JPH01258699A - Bradykinin derivative and determination thereof - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) expressed by the formula [X is H or Lys (Lys is lysin); Arg is arginine; Pro is proline; Hyp is hydroxyproline; Gly is glycine; Phe is phenylalanine; Ser is serine residue]. USE:A diagnostic agent for disease accompanying inflammation of cancer, etc. and rising vascular permeability. PREPARATION:Boc-L-amino acid (Boc is tert.-butyloxycarbonyl) is successively coupled with e.g., p-methylbenzhydrylamine resin used as a solid phase carrier according to the amino acid sequence of peptide and then the peptide is eliminated from the carrier to afford the peptide expressed by the formula, which is then used as a binder and bonded to a protein such as bovine serum albumin to provide the peptide-protein bonded product used as a hapten antigen for preparing an antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention primarily relates to immunological quantification of a peptide represented by the following general formula (I), and is particularly useful in the field of clinical diagnosis.

X-Arg-Pro-11yp-Gly-Phe-9e
r-Pro-Phe-Arg-011(I) (wherein,
X means a hydrogen atom or Lys, Lys is lysine,
Arg means arginine, Pro means proline, 1I'P means hydroxyproline, Gly means glycine, Phe means ), nilalanine, and Ser means serine residue, respectively. ) PRIOR ART AND PROBLEMS TO BE SOLVED Pradykinin (hereinafter referred to as BK) and Calici/(hereinafter referred to as KK) are peptides that have physiological effects such as dilation and increased permeability of small blood vessels, and contraction or relaxation of smooth muscles. - A peptide in which the 7th nyp from the C-terminus in Tsuzuriyo (I) is substituted with Pro, and when X is a hydrogen atom, it is BK, and when X is Lys, it is KK, and both It is a typical kinine compound.

A kit for quantifying UK in human body fluids by enzyme immunoassay (EIA) is commercially available from Dainippon Pharmaceutical Co., Ltd., but with this kit, not only BK but also KK and peptide (I) are quantified as 6BK, and peptide ( I) could not be separately quantified.

There is no report mentioning a method for quantifying a trace amount of peptide (1).

The present inventors have demonstrated the presence of peptide (I) in human body fluids such as urine, blood, and ascites, and that the concentration of these peptides in human body fluids is useful as a diagnostic indicator for diseases such as cancer that are accompanied by inflammation and increase vascular permeability. Based on the knowledge that this is possible, they succeeded in creating an antibody with excellent specificity for peptide (I), and completed the present invention.

Means for Solving the Problems The present invention provides peptide (I) or its analogues as hapten antigens for the preparation of antibodies that recognize them, or as EIA
The present invention relates to a conjugate of peptide (I) and protein that is useful as an enzyme-labeled antigen.

Peptide (1) and its salts can be produced by condensing amino acids one by one, by condensing condensates of a plurality of amino acids, or by a combination of these methods. Such condensation can be carried out 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, or an active ester method.

In the solid-phase method, an amino acid with a protected functional group that does not need to be involved in the reaction is bonded to an insoluble carrier such as Bam (PA) resin through the carboxyl group of the amino acid, the protecting group is removed, and this An amino acid with a protected functional group that does not need to be involved is coupled, and this operation is repeated until the desired peptide chain is obtained.Then, the bond with the insoluble carrier is cleaved by treatment with hydrogen heptadide, etc., and when the protecting group remains, is a method to obtain the target peptide by eliminating this. Since most of the protecting groups are usually removed by the hydrofluorination treatment, there is usually no need to carry out the protective film m operation independently.

Proteins to be bound to peptide (I) include albumin, globulin, thyroglobulin, shellfish hemocyanin,
Examples include immunogenic proteins such as edestin and enzymes such as β-galactosidase, glucose-6-phosphate dehydrogenase, and alkaline phosphatase.

The conjugate of peptide (I) and immunogenic protein B can be used as a hapten to prepare antibodies.
) and an enzyme can act as an enzyme-labeled antigen in the EIA method. In the EIA method, [peptide (I)
-enzyme] and [peptide (I)-immunogenic protein] may be the same or different.

The binding of peptide CI> to a protein can be carried out according to a conventional method using a binding agent. As a binding agent, peptide (I)
Gluculaldehyde and toluene diincyanate, which chemically bond between the amino group of the protein and the amino group of the protein,
Dihalogenated dinitrobenzene, or the S11 group of the snug peptide (I) and the amino group of the protein, or conversely, the 511 group of the protein and the amine 7 group of the peptide (I) are linked together, for example, as described in Japanese Patent Publication No. 58-8395. m-MI3
Examples include maleimide derivatives abbreviated as S. Peptide (I) or a protein that does not have a Sl+ group can be subjected to binding after introducing a Sl+ group using, for example, N-(acetylmercaptoacetoxy)succinimide as disclosed in JP-A-57-142H7. .

A preferable binding mode between peptide (I) and protein is binding at the C-terminus of peptide (I).

Such a bond can be achieved by synthesizing peptide (I) in which cysteine (cys) or a peptide containing Cys is further bonded to the C-terminus of peptide (I), and then attaching the above maleimide derivative to the protein through the Sl+ group in Cys. This can be achieved by combining.

The present invention also relates to antibodies that specifically recognize peptide (I) or its analogs. The peptide here (
Analogues of I) are peptides that have an amino acid sequence partially in common with peptide (I), and have more or less number of constituent amino acids than peptide (I), or peptides containing amino acids that are constituents of peptide (I). is substituted with another amino acid, or has physiological activity equivalent to that of peptide (I). The antibody of the present invention has the general formula (I)
A conjugate of a peptide represented by and an immunogenic protein (
Parenteral administration (immunization) of Habten antigen) to animals such as rabbits, guinea pigs, goats, and sheep with an appropriate adjuvant.
It can be easily obtained by collecting the serum and subjecting it to known treatments. Monoclonal antibodies can be created by collecting antibody-producing cells from the lungs of animals thus immunized and performing operations such as fusion with myeloma cells and screening for clonal cells according to conventional methods.

The present invention also relates to a kit for immunological quantification of peptide (I) or its analogs using the above-mentioned antibody. Quantification of peptide (I) or its analogs is carried out using the following reagents (a): Peptide (b) of general formula (I) labeled with a labeling substance;
) The above-described antibody itself or its insolubilized product (c) When the reagent (b) is the antibody itself, a reagent for separating the reaction product and the residue when this and the reagent (a) are reacted. This can be easily carried out using a kit consisting of the following.

Reagent (a) is a conjugate (labeled antigen) of a labeled substance and a peptide represented by general formula (I), and is produced according to a conventional method. Labels include enzymes, radioactive substances, fluorescent substances,
Subtleties/compounds may be mentioned, and enzymes are particularly preferred. Enzymes include β-galactosidase, alkaline phosphatase, lipase, peroxidase, glucose-6
- Phosphate dehydrogenase and the like are used. The method of binding the enzyme and peptide (I) is as described above.

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. Binding of an insoluble carrier and an antibody can also be carried out in the same manner as described above. As the insolubilizing carrier, any carrier used in this field may be used, but bacterial cell wall fragments disclosed in US Pat. No. 166.767 are particularly preferably used.

: is usually used as a drug (c) for the test ``g (b
), which is an antibody (second antibody) that has been insolubilized (insolubilized second antibody). The second antibody uses, for example, an IgG fraction obtained from rabbit serum as an antigen,
It can be prepared by immunizing other animals, such as guinea pigs. The insoluble second antibody using bacterial cell wall fragments as a carrier is
It is commercially available from Dainippon Pharmaceutical Co., Ltd. under the name Marcela.

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

Quantitation of peptide (I) or its analogs using these reagents involves: ① Reacting the sample with reagent (a) and reagent (b);
■ When an insolubilized antibody is used as reagent (b), the reaction mixture is centrifuged to separate the free reagent (a) (supernatant) and other substances (precipitate) (B/P separation), and ■ reagent When the antibody of the present invention itself is used as (b), B/F 3 is further reacted with reagent (c).
), and the activity of the labeled substance in the supernatant or precipitate separated in step 2 or 2 is measured. The conditions for carrying out these steps (1) to (2) are the same as in the ordinary EIA method. When the specimen contains kininase, such as human body fluids, it is usually better to pre-decontaminate the specimen with trichloroacetic acid or the like.

Specific Examples Next, the present invention will be explained in more detail with reference to Examples. In addition, below, the peptide in which X in general formula (I) is a hydrogen atom is abbreviated as IIYP-IIK, and the peptide in which X is Lys is abbreviated as IIYI'-ini.

Example 1 Synthesis of peptide (f) (A)
IIYP-BK is a solid phase peptide synthesizer model 990
J using E (Bepkumann Instrument Co.)
,^m,chem,soc,, 86.304-305
(No. 1911). That is, HYP-BK was synthesized by sequentially cappurating each Boc-L-amino acid onto p-medilbe/thuhydrylamine resin according to the procedure. As wJ mixture,
In the case of Boa-arginine, dicyclohexylcarbodiimide and 1-hydroxybenztriazole were used, in the other cases only the former was used. The ami/il sequence of IIYP-BK obtained was confirmed using 47θA protein sequencer (Applied Biosystems).

(B) Obtain the next peptide in the same manner as in (^).

-KK to +1'/ -Cys to -B to 11Y (peptide with Cys condensed to the C-terminus of HYP-B) 11YK- to -Cys
() IYP- to Kf) Peptide with Cys condensed to the C-terminus) Example 2 Binding of peptide and protein 0.05 containing 50 mg of bovine serum albumin (BS^)
M phosphate buffer (PI(7) m-M in 50ij!
Dioxane 5■ containing 2.5 g of BS! Add PI430, stir for 1 hour at room temperature, dilute 2 times with the same buffer, and add PI430.
(Amicon) Filter and wash with Kai. Transfer the membrane material to the same buffer solution 5.
5 soda of the same buffer containing 51 g of IIYPJK-Cys dissolved in 1 soda! and further stirred at room temperature for 2 hours.

This mixture was dialyzed twice with 0.9% NaCl 51 and H”/
A P-BK-Cys-[+58 conjugate (hapten antigen solution) is obtained.

Similarly, IIYP-ni-Cys-IIS^ conjugate (
hapten anti-cA solution) is obtained.

Example 3 Preparation of antibodies (A> Polycinal antibody 2!L of the hapten antigen solution obtained in Example 2 was diluted with 0.9% so that the absorbance at 280n was approximately 2.
Dilute with NaCl, mix with an equal volume of Freund's complete adipose tissue, and apply the emulsified mixture to two 0.2 ml areas per rabbit in the hind paws and 8 0.2 ml areas subcutaneously on the back. Local injection (immunization), from next time, every two weeks,
Inject 0.2 mjt subcutaneously in 10 locations on the back. After 5 immunizations, one week later, whole blood from the carotid artery was collected under anesthesia to obtain anti-blood samples 1 to 1 for each animal.

(B) Monoclonal antibody BALB/C For each mouse, 0.5 ml of the emulsion prepared in (^) was divided into 4 to 5 areas and incubated for 2 weeks.
1. Inject subcutaneously. One week after the third immunization, the pancreas is collected and antibody-producing cells are obtained by a conventional method.

A hybridoma is prepared by fusing these cells (7 x 10' cells) with mouse myeloma cells (P3X (f3Ag-01, 2, 10' cells) using the polyxq-v glycol method. Place them in a 98-well titer plate. distribute,
Cultivate using conventional methods. Screening for antibody-producing cells is carried out using the enzyme-labeled antigen of Example 5, insolubilized anti-goat IgG rabbit antibody (Marcera 30), enzyme substrate, and enzyme reaction terminator. Finally 3! 1 of monoclonal antibody (IgG type) producing hybridomas were obtained. Of these, 2u produced monoclonal antibodies suitable for EIA.

Example 4 Binding of peptide and enzyme β-derived from Escherichia coli
Galactosidase (hereinafter referred to as β-G).

It is called al. 2■ of water containing 1 g of Boehringer Mannheim)! 1 soda of water containing 500 μg of maleimide! and stirred at room temperature for 1 hour. This is dialyzed twice against 0.02M phosphate buffer (pH 7) 51 to obtain β-Gal with protected SH groups.

0.4 ml of dimethylformamide containing 380 μm of m-MB was added to the dialyzed content 3.j and stirred at room temperature for 2.5 hours. This solution 1■! It obtained in Example 1(B)
'/P-BK-Cys or IIYP-KK-Cys (
7) 200μ of water containing 500μg! 2 at room temperature.
Stir for an hour and use a Biogel P4 column (Bio-Rad, 2
．． 5X 32 c soda, 100-200 mesh) and 5 so! The enzyme-active portion was collected and treated with enzyme-labeled antigen (IIYI'-8 to -Cys-β-Gal or HY
A P-Cys-β-Gal) solution is obtained.

Example 5 Quantification of IIYP-13 by EIA Reagent standard vC milk 0-1000 ng/■! Example 1 (^)
A solution obtained by dissolving I+'/P-BK obtained in the following buffer A.

A solution obtained by diluting the serum obtained in Antibody Example 3 (^) (polyclonal antibody to anti-11YP-B) 5000 times with buffer A.

-Cys-β-G a 1 to HYP-B obtained in enzyme-labeled antigen Example 4
2 *A solution obtained by diluting the solution 20 times with buffer A.

Insolubilized second antibody Marcella 10 (insolubilized anti-hera IgG goat antibody) (Dainippon Pharmaceutical ■) 0.9% NaCl Substrate 271M2-nitrophenyl-β-D-galactoside 1-MMgCI2-40% ethylene glycol-〇, 1
%N a N 3 Enzyme reaction terminator 0.1 M K2HPO4-NaOH (pH 11) Buffer A 0.1%BS^-0.1%NaN3-0.9%NaCl
-0,04M IJ 71! It 1mm liquid liquid pl+7
) Buffer B 0.5M Tris-HCl -0.2% tLatin-0.9%
Nshin CI (pl+8) 20% trichloroacetic acid (protein removal agent) operation (O pretreatment (protein removal) 500μr sample (serum, etc.) in a plastic tube
, mix with 100 μl of 20% trichloroacetic acid, and centrifuge to remove the raw sill precipitate. It(15000X g, 1
0 minutes) and supernatant 250μ! Add the same amount of Buffer B to the sample and use it as a sample.

(2) Quantification method: 100μ of standard antigen or pretreated sample in a test tube (I x 10ew)! and then 200μ of antibody! Add 37
Incubate at ℃ for 5 days. After 30 minutes, enzyme-labeled anti-R200μ2 was mixed and incubated at 37°C for 5 minutes. 30 minutes bale Marcella IO 20
Add 0 μl and incubate at the same temperature for 15 minutes. then 2 m
Add 0.9% NaCl and centrifuge (1500x 10
min) and remove the supernatant. This wash? ? Repeat the operation once again. 500μ for precipitation! Add Buffer A and stir to make it homogeneous. 100μ of substrate for this! Add and stir, 3
Incubate at 7°C for 30 minutes and add 1.5mj7 of enzyme reaction terminator. Centrifuge this (1500Xg, 10 minutes)
L, measure the absorbance of the supernatant at 410 nm. HYP-I in the sample from the separately prepared calibration 1a (Figure 1).
Find IKffi.

Example 6 Cross-reactivity samples include BK, Des 9BK (BK lacking the 9th amino acid from the c-terminus, the same applies hereinafter), and Des8.9
The same operation as in Example 5 was performed except that K was used for 8 and 1 (YP-). As a result, no cross-reactivity was observed at 500 ng/■j or less, and at 1000 ng lll, BK and Des 8 0.39 for .9BK
%, 0.39% for Des9 D K and KK
In the case of IIYP-Ni, only 0.72% of crossover was observed.

Example 7 EIA Example 3 using monoclonal antibodies
11VP-IIK was quantified in the same manner as in Example 5, except that the monoclonal antibody obtained in (B) and Marcella 30 were used.

Example 8 EIA of HYP-KK Buffer A containing HYP-KK obtained in Example 1 (B) was used as the standard antigen, and anti-11YP prepared in Example 3 (^) was used as an antibody.
EIA of I(YP-KK) was carried out in the same manner as in Example 5, except that a buffer containing -KK antibody was used.

(Hereafter, extra white)

[Brief explanation of the drawing]

FIG. 1 is a calibration curve for HYP-BKs. In Fig. 1, BX/BO is the absorbance when the standard antigen concentration is 00 (Ilo) and the absorbance when the standard antigen concentration is X (Bχ).
The ratio (%) is shown. Patent applicant Dainippon Pharmaceutical Co., Ltd.

Claims (5)

[Claims] (1) General formula (I) X-Arg-Pro-Hyp-Gly-Phe-Ser
-Pro-Phe-Arg-OH (I) (wherein, X means a hydrogen atom or Lys, Lys is lysine, Ar
g means arginine, Pro means proline, Hyp means hydroxyproline, Gly means glycine, Phe means phenylalanine, and Ser means serine. ) A combination of a peptide and a protein. (2) A peptide represented by general formula (I) or a salt thereof. (3) An antibody that recognizes a peptide represented by general formula (I) or an analog thereof. (4) General formula consisting of at least the following reagents (
A kit for immunological quantification of the peptide represented by I) or its analog; (a) the peptide represented by the general formula (I) labeled with a label; (b) the antibody itself according to claim 3 or its insolubilized form; (c) When the reagent (b) is the antibody itself, a reagent for separating the reaction product and the residue when the antibody is reacted with the reagent (a). (5) The reagent (a) is an enzyme-labeled substance, and the reagent (b) is an antibody prepared using a peptide represented by the general formula (I) bound to an immunogenic protein at the C-terminus as an antigen; 5. The kit according to claim 4, wherein reagent (c) is an insolubilized second antibody against the antibody of reagent (b).