JPH06157592A - Peptide or its derivative, combination thereof with protein and production of antiendothelin-1 antibody using the same as immunogen - Google Patents

Abstract

PURPOSE:To obtain an immunogenic peptide (protein) for obtaining an antiendothelin-1 antibody binding to only a specific antigenic determinant (a loop structure between cysteines at the 3rd and 11th positions) of the endothelin-1 molecule and to provide a method for producing the antiendothelin-1 antibody using the immunogenic peptide as the immunogen. CONSTITUTION:A chemically synthesized peptide Ac-Ser-Ser-Leu-Met-Asp-Lys- Glu (Ac indicates that the amino group at the N-terminal serine is acetylated) is bound to a protein [keyhole limpet hemocyanin(KLH) or bovine serum albumin(BSA)] according to a carbodiimide method. The peptide-binding protein is bound to an antiendothelin-1 polyclonal antibody without binding to a commercially available murine monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention has a partial sequence of the amino acid sequence of endothelin-1, but does not have the physiological activity specific to endothelin, and has immunogenicity for producing anti-endothelin-1 antibody in animals. The present invention relates to a peptide or derivative thereof, a conjugate thereof with a protein, and a method for producing an anti-endothelin-1 antibody using these. The peptide and its derivative of the present invention serve as an immunogen for producing anti-endothelin-1 antibody, and are also useful as a ligand for the purification of anti-endothelin-1 antibody or a detection reagent.

[0002]

BACKGROUND OF THE INVENTION Endothelin-1 is a peptide consisting of 21 amino acid residues, as shown in FIG. 1, which has the strongest vascular smooth muscle contracting activity among the physiologically active substances discovered so far. (Yanagisawa, M. et a
l.:Nature, 1988, 332 , 373).

It is known that the blood level of endothelin-1 has a strong correlation with pathological conditions / diseases such as hypertension, renal failure and respiratory diseases (Shichiri, M. et.
al.:Hypertension, 1990, 15 , 493; Kohno, M. et al .:
Am. J. Med., 1990, 88 , 614).

Conventionally, there have been known methods for measuring endothelin-1 in blood, such as a radioimmunoassay method and an enzyme immunoassay method, all of which use an anti-endothelin-1 antibody as a specific detection reagent. Of the latter enzyme immunoassay methods, those using an enzyme-labeled antibody often use a method called a sandwich method. In this method, endothelin-1 in a sample is captured by an anti-endothelin-1 antibody (primary antibody) immobilized on a solid phase such as an immunoplate, and then another enzyme-labeled anti-endothelin-1 antibody (secondary antibody) is used. (Antibody) is added and sandwiched, a color-forming or luminescent substrate is added, and the generated color or luminescence amount is measured.

In order to prepare an anti-endothelin-1 antibody, conventionally, endothelin-1 itself having a complete amino acid sequence as shown in FIG. 1 or its binding to a protein such as albumin is used as an antigen. A method of immunizing animals has been adopted (JP-A-2-150617, JP-A-2-238894).

[0006]

However, endothelin-1 itself having the complete amino acid sequence as shown in FIG. 1, or a method of binding this to a protein and immunizing an animal with this as an antigen, endothelin- An antibody reactive with a plurality of antigenic determinants in one molecule was obtained, and an antibody reactive only with a specific antigenic determinant could not be obtained. Therefore, in order to obtain an antibody that reacts only with a specific site of endothelin-1, it is necessary to perform complicated separation and purification in which only the desired antibody is extracted in the stage after antibody production. Another problem was that endothelin-1 itself or, when bound to a protein and used as an antigen, often killed an immunized animal due to the strong pressor action of endothelin-1.

The present invention uses an immunogenic peptide for obtaining an anti-endothelin-1 antibody which reacts only with a specific antigenic determinant in an endothelin-1 molecule, and uses it as an immunogen (no need for complicated separation and purification). It is an object to provide a method for producing an anti-endothelin-1 antibody.

[0008]

In order to achieve the above object, first of all, the inventors of the present invention, in order to find a highly antigenic region in an endothelin-1 molecule, an endothelin-1 partial sequence peptide consisting of 6 amino acid residues. Was synthesized on a polyethylene rod while shifting each residue from the amino terminal side, and the synthesized peptide was synthesized as a known anti-endothelin-1.
Ser-Leu-Met-Asp- was detected by reacting with a polyclonal antibody and examining peptides with high affinity for the polyclonal antibody.
It was found that peptides having the sequences Lys-Glu and Ser-Ser-Leu-Met-Asp-Lys-Glu have the highest affinity for the antibody. The present invention has been completed based on this finding.

That is, the present invention provides the following (1) to (6):
(1) A peptide represented by Ser-Ser-Leu-Met-Asp-Lys-Glu (I). (2) A peptide represented by Ser-Ser-Leu-Met-Asp-Lys (II). (3) A peptide represented by Ser-Leu-Met-Asp-Lys-Glu (III). (4) A part or all of the amino group or carboxyl group at the terminal of the peptide of any one of (1) to (3) above, or the amino group, the carboxyl group or the hydroxyl group of the side chain is optionally blocked or protected. Peptide derivative. (5) An anti-endothelin-1 antibody-inducible protein obtained by binding the peptide according to any one of (1) to (3) above or the peptide derivative according to (4) above to the protein. (6) An anti-endothelin-containing method comprising immunizing an animal with the peptide according to any one of (1) to (3) above, the peptide derivative according to (4) above or the anti-endothelin-1 antibody-inducible protein according to (5) above. 1 Method for producing antibody. In addition, in the present specification, unless otherwise specified, an amino acid is a conventional 3
Each amino acid is represented by a letter, each amino acid is in the L-form, and the peptide is shown with the N-terminal on the left side and the C-terminal on the right side according to the conventional notation.

Ser-Ser-Leu-Met-Asp-Lys-Glu of the present invention
The peptide represented by (I) is endothelin-1 (Fig. 1).
Is a peptide having a partial sequence from the 4th serine to the 10th glutamic acid. Similarly, the Ser-Se of the present invention
The peptide represented by r-Leu-Met-Asp-Lys (II) is a peptide having a partial sequence from the 4th serine to the 9th lysine of endothelin-1 and is the Ser-Leu-Met- of the present invention.
The peptide represented by Asp-Lys-Glu (III) is a peptide having a partial sequence from the 5th serine of endothelin-1 to the 10th glutamic acid.

As is clear from FIG. 1, the 3rd cysteine and the 11th cysteine are cross-linked by an SS bond.
It corresponds to the partial sequence of the loop structure made from one cysteine. Therefore, it is expected that the antibody prepared by using the peptides (I) to (III) of the present invention as an immunogen is an antibody having a specific affinity for the loop structure of endothelin-1.

The peptide of the present invention can be synthesized by a conventional peptide chemical synthesis method such as a solid phase method or a liquid phase method.
In these peptides, a part or all of the terminal amino group or carboxyl group, or side chain amino group, carboxyl group or hydroxyl group is optionally blocked by a solid phase or protected by a protective group such as an acetyl group. Good.

As a protein to which the peptide or peptide derivative of the present invention is bound, a protein which enhances the antigenicity of these peptides is selected. Examples of such proteins include proteins such as albumin, keyhole limpet hemocyanin (hereinafter abbreviated as KLH), egg white lysozyme, and thyroglobulin.

As a method for binding the peptide or peptide derivative of the present invention to a protein, a carbodiimide method (Lewis, JE et al .: Antimicrob. Agents Chemot
her., 1975, 7 , 42 .; Broughton, A. et al .: Antimicro
b. Agents Chemother., 1976, 10 , 652), N-hydroxysuccinimide method (Kitagawa, T. et al .: Chem.Parm.
Bull., 1972, 20 , 2176 .; Hoshida, H. et al .: Chem.
There are known methods such as Parm. Bull., 1983, 31 , 4001).

To obtain the anti-endothelin-1 antibody (polyclonal antibody), the peptide, peptide derivative of the present invention or a conjugate of these and a protein is administered to an animal such as mouse, rat, rabbit, goat, or sheep multiple times. Immunization is performed and the serum of the immunized animal is collected.
At this time, as the immunogen, it is preferable to administer a conjugate of the protein and the peptide, as compared with the peptide or the peptide derivative thereof, because the production amount of the antibody is increased. Animals such as mice are immunized in the same manner as described above, and a known cell fusion method, that is, cell fusion of the spleen cells and myeloma cells, cloning, collection of antibody-producing hybridomas, and culturing of these antibodies ( Monoclonal antibody) may be obtained.

The peptide or peptide derivative of the present invention bound to a protein such as albumin and bound to a solid phase such as an immunoplate is the anti-endothelin-1.
It can be used as a material for detecting antibodies. Physical adsorption may be used for binding the peptide to a solid phase such as an immunoplate, or a plate having a functional group exposed on the surface may be used for covalent binding.

Next, an experimental example will be described. The "washing solution 1" used in the following experimental examples or examples is Dulbecco's modified phosphate buffered saline containing 0.1% Tween 20, and "buffer solution 1" is 0.1% skim milk powder and Dulbecco's modified phosphate saline containing 0.1% Tween 20.

Experimental Example 1 Screening for antigenic determinant of endothelin-1 Screening for antigenic determinant of endothelin-1
Report by ysen et al. (Geysen, HM et al .: Proc. Natl. A
cad. Sci. USA, 1984, 81 , 3998).

(A) Synthesis of peptide for screening Mimotope Design Kit [Cambridge Research Biochemicals In
c.)] was used to synthesize a peptide having a partial amino acid sequence of endothelin-1 on the pin block. There are 10 kinds of peptides synthesized below. (1) Cys-Ser-Cys-Ser-Ser-Leu (2) Ser-Cys-Ser-Ser-Leu-Met (3) Cys-Ser-Ser-Leu-Met-Asp (4) Ser-Ser-Leu -Met-Asp-Lys (5) Ser-Leu-Met-Asp-Lys-Glu (6) Leu-Met-Asp-Lys-Glu-Cys (7) Met-Asp-Lys-Glu-Cys-Val (8) ) Asp-Lys-Glu-Cys-Val-Tyr (9) Lys-Glu-Cys-Val-Tyr-Phe (10) Glu-Cys-Val-Tyr-Phe-Cys

The peptide synthesis method is according to the manual attached to the kit.
9-fluorenylmethyloxycarbonyl (hereinafter abbreviated as Fmoc) -L-
Performed with amino acids. The amino acid active esters used were all protected by Fmoc except for serine.
It is a pentafluorophenyl ester (hereinafter abbreviated as -Opfp), and only serine is dihydrox with the amino group protected by Fmoc.
It is a y-benzotriazine ester (hereinafter abbreviated as -ODhbt). The side chain protecting group of aspartic acid and glutamic acid is t-butyl ester (hereinafter abbreviated as -OtBu), the side chain protecting group of cysteine is trityl- (hereinafter abbreviated as Trt), and the side chain protecting group of lysine. Is t-butyloxycarbonyl (hereinafter Boc
Abbreviated), and the side chain protecting groups of serine and tyrosine are t-bu
It is tyl ether (hereinafter abbreviated as -tBu). The surface of the tip of the polyethylene pin used for the synthesis is a graft-polymerized acrylic acid polymer, to which Fmoc-β-alanine is bonded via a hexamethylenediamine spacer.

The pin block to which Fmoc-β-alanine was covalently bonded was immersed in a piperidine: N, N-dimethylformamide (hereinafter abbreviated as DMF) mixed solution 1: 4 (volume ratio) for 30 minutes to remove the Fmoc group. Then, with DMF for 5 minutes (2
(2 times), washed with methanol for 2 minutes (1 time), washed with methanol for 5 minutes (2 times), and further air-dried to completely evaporate methanol. After the pin block was immersed in DMF for 5 minutes, the tip of each pin was immersed in an Fmoc-amino acid solution at 30 ° C. for 18 hours to add an amino acid. At this time, the amino acid solution contains 20 mM each of the carboxyl-terminal Fmoc-amino acids of the peptide to be synthesized on each pin.
It was dissolved in a DMF solution containing 2.5 mM 1-hydroxy-benzotriazole (hereinafter abbreviated as HOBT). After the amino acid was added, it was washed with DMF for 2 minutes and with methanol for 2 minutes (three times). The above-mentioned addition reaction of amino acids was carried out one by one from the amino acid at the carboxyl terminal to the amino terminal side in order to synthesize a peptide consisting of 6 amino acid residues on each pin block.

After coupling the last amino acid, the pin block was added to DMF: acetic anhydride: triethylamine 5: 2: 1.
(Volume ratio) The mixture was immersed in the mixed solution at 30 ° C. for 90 minutes to acetylate the amino groups. After the reaction, it was washed with DMF for 2 minutes and with methanol for 2 minutes (three times), and dried in air. In order to remove the side chain protecting group, the pin block was immersed in a mixed solution of trifluoroacetic acid: anisole: ethanedithiol 95: 2.5: 2.5 (volume ratio) for 4 hours at room temperature, and then 0.1
% (W / V) hydrochloric acid, 50% (V / V) methanol solution was immersed in the solution, and after ultrasonic treatment, this was used for the following experiments.

(B) Screening of high affinity region An amino acid sequence having high affinity for a known anti-endothelin-1 polyclonal antibody was examined as follows. To prevent non-specific adsorption of anti-endothelin-1 antibody to pin blocks and immunoplates, 96% immunoplates were supplemented with 0.2% skim milk powder (Kirkegaard and Perr).
(manufactured by y Laboratories. Inc.) was taken in an amount of 400 μl per well, and each pin of the pin block was set so as to enter each well, and immersed at 4 ° C. overnight.

After thoroughly washing the pin block and plate with Washing Solution 1, anti-endothelin-diluted 1/1000.
175 μl of buffer solution 1 containing 1 polyclonal antibody (manufactured by Peptide Institute Co., Ltd.) was taken per well, and pins were immersed in the wells at 37 ° C. for 2.5 hours. At this time, wells containing only buffer solution 1 were also prepared at the same time. The plate and the pin block were thoroughly washed with the washing solution 1, and then diluted to 1/2000 with an alkaline phosphatase-labeled anti-rabbit antibody goat antibody (Kirkegaard and Perry Laboratories. Inc.
Buffer solution 1) containing (secondary antibody) 175 per well
μl was added. Dip the pins of the pin block into the wells and
Incubated at 0 ° C for 1 hour. After 1 hour, the pin block and plate were thoroughly washed with a washing solution, and then p-nitrophenyl phosphate (hereinafter abbreviated as PNPP. Alkaline phosphatase substrate, Kirkegaard and Perry Laborator
ies. Inc.) solution was added in an amount of 175 μl per well, and pins were immersed in the wells and reacted at 37 ° C. for 1 hour.

Stop the reaction by removing the pin block,
Take 100 μl from each well and collect in each well of another plate. Microplate reader [MTP-
22 type microplate photometer, Corona Denki Co., Ltd.] absorbance of solution in each well A _405/492 (405 nm
And the difference in absorbance at 492 nm). The difference between the absorbance of the well to which the anti-endothelin-1 polyclonal antibody was added and the absorbance of the well to which the anti-endothelin-1 polyclonal antibody was not added (control) was determined and defined as the antibody binding ability.

The results are shown in Table 1. From the results in Table 1, Ser-Ser-Leu-Met-Asp-Lys and Ser-Leu-Met-Asp-Lys
-A peptide having the amino acid sequence of Glu is anti-endothelin-
It can be seen that it has a high binding ability to one polyclonal antibody.

[Table 1] Table 1 Binding ability of a peptide having a partial amino acid sequence of endothelin-1 and an anti-endothelin-1 polyclonal antibody ━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━ No. Peptide (amino acid sequence) Binding capacity (Absorbance: A _405/492 ) ━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━ 1 CSCSSL 0.013 2 SCSSLM 0.520 3 CSSLMD 0.474 4 SSLMDK 0.772 5 SLMDKE 0.794 6 LMDKEC 0.189 7 MDKECV 0.133 8 DKECVY 0. 012 9 KECVYF 0.010 10 ECVYFC 0.010 11 Control (pin block only) 0.000 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━ In Table 1, Pep The amino acid of tide is represented by a single letter, C for Cys, D for Asp, E for Glu, F for Ph.
e, K are Lys, L is Leu, M is Met, S is Se
r and V represent Val and Y represents Tyr.

[0027]

EXAMPLES Example 1 Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu
Synthesis of peptide represented by Ser-Ser-Leu-Met-Asp-Lys (II) and Ser in Experimental Example 1
-Leu-Met-Asp-Lys-Glu (III) peptide was found to have high affinity with anti-endothelin polyclonal antibody, so the heptapeptide Ac-Ser-Ser- containing the amino acid sequence of the above peptide in common. Leu-Met-Asp-Lys-Glu (wherein Ac-indicates that the amino group of the N-terminal serine is acetylated) was used as a peptide synthesizer (Milligen /
Biosearch, fully automatic peptide synthesizer 90
Type 50, solid phase resin was synthesized by Fmoc-Glu-pepsin KA).

Fmoc-Lys (Bo
c) -OPfp, Fmoc-Asp (OtBu) -OPfp, Fmoc-Met-OPfp, Fmoc-
Leu-OPfp, Fmoc-Ser (tBu) -ODhbt, Fmoc-Ser (tBu) -ODhbt
After the reaction, the peptide chain was extended to remove the Fmoc group remaining at the amino terminus by piperidine treatment,
The amino terminus was acetylated with a DMF solution containing 0.5 M acetic anhydride. The solid phase resin to which the synthetic peptide was bound was taken out from the column and immersed in a mixed solution of trifluoroacetic acid: thioanisole: ethanedithiol: m-cresol (volume ratio 40: 6: 3: 1) to remove the peptide from the resin. Upon release, the side chain protecting group was removed.

The obtained peptide was separated and purified by high performance liquid chromatography (655 Liquid Chromatography, Hitachi Ltd.) using a reverse phase column (D-ODS-5, manufactured by YMC).

Example 2 Ac-Ser-Ser-Leu to protein
-Met-Asp-Lys-Glu bond Ac-Ser-Ser-Leu-Met-Asp-Lys by carbodiimide method
-Glu was chemically bonded to KLH and bovine serum albumin (hereinafter abbreviated as BSA). Ac-Ser-Ser-L obtained in Example 1
eu-Met-Asp-Lys-Glu 240 nmol, N-hydroxysulfosuccinimide (hereinafter abbreviated as Sulfo-NHS) 800 nmo
l and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (hereinafter abbreviated as EDC) 800 nmol
64 mM of 50 mM potassium phosphate buffer (pH 7.0)
It was dissolved in 1 and left at 0 ° C. for 30 minutes. KLH 4 nmol is added to 50 mM potassium phosphate buffer (pH 7.0) 2.2 m
1 l, add this to the above solution and at room temperature for 1 hour,
It was then left overnight at 4 ° C. Then, this reaction solution is mixed with 50
Dialyzed against mM potassium phosphate buffer (pH 7.0), unreacted Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu, Sulfo-
NHS and EDC were removed.

Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu was added to BSA.
Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu
Amount 4.1 μmol, Sulfo-NHS amount 12.3 μmo
1, EDC amount of 12.3 μmol and BSA amount of 0.41
The procedure was the same as in the case of binding to KLH except that the concentration was μmol.

As a result of protein quantification, almost 100% of any protein (KLH or BSA) was recovered. Below, the peptide Ac-Ser-Ser-Leu-Met-Asp-Lys-
Ac-Ser-Ser-Leu-M is obtained by chemically binding Glu to KLH.
Those chemically bound to et-Asp-Lys-Glu-bound KLH and BSA are referred to as Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu-bound BSA.

Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu conjugated tan
Affinity test of anti-endothelin-1 antibody in protein (1) Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu binding KLH KLH, Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu binding KLH,
And KLH bound with endothelin-1 (hereinafter referred to as endothelin-1-binding KLH) are adjusted to a final concentration of 10 μg / ml with 50 mM sodium carbonate: 50 mM sodium hydrogen carbonate 1:24 (volume ratio).
After being dissolved in a solution, 100 μl of each was placed in a well of an immunoplate (MaxiSorpF96, manufactured by Nunc) and left at 4 ° C. overnight to immobilize each protein on the immunoplate. The plate was washed with distilled water, 400 µl of 0.2% skim milk powder was added, and the plate was incubated at 37 ° C for 2.5 hours. After washing each well with washing solution 1, anti-endothelin-1 mouse monoclonal antibody (Cat.
No.ET-01, made by Yamasa Shoyu, diluted 1/1000 with buffer 1), normal mouse serum (diluted 1/1000), anti-endothelin-1 rabbit polyclonal antibody (Peptide Research Institute, 1 / 1000 diluted solution) and normal rabbit serum (1/1000 diluted solution) were added in an amount of 200 μl per well,
Incubated at 37 ° C for 2 hours.

After washing the wells with washing solution 1, peroxidase-labeled anti-mouse antibody goat antibody (Kirkegaard and Perry Laboratories.
(Manufactured by Inc.) and a rabbit antibody-added well, an alkaline phosphatase-labeled anti-rabbit antibody goat antibody was diluted 1/1000 with buffer 1 and 200 μl thereof was added, and the mixture was incubated at 37 ° C. for 1 hour. Each well was washed with washing solution 1, and the former was 3,3 ′, 5,5′-tetramethylbenzidine (hereinafter abbreviated as TMB, a peroxidase substrate, Kirkegaard and Perry Laboratories.
(manufactured by nc.), and 200 μl of PNPP was added to the latter and reacted at 37 ° C. for 30 minutes. After adding 100 μl of 1M phosphoric acid to the wells containing MTB, the absorbance of each well was determined using a microplate reader. At this time, the value obtained by subtracting the absorbance at 610 nm from 492 nm was calculated for the well to which TMB was added, and 405 for the well to which PNPP was added.
The value obtained by subtracting the absorbance at 492 nm from nm was determined.

Table 2 shows the results (absorbance is expressed as the above difference in absorbance).

[Table 2] Table 2 Binding ability to peptide-binding protein (KLH) (unit: absorbance) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━ Serum or antibody Peptide-binding protein Mouse serum Mouse antibody Rabbit serum Rabbit antibody ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━ KLH (negative control) 0.009 0.005 0.003 0.023 Endothelin-1 binding KLH 0.009 0.649 0.007 1.61
3 Heptapeptide bond KLH 0.008 0.004 0.007 0.907 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

In Table 2, mouse serum is normal mouse serum, mouse antibody is anti-endothelin-1 mouse monoclonal antibody, rabbit serum is normal rabbit serum, rabbit antibody is anti-endothelin-1 rabbit polyclonal antibody, heptapeptide. The bound KLH represents Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu-bound KLH, respectively.

Looking at the binding ability between the peptide binding protein (KLH) and the rabbit polyclonal antibody, the binding ability of the heptapeptide-bound KLH to the rabbit polyclonal antibody is the endothelin-1 binding KL to the rabbit polyclonal antibody.
It was about 60% of the binding capacity of H 2. This means that about 60% of the anti-endothelin-1 rabbit polyclonal antibody bound to the partial sequence Ser-Ser-Leu-Met-Asp-Lys-Glu of endothelin-1. On the other hand, the mouse monoclonal antibody used bound to endothelin-1 (probably bound to the N-terminal site of endothelin-1), but surprisingly
It was found from Table 2 that it did not bind to Ser-Ser-Leu-Met-Asp-Lys-Glu. The reason for this is that the mouse monoclonal antibody used was Ser ⁴ -Ser-Leu-Met-Asp-Lys of endothelin-1.
-This is probably because it recognizes an N-terminal site different from the Glu ¹⁰ site.

(1) Ac-Ser-Ser-Leu-Met-Asp-Lys-Glu-conjugated BSA BSA, Ac-Ser-Ser-Ser-Leu-Met-Asp-Lys-Glu-conjugated BSA,
And BSA bound with endothelin-1 (hereinafter referred to as BSA bound with endothelin-1) are each adjusted to a final concentration of 10 μg / ml with 50 mM sodium carbonate:
After being dissolved in a 50 mM sodium hydrogen carbonate 1:24 (volume ratio) solution, 100 μl thereof was placed in each well of the immunoplate and left overnight at 4 ° C. to immobilize each protein on the immunoplate. After washing each well with distilled water, 0.2% non-fat dry milk was added to each well at 400 μl.
Each was added and incubated at 37 ° C. for 2.5 hours. After washing each well with the washing solution 1, the anti-endothelin-1 rabbit polyclonal antibody diluted with the buffer solution 1 (1 /
(1000 dilution) and normal rabbit serum (1/1000 dilution) were added at 200 μl per well and incubated at 37 ° C. for 2 hours.

After washing the wells with washing solution 1, the wells are washed with buffer solution 1
Alkaline phosphatase-labeled anti-rabbit antibody goat antibody diluted at 1000/1000 was added to each well in an amount of 200 μl.
Incubated at 7 ° C for 1 hour. After washing each well with Washing Solution 1, 200 μl of PNPP was added to each well and reacted at 37 ° C. for 30 minutes. Then, using a microplate reader, the absorbance (40
The difference in absorbance between 5 nm and 492 nm was measured.

Table 3 shows the results.

[Table 3] Table 3 Binding ability to peptide-binding protein (BSA) (unit: absorbance) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━ serum or antibody peptide binding protein rabbit serum rabbit antibody ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ BSA ( Negative control) 0.002 0.022 endothelin-1 binding BSA 0.007 1.563 heptapeptide binding BSA 0.005 0.895 ━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━ In Table 3, rabbit serum is normal rabbit serum, rabbit antibody is anti-endothelin-1 rabbit polyclonal antibody, and heptapeptide-conjugated BSA is Ac-Ser-Ser-. Leu-Met-Asp-Lys-G
The lu binding BSAs are shown respectively.

The binding ability of the peptide-bound BSA to the rabbit polyclonal antibody was about 60% of that of endothelin-1-bound BSA to the rabbit polyclonal antibody. Similar to the above, about 60% of the anti-endothelin-1 rabbit polyclonal antibody was a partial sequence Ser of endothelin-1.
-It means that it recognizes Ser-Leu-Met-Asp-Lys-Glu.

[0042]

The peptides of claims 1 to 3 and the peptide derivative of claim 4 bind only to a specific antigenic determinant of the endothelin-1 molecule (loop structure between the 3rd and 11th cysteines). So anti-endothelin-1
It serves as a ligand for antibody purification or a detection reagent.
It is a raw material of an immunogenic protein for obtaining anti-endothelin-1 antibody. The anti-endothelin-1 antibody-inducible protein according to claim 5 obtains an anti-endothelin-1 antibody which reacts only with a specific antigenic determinant (loop structure between the 3rd and 11th cysteines) of the molecule of endothelin-1. Is an immunogenic protein for. According to the method of claim 6,
An anti-endothelin-1 antibody that recognizes a specific antigenic determinant (loop structure between the 3rd and 11th cysteines) of the molecule of endothelin-1 can be produced without requiring complicated separation and purification.

[0043]

[Sequence list]

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the secondary structure of the molecule of endothelin-1.

Claims (6)

[Claims] 1. A peptide represented by Ser-Ser-Leu-Met-Asp-Lys-Glu (I). 2. A peptide represented by Ser-Ser-Leu-Met-Asp-Lys (II). 3. A peptide represented by Ser-Leu-Met-Asp-Lys-Glu (III). 4. A part or all of the terminal amino group or carboxyl group or the side chain amino group, carboxyl group or hydroxyl group of the peptide of claim 1, claim 2 or claim 3 is optionally blocked or protected. Peptide derivative. 5. An anti-endothelin-1 antibody-inducible protein obtained by binding the peptide of claim 1, claim 2 or claim 3 or the peptide derivative of claim 4 to a protein. 6. An anti-endothelin-comprising immunization of an animal with the peptide of claim 1, claim 2 or claim 3, the peptide derivative of claim 4 or the anti-endothelin-1 antibody-inducible protein of claim 5. 1 Method for producing antibody.