JP2648855B2 - Quantitation of peptides, their antibodies and endothelin - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a novel peptide, an antibody thereto, and a kit for immunological quantification of endothelin and its analogs, and is particularly useful in the field of clinical diagnosis. It is.

2. Prior Art and Problems to Be Solved Masaki et al., One of the inventors of the present invention, isolated and purified a vasoconstrictive peptide found in the culture supernatant of human epithelial cells and named it endothelin [Vascular Volume 11, 1 issue
BS page 2, 1988]. Later, Masaki et al. Gained new knowledge that endothelin has a structure represented by the following formula.

Endothelin is expected to be an endogenous factor involved in circulatory regulation, and its pharmacokinetics is thought to play a part in pathophysiology such as essential hypertension. In addition, it is considered that the expression of such a physiological activity of endothelin requires a cyclic portion from the N-terminus to the central portion and all of the hydrophobic site near the C-terminus.

It would be possible to diagnose such lesions if the amount of endothelin in the body fluid could be tracked. However, there is no report on the quantification of endothelin. Thus, the present inventors have produced endothelin and its analogs, created antibodies thereto, established a method for quantifying endothelin or its analogs using this antibody, and completed the present invention.

Means for Solving the Problems The present invention provides the following formula (1-1) Cys-His-Leu-Asp-Ile-Ile-Trp (1-1) or the following formula (2-1) Cys-Ser-Ser- The present invention relates to a peptide represented by Leu-Met-Asp-Lys-Glu (2-1) or a salt thereof.

These peptides are useful as haptens or the like for preparing antibodies that recognize endothelin or its analogs.

In addition, the symbol used in this specification means the following amino acid residue.

Cys: cysteine Ser: serine Leu: leucine Met: methionine Asp: aspartic acid Lys: lysine Glu: glutamic acid Val: valine Tyr: tyrosine Phe: phenylalanine His: histidine Ile: isoleucine Trp: tryptophan Formula (1-1) or (2- The peptide represented by 1) and a salt thereof can be produced by a method of condensing amino acids one by one, a method of condensing condensates composed of a plurality of amino acids, or a method of combining these. Such condensation can be performed in a liquid phase or a solid phase, preferably in a solid phase, by a conventional method such as an azide method, a mixed acid anhydride method, a dicyclohexylcarbodiimide method, and an active ester method. In the solid phase method, an amino acid having a protected functional group which does not need to be involved in the reaction is bonded to an insoluble carrier such as a pam resin through a carboxyl group of the amino acid, the protecting group is removed, and the reaction is carried out. When a functional group that does not need to be involved is coupled with a protected amino acid, this operation is repeated until a desired peptide chain is obtained, and then the bond with the insoluble carrier is cleaved by hydrogen fluoride treatment or the like. This is a method for obtaining a target peptide by removing the compound. Usually, most of the protecting groups are eliminated by the treatment with hydrogen fluoride, so that it is not usually necessary to carry out the elimination operation of the protecting groups independently.

Functional groups that do not need to be involved in the reaction include amino groups, hydroxyl groups, carboxyl groups, SH groups, and NH groups in indole and imidazole rings. These groups are usually protected with protecting groups. The amino protecting group is Bo
t-butyloxycarbonyl abbreviated as c, 2-chlorobenzyloxycarbonyl abbreviated as ClZ,
Benzyl, abbreviated as Bzl, or BrZ
P-bromobenzyloxycarbonyl, etc., a cyclohexyl ester, abbreviated as OcHex, as a carboxyl protecting group, and acetamide methyl, abbreviated as Acm, or 4-MeBzl, as an SH protecting group.
Examples of the protecting group include methylbenzyl and the like, and further, as the NH protecting group in the indole ring or the imidazole ring, formyl abbreviated as For and p-toluenesulfonyl abbreviated as Tos.

Endothelin can be produced according to the description in Example 1 described later. It also mentions how to form two sets of -SS- bonds at specific sites of endothelin.

The present invention also relates to the peptide (1-1) or (2)
-1). The antibody of the present invention recognizes not only the peptide (1-1) or (2-1) but also a peptide containing these peptide residues as components of the molecule. Examples of such a peptide recognized by the antibody of the present invention include endothelin, those having a larger or smaller number of constituent amino acids than those of endothelin, those in which the constituent amino acids of endothelin are substituted with other amino acids, and the like. In the present specification, these peptides recognized by the antibody of the present invention are referred to as the peptides (1-1) and (2).
-1) and are collectively referred to as "endothelin or an analog thereof".

The antibody of the present invention comprises a conjugate of a peptide represented by the formula (1-1) or (2-1) and an immunogenic protein (hapten antigen), together with an appropriate adjuvant, which is used in rabbits such as guinea pigs, goats and sheep. Parenteral administration (immunization) to animals
It is easily obtained by collecting the serum and performing a known treatment. Examples of the immunogenic protein include albumin, globulin, thyroglobulin, shellfish hemocyanin, edestin and the like. Binding of these immunogenic proteins to the peptide represented by the formula (1-1) or (2-1) can be performed according to a conventional method using a binding agent. As the binder, peptide (1-1) or (2)
Glutaraldehyde, toluene diisocyanate, dihalogenated dinitrobenzene, or the SH group of peptide (1-1) or (2-1) which chemically bonds between the amino group of -1) and the amino group of the immunogenic protein. Crosslinking with an amino group of an immunogenic protein, for example, JP-B-58-8395
Maleimide derivatives abbreviated as m-MBS described in the specification, and carbodiimide derivatives such as water-soluble carbodiimides that bind between the amino group or carboxyl group of the peptide and the carboxyl group or amino group of the immunogenic protein. Is mentioned. Immunogenic proteins in which no SH group is present are disclosed in Japanese Patent Application Laid-Open No. Sho 57-1442967, for example, N- (acetylmercaptoacetoxy)
Using succinimide, the SH group can be introduced before binding. The peptide thus obtained (1-
A desired antibody can be obtained by sufficiently immunizing an animal with a conjugate of 1) or (2-1) with an immunogenic protein. Monoclonal antibodies were collected from the spleen of the antibody-producing cells of the animal immunized in this manner, and then, according to a conventional method,
It can be created through operations such as fusion with myeloma cells and screening of clonal cells.

The present invention also relates to a kit for immunological quantification of endothelin or an analog thereof using the above antibody. The quantification of endothelin or an analog thereof is at least carried out by the following reagents: (a) endothelin or an analog thereof labeled with a label, (b) an antibody against a peptide represented by the formula (1-1) or (2-1) (C) When the reagent (b) is the antibody itself, a kit comprising a reagent for separating the reaction product obtained when the reagent (b) is reacted with the reagent (a) from the residue. Can be easily implemented.

The reagent (a) is a conjugate (labeled antigen) of a label with endothelin or an analog thereof, and binds the peptide (1-1) or (2-1) with the immunogenic protein as described above. Can be prepared in the same manner as in the case of Examples of the label include an enzyme, a radioactive substance, a fluorescent substance, and a spin compound, and an enzyme is particularly preferable. As the enzyme, β-galactosidase, alkaline phosphatase, lipase, peroxidase, glucose-6-phosphate dehydrogenase and the like are used.

The reagent (b) is the antibody of the present invention itself or an insolubilized antibody obtained by binding the antibody of the present invention to an insoluble carrier.
The binding between the insoluble carrier and the antibody can be performed in the same manner as described above.
Any insoluble carrier may be used as long as it is used in this field, but the bacterial cell wall disclosed in US Pat. No. 4,166,767 is particularly preferably used.

Commonly used as the reagent (c) is the reagent (b)
It is an antibody (second antibody) to the antibody that is insolubilized (second insolubilized antibody). The second antibody can be prepared, for example, by immunizing another animal, for example, a guinea pig using the IgG fraction obtained from rabbit serum as an antigen.

In addition, reagents such as a buffering agent, a reagent for measuring a labeling activity, and a standard antigen solution for preparing a calibration curve are used.

For quantification of endothelin or an analog thereof using these reagents, the sample is reacted with the reagent (a) and the reagent (b). When an insolubilized antibody is used as the reagent (b), the reaction mixture is centrifuged. When the free reagent (a) (supernatant) and the other (precipitate) are separated (B / F separation) and the antibody itself of the present invention is used as the reagent (b), the reagent (c) is further added. After the reaction, B / F separation is performed, or the activity of the supernatant separated by the or the activity of the label in the precipitate is measured.

Specific Example Next, the present invention will be described in more detail with reference to examples.

Example 1 peptide of 1-A endothelin producing _{Boc-Trp (For) -OCH 2} - Pam resin (0.50 mM) was used as a starting material, and using an Applied Biosystems Inc. Boc- amino acid derivative cartridges (2.0 mM) , After elimination of Boc with trifluoroacetic acid, C
Extend the peptide chain from the terminal side. However, Boc-Asp (Oc
Hex), Boc-Glu (OcHex), Boc-His (Tos), Boc-Cy
s (Acm) was used after encapsulating a powder manufactured by Peptide Laboratories Inc. in a dedicated cartridge. Thus, a peptide-resin conjugate represented by the following formula is obtained.

Boc-Cys (Acm) -Ser (Bzl) -Cys (4-MeBzl) -Ser (Bzl) -Ser (Bzl) -Leu-Met-Asp (OcHex) -Lys (ClZ) -Glu (OcHex) -Cys ( 4-MeBzl) -Val-Tyr ( BrZ) -Phe-Cys (Acm) -His (Tos) -Leu-Asp (OcHex) -Ile-Ile-Trp (For) -OCH 2 - resin the peptide-bound resin 1.0g Was treated with 12 ml of anhydrous hydrogen fluoride at −2 ° C. for 1 hour in the presence of 3.0 ml of p-cresol to obtain Ac.
All protecting groups except for m and For are eliminated. Hydrogen fluoride is distilled off under reduced pressure, and the residue is solidified by adding diethyl ether. The solid is collected by filtration, washed with ether and dried under reduced pressure. This is dissolved in 20 ml of trifluoroacetic acid, the resin is filtered off and concentrated.
Ether was added to the residue, and the resulting precipitate was collected by filtration, washed with ether, dried under reduced pressure, and dried at 2 Acm, 2 SH groups, 1 T
A peptide having rp (For) is obtained. This was dissolved in a 0.1 M ammonium acetate (pH 7.2) -8 M urea buffer so as to have a concentration of 0.5 mM, and an aqueous solution of potassium ferricyanide (1.
5 equivalents). After 40 minutes, the reaction mixture is added to a 200 ml DIAION HP-20 column, washed with water, and eluted with acetonitrile / trifluoroacetic acid / water (75: 0.1: 1: 25). The Pauli reaction-positive fraction is concentrated under reduced pressure and freeze-dried to obtain a peptide protected by Acm of Cys at positions 1 and 15 of endothelin and having a disulfide bond between positions 3 and 11.
Methanol-water (4: 1) containing 1N hydrochloric acid (15 equivalents)
And add 0.05M iodine-methanol (16 equivalents) at once with vigorous stirring. After reacting for 1 hour at room temperature, 0.05M ascorbic acid aqueous solution (16 equivalents)
To stop the reaction. The reaction mixture is concentrated, adsorbed on Diaion HP-20, washed thoroughly with water, and washed with acetonitrile /
Elution with trifluoroacetic acid / water (75: 0.1: 25) gives the endothelin Trp protected by For. This is concentrated and the residue is dissolved in 50 ml of water, and under ice cooling, 5 ml of a 1N aqueous sodium hydroxide solution is added to desorb For. Two minutes later, the mixture was neutralized with 5 ml of a 1N aqueous hydrochloric acid solution, and acetonitrile / trifluoroacetic acid / water (20: 0.1: 80) was used (45:50) using a Waters Prep / LC system 500 and a cartridge column (C18, 300A).
0.1: 55) and purified by endothelin.
Get 30 mg.

This product elutes at 18.0 minutes when subjected to high performance liquid chromatography (gradient) using 10-60% acetonitrile-0.1% trifluoroacetic acid as an eluent. Also, 30
Isocratic elution with 1% acetonitrile-0.1% trifluoroacetic acid eluted at 16.8 minutes, the elution position was completely identical to human endothelin, and the contractile activity using rat pulmonary artery specimens was equivalent to human endothelin. Amino acid analysis values after hydrolysis with 6N hydrochloric acid at 100 ° C. for 22 hours are as follows. (Theoretical values in parentheses) Trp0.28 (1), Lys0.97 (1), His1.01 (1), Asp2.
00 (2), Ser2.55 (3), Glu1.04 (1), 1/2 (Cys)
2 1.92 (4), Val 0.93 (1), Met 0.86 (1), Ile 0.98
(2), Leu1.92 (2), Tyr0.97 (1), Phe1.00
(1) Production of 1-B endothelin isomer According to 1-A, a peptide in which only the position of the bond between two disulfides is different from that of endothelin is obtained.

This peptide is eluted at the same position (18 minutes) as endothelin when subjected to high performance liquid chromatography (gradient) using 10-60% acetonitrile-0.1% trifluoroacetic acid as an eluent, but 30% acetonitrile-0.1
% Trifluoroacetic acid was eluted at 15.4 minutes under isocratic conditions, the elution position did not coincide with the human endothelin obtained in 1-A, and its bioactivity was less than 1/10 of that of human endothelin.

Production of 1-C peptide (1-1) or (2-1) The following peptide is obtained in the same manner as in 1-A. Amino acid analysis of these peptides was consistent with theoretical values.

In the following, Cys (SH) means Cys having an SH group, and the SH group of Cys with Acm is protected.

Cys (SH) -His-Leu-Asp-Ile-Ile-Trp (1-1) Tyr-Phe-Cys (SH) -His-Leu-Asp-Ile-Ile-Trp
(1-2) Clu-Cys (SH) -Val-Tyr-Phe-Cys (Acm) -His-Leu-Asp-Ile-Ile-Trp (1-3) Met-Asp-Lys-Glu-Cys (SH ) -Val-Tyr-Phe-Cys (Acm) -His-Leu-Asp-Ile-Ile-Trp (1-4) Cys (SH) -Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys ( (Acm) -Val-Tyr-Phe-Cys (Acm) -His-Leu-Asp-Ile-Ile-Trp (1-5) Cys (SH) -Ser-Ser-Leu-Met-Asp-Lys-Glu (2 −
1) Example 2 Preparation of Hapten Antigen 7.8 mg of 2-A porcine thyroglobulin and 3.0 mg of endothelin prepared in Example 1 are dissolved in 1 ml of a 0.1 M phosphate buffer (pH 7). To this mixture is added 250 μm of a 1% glutaraldehyde aqueous solution while stirring, and the mixture is stirred at room temperature for 2 hours. This reaction mixture was dialyzed against 0.9% NaCl 2 at 4 ° C. for 48 hours, and the entire amount of the dialysate was diluted and suspended in 10% with 0.9% NaCl.
Aliquot into aliquots and store frozen.

2-B endothelin 2.0 mg and porcine thyroglobulin 6.7 mg
Dissolve in 1 ml of 0.1 M phosphate buffer (pH 7) and add 30 mg of water-soluble carbodiimide hydrochloride while cooling at 4 ° C. 10 ℃
After overnight reaction, the reaction mixture is dialyzed against 0.9% NaCl at 4 ° C., and the entire amount of the dialysate is diluted and suspended in 10 ml with 0.9% NaCl, and each 1 ml is divided into small portions and stored frozen.

14 mg of 2-CBSA (bovine serum albumin) was dissolved in 1 ml of 0.1 M phosphate buffer (pH 7), and a solution of m-MBS in tetrahydrofuran (11.6 mg / 0.5 ml) was added thereto while cooling under stirring. And react for 2 hours. Wash the reaction mixture with ether,
Remove excess m-MBS. The aqueous layer was freeze-dried and dissolved in 2 ml of pure water to obtain Example 1-C. 11.
5 mg of peptide (2-1) is added thereto, and reacted at 10 ° C. overnight. Then, the reaction mixture is sufficiently dialyzed against pure water and lyophilized to obtain 19.0 mg of a conjugate of peptide (2-1) and BSA. To prepare a conjugate of peptide (1-1) and BSA.

Example 3 Preparation of Antibody 3-A Anti-endothelin Antibody The endothelin-porcine thyroglobulin conjugate produced in Example 2-A was dissolved in 0.9% NaCl solution to a 1% concentration, and an equal volume of Freund's complete adjuvant was added. hand
A W / O emulsion is prepared, and 0.1 ml is injected into two rabbit footpads and eight subcutaneous backs. Two weeks later, 0.1 ml is injected subcutaneously in 5 places on the back. Thereafter, the same booster is performed six times every two weeks. On the 10th day after the final immunization, whole blood is collected from the carotid artery. To 5 ml of this serum, an equal volume of 0.1 M phosphate buffer (pH 7) was added, and under ice-cooling, 10 ml of a saturated ammonium sulfate solution was added.
Stir for 0 minutes. This is centrifuged (12000 × g, 10 minutes)
Separate the precipitate. This precipitate is dissolved in 5 ml of the same phosphate buffer, an equal amount of saturated ammonium sulfate is added, and the precipitate is centrifuged in the same manner. Dissolve the precipitate in 5 ml of 0.1 M phosphate buffer (pH 7)
4 for 2 in 0.02M phosphate buffer (pH 7) containing 5% NaCl
Dialysis at 24 ° C. for 24 hours to obtain an antibody-containing solution.

3-B Antibody against peptide (1-1) or (2-1) The respective antibodies are obtained in the same manner as in 3-A except that the conjugate of Example 2-C is used.

Example 4 Preparation of Insolubilized Secondary Antibody 40 mg of bacterial cell wall pieces of Lactobacillus plantarum ATCC 8014 (hereinafter referred to as LP cell wall pieces) were suspended in 1 ml of purified water, and after sufficiently homogenized, 1 mg of anti-rabbit IgG guinea pig antibody And, with stirring, 15 μM of a 1 M sodium acetate buffer solution (pH 4), 5% aqueous carbodiimide aqueous solution of 30 μM
And 15% of 25% glutaraldehyde are sequentially added, and the mixture is stirred at room temperature for 1 hour. The reaction mixture was centrifuged (1500 × g, 10 minutes), the supernatant was discarded, and 5 ml of buffer A (0.1% BSA
Add −0.1% NaN ₃ −0.9% NaCl-0.04M phosphate buffer (pH 7) and wash by centrifugation. This is repeated 5 times and suspended in 2000 ml of buffer A containing 0.5% of LP cell wall pieces.

Example 5 Preparation of Oxygen-Labeled Antigen (A-1) Binding of Endothelin to m-MBS 100 μg of dimethylformamide solution containing 100 μg of m-MBS was added to 1 ml of 0.1 M phosphate buffer (pH 7) containing 100 μg of endothelin, and room temperature was added. For 30 minutes (Solution I).

(A-2) Binding On the other hand, 500 μg of β-galactosidase (hereinafter, β-G
Abbreviated as al. 400μm 0.1M phosphate buffer (pH7) containing Boehringer Mannheim, E. coli and saturated ammonium sulfate solution
A mixed solution of 400 μm is prepared, mixed and stirred with the above solution I, and stirred at room temperature for 1 hour (solution II).

The above solution II was passed through Sepharose 6B (Pharmacia, 2.7 × 40 cm) thoroughly washed with buffer A, eluted with buffer A, fractionated in 4 ml portions, and pooled fractions of Nos. 13 to 15 Is diluted with buffer A to obtain an enzyme-labeled antigen.

(B) 1 ml of water containing 500 μg of maleimide was added to 10 ml of 0.02 M phosphate buffer (pH 7) containing 5 mg of β-Gal, stirred at room temperature for 1 hour, and dialyzed twice with 5 ml of the same buffer. 250 μl of a dimethyl sulfoxide solution containing 100 μg of m-MBS in 3 ml of the internal solution
And stirred at room temperature for 2 hours. To this was added 250 μl of an aqueous solution containing 100 μg of the peptide (2-1) obtained in Example 1-C.
, And further stirred for 2 hours.
Elute with buffer A over a 6B column (1.6 x 920 cm).
Fraction 24 containing β-Gal-peptide (2-1)
get ml.

Similarly, the peptide obtained in Example 1-C (1-
Prepare the enzyme-labeled antigen in 1).

Example 6 Quantification of Endothelin (1) Reagent Standard antigen solution Endothelin buffer A solution at various concentrations Enzyme-labeled antigen solution Anti-endothelin antibody (first antibody) solution Insolubilized second antibody suspension 0.9% NaCl solution Buffer A Enzyme substrate Solution 0.3 mM M4-Methylumbelliferyl-β-D-galactoside-1 mM MgCl ₂ -40% Ethylene glycol-0.1% Na
N ₃ enzyme reaction stop solution 0.1MK ₂ HPO ₄ -NaOH (pH11) (2) Quantitative operation 1st antigen-antibody reaction Standard antigen solution or sample (serum etc.) ) Add 100μ, add the first antibody, stir, and incubate at 37 ° C for 60 minutes. Next, 100 μl of an enzyme standard antigen solution is added and incubated at 37 ° C. for 30 minutes.

Second antigen-antibody reaction and B / F separation Add 200 μL of the insolubilized second antibody suspension to the reaction solution, and add
After incubating for 15 minutes at, 2 ml of 0.9% NaCl solution is added to all the test tubes, and the mixture is centrifuged (1500 × g, 10 minutes) to remove the supernatant. This washing operation is repeated one more time.

Enzyme reaction 0.5 ml of buffer A is added to the precipitate, and the mixture is stirred with a mixer to completely disperse the precipitate. The precipitate is preheated at 37 ° C. for 3 minutes, and 100 μl of the fluorescent substrate solution is added thereto. Incubate at the same temperature, 20
After one minute, add 1.5 ml of the enzyme reaction stop solution and stir.

 Measurement of enzyme activity The fluorescence intensity of the reaction mixture is measured.

Calculation of the amount of endothelin The relationship between the amount of standard antigen and the absorbance is plotted on a semilogarithmic graph, and the amount of endothelin in the sample is determined from the obtained calibration curve.

Example 7 Calibration Curve A calibration curve for endothelin in FIG. 1 was prepared according to Example 6, except that the following reagents were used.

Enzyme-Labeled Antigen-Endothelin-β-Gal Conjugate Standard Antigen-Endothelin Antibody-Antibody Prepared from Endothelin Example 8 Calibration Curve A calibration curve of the peptide shown in FIG. 2 was prepared according to Example 6 except that the following reagents were used.

Enzyme-Labeled Antigen to Peptide (2-1) -β-Gal Conjugate Standard Antigen to Peptide (2-1) Antibody Prepared from Antibody to Peptide (2-1) Example 9 Calibration Curve Example Except for Using the Following Reagents According to 6, the same calibration curve of endothelin as shown in FIG. 1 was prepared.

Enzyme-labeled antigen-(1-1) -β-Gal conjugate Standard antigen-endothelin Antibody-antibody prepared from peptide (1-1)

[Brief description of the drawings]

 1 and 2 show the calibration curves of the peptides.

Claims (4)

(57) [Claims] 1. A peptide represented by the following formula (1-1): Cys-His-Leu-Asp-Ile-Ile-Trp (1-1) or a salt thereof. 2. A peptide represented by the following formula (2-1): Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu (2-1) or a salt thereof. 3. An antibody against the peptide according to claim 1 or 2. 4. A kit for immunological determination of endothelin or an analog thereof comprising at least the following reagents: (a) endothelin or an analog thereof labeled with a label; (C) when the reagent (b) is the antibody itself, a reagent for separating a reaction product obtained when the reagent (b) is reacted with the reagent (a) from a residue.