring. The precipitate was dissolved in BBS, dialyzed against 0.01 M phosphate buffer pH 8 containing 0.01 M sodium chloride, and then applied to a DEAE-cellulose column (20 mmφ × 100 mm). By eluting the antibody with a 0.01M-0.35M saline concentration gradient, 7 ml to 70 mg of ascites monoclonal antibody was obtained as a purified preparation.

(13) Examination of neutralizing activity of monoclonal antibody About 2cm spiral strips extracted from rat myometrium are subjected to Krebs-Henseleit solution (hereinafter abbreviated as nutrient solution) under aeration of mixed gas (95% O2 + 5% CO2). Suspended in a Magnus tube filled with After standing at 37 ° C. for 3 hours, the tension generated by contraction of intestinal smooth muscle was measured with an isometric transducer (Polygraph, manufactured by NEC Sanei Co., Ltd.). The tension generated even when an endothelin-2 / VIC solution (final endothelin-2 / VICI concentration 1 × 10-8M) reacted with the sample at 4 ° C. for 3 hours is 8 tensions induced by 60 mM KCl. % (N = 4), as a control, when endothelin-2 / VIC solution (final 1 × 10-8M) was added, tension due to contraction almost equal to 60 mM KCl was observed. .
From the above, it was revealed that endothelin-2 / VIC neutralizes intestinal smooth muscle contractile activity.

(14) Competitive method-EIA
1) EIA using polyclonal antibody against immunogen (I)
On an anti-rabbit IgG-binding microplate, 50 μl of rabbit anti-endothelin-2 / VIC serum (see FIG. 1) against immunogen (I) finally diluted 200,000 times with buffer A, and 50 μl of endothelin-2 / VIC standard solution, Polypeptide Cys His Len Asp Ile Ile Trp standard solution 50 μl or endothelin-1 (purchased from Peptide Institute, Inc.) standard solution 50 μl was added and reacted at 4 ° C. for 16 hours. After that, 50 μl of peroxidase-labeled endothelin-2 / VIC (diluted 300-fold with buffer A) was added and reacted at room temperature for 4 hours. After the reaction, the plate was thoroughly washed with PBS, and the enzyme activity on the solid phase was measured by the method described above. Results are shown in FIG. 2C .
In the figure,-●-indicates the standard curve of endothelin-2 / VIC,-▲-indicates the standard curve of the polypeptide Cys His Leu Asp Ile Ile Trp, and -O- indicates the standard curve of endothelin-1. B on the vertical axis represents the enzyme activity on the solid phase in the presence of an antigen such as endothelin-2 / VIC, endothelin-1, etc., and B0 represents the enzyme activity on the solid phase in the absence of antiserum. The conventional measurement method using biological activity as an index is expected to be affected by other vasoconstrictors such as endothelin-1, but the above results use the anti-endothelin-2 / VIC antibody of the present invention. The immunoassay shows that endothelin-2 / VIC can be specifically measured without being affected by endothelin-1.

(15) Sandwich method-EIA (Part 1)
The sandwich method for measuring endothelin-2 / VIC-EIA is described below.
i) Preparation of enzyme-labeled antibody From the affinity-purified anti-Cys His Leu Asp Ile Ile Trp antibody described in (7) ii) above, the method of Ishikawa et al. [Journal of Applied Biochemistry (J. Appl. Biochem)., 6: 56-63 (1984)], Fab′-peroxidase-labeled product was prepared. Specifically, 160 μg of pepsin (Sigma, double crystal) was added to 6.4 mg of a specific antibody dissolved in 0.1 M acetate buffer solution, pH 4.5, reacted at 37 ° C. for 16 hours, and then superrose 12 equilibrated with BBS. F (ab ′) 2 fraction was purified by FPLC (Pharmacia) using a column. The fraction was dialyzed against 0.1 M acetate buffer, pH 5, and finally 20 mM β-mercaptoethylamine was added and left at 37 ° C. for 90 minutes. The reaction solution was separated by FPLC using a Superose 12 column equilibrated with 0.1 M phosphate buffer containing 2.5 mM EDTA, pH 6.0 to obtain Fab ′ fraction. On the other hand, horseradish peroxidase (5 mg) was dissolved in 0.9 ml of 0.1 M phosphate buffer, pH 7, and 1.05 mg of GMBS dissolved in 50 μl of DMF was added and reacted at room temperature for 40 minutes. The reaction solution was separated with a Sephadex G-25 column (eluent 0.1 M phosphate buffer, pH 6.8), and the resulting maleimidated peroxidase 3.5 mg and the Fab ′ fraction 0.8 mg were mixed together and collodion pack. After concentrating to about 0.3 ml with MS Equipment Co., Ltd., it was left at 4 ° C. for 16 hours. The reaction solution was applied to a Ultrogel AcA44 column (10 mmφ × 40 mm) using 0.1 M phosphate buffer, pH 6.5 as an eluent, and the Fab′-peroxidase complex fraction was purified.

ii) Sandwich method-EIA (colorimetric method)
100 μl each of 0.1 M carbonate buffer solution and pH 9.6 solution containing 20 μg / ml of purified monoclonal antibody AwETN40a was dispensed to a 96-well microplate and left at 4 ° C. for 24 hours. The excess binding sites in the wells were inactivated by adding 300 μl of Block Ace (manufactured by Snow Brand Milk Products, sold by Dainippon Pharmaceutical Co., Ltd.) diluted 4-fold with PBS. 100 μl of endothelin-2 / VIC standard solution diluted with buffer A was added to the plate prepared as described above, and reacted at room temperature for 5 hours. After the plate was washed with PBS, 100 μl of anti-Cys His Leu Asp Ile Ile Trp antibody Fab′-peroxidase labeled (diluted 300-fold with buffer A) was added, and reacted at room temperature for 3 hours. After the plate was washed with PBS, the enzyme activity on the solid phase was measured by the method described in (5) above. The results are shown in FIG . Based on the above results, the anti-endothelin-2 / VIC monoclonal antibody that recognizes other than the C-terminal of endothelin-2 / VIC was used as a solid phase, and the polyclonal antibody Fab ′ that recognizes the C-terminal of endothelin-2 / VIC was used. It was revealed that 100 pg / ml, 50 fmole / well of endothelin-2 / VIC can be measured by the sandwich method-EIA (colorimetric method) used for the label.

It is the figure which showed the hapten of this invention, the amino acid sequence of an immunogen, and its structure with the amino acid sequence and structure of endothelin-2, VIC, endothelin-1, and endothelin-3. It is a graph which shows the specificity of ET-2 / VIC antiserum by RIA (radioimmunoassay), and the graph which shows the specificity of the ET-2 / VIC purified antibody by ELISA (Eliza). It is a graph which shows the result of having performed the quantitative analysis of the gene expression of ET-2 / VIC and ET-1 in a mouse | mouth digestive tract. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the colon. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the colon. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the ileum. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the ileum. It is a microscope picture which shows the specific distribution of ET-2 / VIC in a Peyer's board. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the duodenum. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the duodenum. It is a microscope picture which shows the specific distribution of ET-2 / VIC in the stomach. 2 is a graph showing the localization of ET-2 / VIC producing cells and macrophages (CD-68 positive cells).

Explanation of symbols

In FIG.
St stomach, Du duodenum, Je jejunum, Il ileum Co colon, Re rectum

In FIG.
(A, C, E) Distribution of ET-2 / VIC (ET-2 / VIC antibody staining)
(A) Villi, (C) Muscle layer, (E) Cross section of villi
(B, D, F) Distribution of ET-1 (ET-1 antibody staining)
(B) Villi, (D) Muscle layer, (F) Cross section of villi
(GO) ET-2 / VIC partially co-localizes with VIP:
(G, J, M) ET-2 / VIC antibody staining, (H, K, N) VIP antibody staining, (I, L, O) Co-staining of both (colocalization)

In FIG.
(A, C, E) Distribution of ET-2 / VIC (ET-2 / VIC antibody staining)
(A) Villi, (C) Muscle layer, (E) Cross section of villi
(B, D, F) Distribution of ET-1 (ET-1 antibody staining)
(B) Villi, (D) Muscle layer, (F) Cross section of villi
(GO) ET-2 / VIC partially co-localizes with VIP:
(G, J, M) ET-2 / VIC antibody staining, (H, K, N) VIP antibody staining, (I, L, O) Co-staining of both (colocalization)

In FIG.
(A, C, D) Distribution of ET-2 / VIC (ET-2 / VIC antibody staining) (B) Distribution of ET-1 (ET-1 antibody staining)
(E) Localization of ET-2 / VIC in M cells (blue: ET-2 / VIC antibody and red: UEA-I lectin double staining)

In FIG.
(A) ET-2 / VIC distribution (ET-2 / VIC antibody staining), (B) ET-1 distribution (ET-1 antibody staining), (CF) in situ hybridization: (C) Sense probe (control) ) 40 times, (DE) antisense probe (detects mRNA), (D) 40 times, (E) 100 times, (F) 200 times

In FIG.
(A) ET-2 / VIC distribution (ET-2 / VIC antibody staining), (B) ET-1 distribution (ET-1 antibody staining),

In FIG.
(A) Colon, (B) Ileum, ET-2 / VIC antibody staining (green), CD68 antibody staining (red)

Claims (5)

A monoclonal antibody that does not recognize either endothelin-1 or endothelin-3 but has specific reactivity with endothelin-2 and VIC, and recognizes a cyclic peptide consisting of the amino acid sequence of a) or b) below. The antibody described above.

Endothelin-2 and partial peptide of endothelin-2 bound with carrier protein, or VIC and partial peptide of VIC bound with carrier protein are immunized to animals, immune cells are collected, and the immune cells and bone marrow A cell producing an antibody that specifically reacts with endothelin-2 and VIC is selected from the obtained fused cells by fusing the tumor cells, and the selected fused cells are cultured, Wherein the partial peptide of endothelin-2 is a cyclic peptide consisting of the amino acid sequence of a) below, and the partial peptide of VIC is a cyclic peptide consisting of the amino acid sequence of b) below The method for producing an antibody according to claim 1, wherein:

The hapten for producing a specific antibody against endothelin-2 and VIC according to claim 1 , comprising a cyclic peptide consisting of the amino acid sequence of a) or b) below.

  A quantitative reagent for endothelin-2 and / or VIC containing the antibody according to claim 1 or a labeled antibody thereof as a main component.   A carrier for quantification of endothelin-2 and / or VIC, wherein the antibody of claim 1 is bound and insolubilized on a carrier.

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