JP2628336B2 - Bradykinin derivatives and their determination - Google Patents

Description

Description: TECHNICAL FIELD The present invention mainly relates to immunological quantification of a peptide (I) represented by the following general formula (I), and is particularly useful in the field of clinical diagnosis. .

(In the formula, X represents a hydrogen atom or Lys.) In the present specification, Lys is lysine, Arg is arginine, Pr
o means proline, Hyp means hydroxyproline, Gly means glycine, Phe means phenylalanine, and Ser means serine residue.

2. Description of the Related Art Bradykinin (hereinafter, referred to as BK) and kallidin (hereinafter, referred to as KK) are peptides having physiological actions such as expansion of small blood vessels, enhancement of permeability, and contraction or relaxation of smooth muscle. Hy at the seventh position from the C-terminal in the general formula (I)
p is a peptide substituted with Pro, X is BK when X is a hydrogen atom, KK when X is Lys,
All are typical kinin compounds. BK in human body fluids
Is commercially available from Dainippon Pharmaceutical Co., Ltd.
Not only KK but also peptide (I) was determined as BK, and peptide (I) could not be quantified by fractionation.

There are no reports referring to a method for determining trace amounts of peptide (I).

The present inventors have found that peptide (I) is present in human body fluids such as urine, blood, and ascites, and the concentration of these peptides in human body fluids can be used as a diagnostic index for diseases such as cancer that are associated with inflammation and enhance vascular permeability. Based on the finding that the antibody has potential, the inventors succeeded in creating an antibody having excellent specificity for peptide (I), and completed the present invention.

Means for Solving the Problems The present invention relates to an antibody that specifically recognizes peptide (I). More specifically, the present invention relates to a compound of the general formula (I) X-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg
(I) (wherein, X represents the same as described above.) An antibody produced by immunizing a vertebrate with a conjugate of the peptide having the C-terminus of the peptide and an immunogenic protein as an antigen. The cross properties described in the following (a) and (b): (a) an antibody against the peptide (I) wherein X is Lys;
Does not substantially cross peptide (I), bradykinin and kallidin in which X is a hydrogen atom.

(B) An antibody against peptide (I) where X is a hydrogen atom does not substantially cross peptide (I) where X is Lys, bradykinin and kallidin.

With an antibody having the formula:

The peptide (I) here 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 a condensation, for example, azide method, mixed anhydride method, dicyclohexylcarbodiimide method, by a conventional method such as active ester method, in the liquid phase or solid phase,
Preferably, it can be performed in a solid phase.

In the solid-phase method, an amino acid having a protected functional group that does not need to participate 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 eliminated, and the reaction is involved in the reaction. Unnecessary coupling of amino acids having protected functional groups is repeated, and this operation is repeated until a desired peptide chain is obtained.Then, the bond with the insoluble carrier is cleaved by hydrogen fluoride treatment or the like. To obtain the target peptide. 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.

The conjugate of the peptide (I) and the immunogenic protein is useful as a hapten antigen for preparing an antibody. Examples of the immunogenic protein to be bound to the peptide (I) include proteins such as albumin, globulin, thyroglobulin, shellfish hemocyanin, and edestin.

Binding of pepride (I) to the immunogenic protein is performed at the C-terminus of peptide (I). Such a join
Cysteine (Cys) or Cys at the C-terminus of peptide (I)
To synthesize a peptide to which a peptide containing
This can be performed by binding the SH group in s with the amino group of the immunogenic protein using a binding agent. As the binder herein, for example, m- described in JP-B-58-8395 can be used.
A maleimide derivative, abbreviated as MBS, may be used.

The antibody of the present invention comprises a conjugate (hapten antigen) of the peptide represented by the general formula (I) thus prepared and an immunogenic protein together with an appropriate adjuvant, such as rabbits, guinea pigs, goats and sheep. It can be easily obtained by parenteral administration (immunization) to a vertebrate animal, collecting the serum thereof, and performing a known treatment. Monoclonal antibodies can be created by collecting antibody-producing cells of the animal thus immunized from the spleen, and performing operations such as fusion with myeloma cells and screening of clonal cells according to a conventional method.

The thus obtained antibody of the present invention has extremely high specificity for an antigen and does not substantially cross with an analog of peptide (I) such as BK or KK.

The present invention also relates to a peptide (I) using the above antibody.
And a kit for immunological quantification. That is, the kit of the present invention comprises: (a) a peptide (I) labeled with a label at the C-terminal, (b) the antibody itself described above or an insolubilized product thereof, and (c) a reagent (b) comprising the antibody itself. In some cases, it is composed of a reagent for separating a reaction product obtained by reacting this with the reagent (a) and a residue.

The reagent (a) is a conjugate (labeled antigen) in which a label and a peptide (I) represented by the general formula (I) are bonded at the C-terminus of the peptide (I). Examples of the label include an enzyme, a radioactive substance, a fluorescent substance, and a spin compound, and an enzyme is particularly preferable. Β-galactosidase as an enzyme,
Alkaline phosphatase, lipase, peroxidase, glucose-6-phosphate dehydrogenase and the like are used. The binding between the enzyme and the peptide (I) can be carried out in the same manner as the binding method between the immunogenic protein and the peptide (I) described above.

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.
Any insoluble carrier may be used as long as it is used in the art, but bacterial cell wall fragments disclosed in US Pat. No. 4,166,767 are particularly preferably used. The binding between the insoluble carrier and the antibody can be performed by a conventional method using a binder. Examples of the binder include glutaraldehyde, toluene diisocyanate, dihalogenated dinitrobenzene, and the like that chemically bond between the amino group of the antibody and the amino group of the insoluble carrier. Further, a maleimide derivative (m-MBS) which crosslinks the SH group of the antibody with the amino group of the insoluble carrier or, conversely, the amino group of the antibody with the SH group of the insoluble carrier is suitably used as the binder. When the SH group does not exist, the SH group may be introduced using, for example, N- (acetylmercaptoacetoxin) succinimide disclosed in Japanese Patent Application Laid-Open No. Sho 57-1442967 and then subjected to binding.

Usually used as the reagent (c) is an antibody (second antibody) for the antibody serving as the reagent (b), which is insolubilized (insoluble second 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. An insolubilized second antibody using a bacterial cell wall fragment as a carrier is commercially available from Dainippon Pharmaceutical Co., Ltd. under the name of Marcela.

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

For quantification of peptide (I) using these reagents, a sample is reacted with reagents (a) and (b). When an insolubilized antibody is used as reagent (b), the reaction mixture is centrifuged and free When the reagent (a) (supernatant) and the other substance (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 reacted. After that, B / F separation is performed, or the activity of the label in the supernatant or the precipitate separated by is measured. The conditions of such steps or implementation are the same as those of the normal EIA method. When the specimen contains kininase such as human body fluid, it is usually better to remove the protein in advance with trichloroacetic acid or the like.

Specific Example Next, the present invention will be described in more detail with reference to examples. In the following, a peptide in which X in Formula (I) is a hydrogen atom is abbreviated as HYP-BK, and a peptide in which X is Lys is abbreviated as HYP-KK.

Example 1 Synthesis of Peptide (I) (A) HYP-BK is a solid-phase peptide synthesizer model 990E
(Beckman Instruments) using J.Am.Che
m. Soc., 86 , 304-305 (1964). That is, according to the procedure, HYP-BK was synthesized by sequentially coupling each Boc-L-amino acid to P-methylbenzhydrylamine resin. As a condensing agent, Boc
-In the case of arginine, dicyclohexylcarbodiimide and 1-hydroxybenztriazole were used; otherwise, only the former was used. The obtained HYP-B
The amino acid sequence of K was confirmed using a 470A protein sequencer (Applied Biosystems).

(B) The next peptide is obtained in the same manner as in (A).

HYP-KK HYP-BK-Cys (Peptide in which Cys is condensed at the C-terminus of HYP-BK) HYP-KK-Cys (Peptide in which Cys is condensed at the C-terminus of HYP-KK) Example 2 Peptide and protein 5 ml of dioxane containing 2.5 mg of m-MBS in 50 ml of 0.05 M phosphate buffer (pH 7) containing 50 mg of bovine serum albumin (BSA)
And stirred at room temperature for 1 hour, and diluted twice with the same buffer.
Filter and wash with a PM30 (Amicon) membrane. Dissolve the material on the membrane in 5 ml of the same buffer, and add 1 ml of the same buffer containing 5 mg of HYP-BK-Cys.
And stirred for a further 2 hours at room temperature. 0.9% N
It is dialyzed twice with 5 of aCl to obtain a HYP-BK-Cys-BSA conjugate (hapten antigen solution).

Similarly, a HYP-KK-Cys-BSA conjugate (hapten antigen solution) is obtained.

Example 3 Preparation of Antibody (A) Polyclonal Antibody The two hapten antigen solutions obtained in Example 2 were each
It was diluted with 0.9% NaCl so that the absorbance at 280 nm was about 2, mixed with an equal amount of Freund's complete adjuvant, and the emulsion was subcutaneously subcutaneously at the back footpad in two places of 0.2 ml per rabbit. 8 injections of 0.2 ml each at the same time (immunization)
From next time, 0.2 ml subcutaneously on the back every 2 hours.
Inject 0 places. Immunization is performed 5 times, and one week later, whole blood is collected from the carotid artery under urethane anesthesia to obtain each antiserum.

(B) Monoclonal antibody Emulsion prepared in (A) 0.1 per BALB / c mouse.
Inject 5 ml subcutaneously at 4 week intervals in 4-5 locations. One week after immunization three times, the spleen is collected, and antibody-producing cells are obtained by a conventional method. The spleen cells (7 × 10
⁵ ) and mouse myeloma cells (P3 × 63Ag-U1, 2.1 × 10
⁶ ) by the polyethylene glycol method to prepare a hybridoma. Dispense into 96-well titer plates,
Culture by a conventional method. Screening of antibody-producing cells is carried out using the enzyme-labeled antigen of Example 5, an insolubilized anti-goat IgG rabbit antibody (Marcela 30), an enzyme substrate and an enzyme reaction terminator. Finally, three monoclonal antibodies (Ig
G type) Productive hybridoma was obtained. Two of them are E
It produced monoclonal antibodies suitable for IA.

Example 4 Binding of Peptide to Enzyme 1 ml of water containing 500 μg of maleimide was added to 2 ml of water containing 1 mg of β-galactosidase from Escherichia coli (hereinafter referred to as β-Ga1; Boehringer Mannheim), and the mixture was stirred at room temperature for 1 hour. This is dialyzed twice against 0.02M phosphate buffer (pH 7) 5 to obtain β-Ga1 in which the SH group is protected.

0.4 ml of dimethylformamide containing 60 μg of m-MBS is added to 3 ml of the dialyzed solution, and the mixture is stirred at room temperature for 2.5 hours. To 1 ml of this solution, add HYP-BK-Cys or HYP-
200 μl of water containing 500 μg of KK-Cys was added, and the mixture was stirred at room temperature for 2 hours, and a Biogel P4 column (BioRad, 2.5 × 3
2 cm, 100-200 mesh) to fractionate 5 ml each, and collect the enzyme-active portion and collect the enzyme-labeled antigen (HYP-BK-Cys-β-Ga1).
Alternatively, a HYP-KK-Cys-Ga1) solution is obtained.

Example 5 Determination of HYP-BK by EIA Reagent Standard antigen HYP obtained in Example 1 (A) so as to have a concentration of 0 to 1000 ng / ml
-A solution in which BK is dissolved in the following buffer A.

Antibody A solution obtained by diluting the serum (anti-HYP-BK polyclonal antibody) obtained in Example 3 (A) 5000 times with buffer A.

Enzyme-labeled antigen A solution obtained by diluting the HYP-BK-Cys-β-Ga1-containing solution obtained in Example 4 with buffer A 20-fold.

Insolubilized second antibody Mercera 10 (insolubilized anti-rabbit IgG goat antibody) (Dainippon Pharmaceutical Co., Ltd.) 0.9% NaCl substrate 27 mM 2-nitrophenyl-β-D-galactoside-1 mM
GCL ₂ -40% ethylene glycol -0.1% NaN ₃ enzymatic reaction stopper 0.1MK ₂ HPO ₄ -NaOH (pH11) buffer A 0.1% BSA-0.1% NaN 3 -0.9% NaCl-0.04M phosphate buffer (pH 7 ) Buffer B 0.5M Tris-HCl-0.2% Gelatin-0.9% NaCl (pH8) 20% Trichloroacetic acid (protein-free) Procedure (1) Pretreatment (protein-free)
, And 100% of 20% trichloroacetic acid was mixed. The resulting precipitate was centrifuged (15000 xg, 10 minutes), and
A sample obtained by adding the same amount of buffer B to a sample is used as a sample.

(2) Quantitative method Standard antigen or pretreated sample 100 in test tube (1 × 10cm)
Add μ, then add 200μ of antibody and incubate at 37 ° C. After 30 minutes, mix 200 µ of enzyme-labeled antigen and incubate at 37 ° C. 30 minutes later, add 200μ of Marcela 10 and add 15 at the same temperature.
Incubate for a minute. Then add 2 ml of 0.9% NaCl and centrifuge (1500
X 10 minutes) and remove the supernatant. This cleaning operation is another
Repeat. Add 500 μl of buffer A to the precipitate and stir to make it uniform. Add 100μ of substrate to this, stir, 37 ℃
For 30 minutes and add 1.5 ml of enzyme reaction terminator. This is centrifuged (1500 × g, 10 minutes), and the absorbance of the supernatant at 410 nm is measured. The amount of HYP-BK in the sample is determined from a separately prepared calibration curve (FIG. 1).

Example 6 Cross-reactivity The same operation as in Example 5 was performed except that BK, Des9BK (BK lacking the ninth amino acid from the C-terminus, the same applies hereinafter), Des8, 9BK, and HYP-KK were used as specimens. As a result, no cross-reactivity was observed below 500 ng / ml, and 1000 ng / ml
In the case of BK and Des8, 9BK, 0.39% of Des
Less than 0.39% crossing was observed for 9BK and KK, and only 0.72% for HYP-KK.

Example 7 EIA Using Monoclonal Antibody HYP-BK was quantified in the same manner as in Example 5 except that the monoclonal antibody obtained in Example 3 (B) and Marcela 30 were used.

Example 8 EIA of HYP-KK Buffer A containing HYP-KK obtained in Example 1 (B) as a standard antigen was used as an antibody, and anti-HY prepared in Example 3 (A) as an antibody.
Except for using buffer A containing P-KK antibody and buffer A containing HYP-KK-Cys-β-Ga1 prepared in Example 4 as an enzyme-labeled antigen, HYP- E of KK
IA was performed.

[Brief description of the drawings]

FIG. 1 is a calibration curve of HYP-BKs. In FIG. 1, Bx / Bo indicates the ratio (%) of the absorbance (Bo) when the standard antigen concentration is 0 and the absorbance (Bx) when the standard antigen concentration is x.

 ──────────────────────────────────────────────────続 き Continued on the front page (56) References Biochemical and Biophysical Research Communications, 150 (1), 511-516, (January 15, 1988)

Claims (2)

(57) [Claims] 1. A compound of the formula (I) X-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg
(I) (wherein, X represents a hydrogen atom or Lys.) An antibody prepared by immunizing a vertebrate with a conjugate of a peptide represented by the formula: An antibody having the cross-characteristics described in the following (a) and (b). (A) an antibody against peptide (I) wherein X is Lys
Does not substantially cross peptide (I), bradykinin and kallidin in which X is a hydrogen atom. (B) An antibody against peptide (I) where X is a hydrogen atom does not substantially cross peptide (I) where X is Lys, bradykinin and kallidin. 2. A kit for immunological quantification of peptide (I) comprising at least the following reagents: (a) peptide (I) labeled at the C-terminal with a label, (b) (C) when the reagent (b) is the antibody itself, a reagent for separating a reaction product obtained when the reagent (a) is reacted with the reagent (a) from the residue.