JPS62233761A - Enzyme labeling body for catecholamine acidic metabolite - Google Patents

Abstract

PURPOSE:To execute a catecholamine acidic metabolite to a specific enzyme immunological measurement by forming a enzyme labeling body in which the hydroxyl group, methoxyl group and carbroxyl group important as the hapten structure of the catecholamine acidic metabolite are not the coupler with enzyme. CONSTITUTION:This enzyme labeling body for the catechlamine acidic metabolite is expressed by formula I (in formula I, R<1> atom or hydroxyl group). This enzyme labeling body is applicable to the competitive enzyme immunological measurement in which the catecholamine acidic metabolite in a measuring sample and the enzyme labeling body are brought into competitive reaction with each other.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an enzyme i4-labeled substance of acidic catecholamine metabolites useful for enzyme immunoassay of acidic catecholamine metabolites and an aa method thereof.

[Prior art] Homovanillic acid (HV), an acidic metabolite of catecholamines,
A) and vanylmandelic acid (or vanillylmandelic acid,
VMA) is also the final metabolite of catecholamines (hereinafter referred to as catecholamines), and is excreted in the urine at a concentration several hundred to several thousand times higher than that of catecholamines and other intermediate metabolites. The urinary concentration of catecholamines is measured as a diagnostic method for catecholamine-producing tumors such as tumors and pediatric neuroblastomas. In particular, mass screening for neuroblastoma has been carried out for newborns nationwide starting this year.

Currently, the VMA measurement method that is mainly used for primary screening is a spot test method in which coloration due to diazo coupling reaction is determined on filter paper. However, this method is easily influenced by other coexisting phenolic compounds and has a problem of high false positive rate.

Separate and quantitative methods using high performance liquid chromatography (HPLC) have been used to simultaneously measure VMA and HVA, but this method is less convenient than the spot test method and is not suitable for measuring a large number of samples. It is. In addition, VM in gas chromatography-mass spectrometry
Although it is possible to measure A and HVA simultaneously and provides more accurate and sensitive measurements than HPLC, it has the disadvantage of requiring large equipment and skilled techniques.

On the other hand, immunoassays generally have the advantage of being able to measure a large number of samples simply and quickly. Conventionally, a method for immunoassaying catecholamine acidic metabolites has been disclosed in JP-A-60-12376.
Although the enzyme immunoassay method is disclosed in Publication No. 5, there is no description of the enzyme label, and the details of the system are unknown.

The present inventors previously developed a hapten antigen in which a catecholamine acidic metabolite and a carrier protein were combined by Mannich reaction to obtain antibodies against catecholamine acidic metabolites (105th Annual Meeting of the Pharmaceutical Society of Japan). Proceedings, p. 393, 5J11-3), there was no knowledge as to whether the enzyme activity of the 'Jgra enzyme remained when this method was applied to the enzyme labeling method.

[Problems to be Solved by the Invention] An object of the present invention is to develop an enzyme-labeled catecholamine acidic metabolite that can be applied to a method for quantifying catecholamine acidic metabolites by enzyme immunoassay.

Conventionally, the DCC method, mixed acid anhydride method, and glutaraldehyde method have been applied as enzyme labeling methods for haptens.

However, when the DCC method and the mixed acid anhydride method are applied to catecholamine acidic metabolites, it is thought that the catecholamine acidic metabolites bind to the enzyme through the carboxyl group, and the characteristic structural parts of the catecholamine acidic metabolites disappear. do. Therefore, specificity in the competitive reaction between the hapten and the enzyme label against the antibody during enzyme immunoassay is lost.

Furthermore, the glutaraldehyde method cannot bind enzymes to catecholamine acidic metabolites.

Therefore, in order to establish a highly specific enzyme immunoassay for catecholamine acidic metabolites, it has been desired to prepare an enzyme eAm that does not lose the structural characteristics of catecholamine acidic metabolites.

[Means for Solving the Problems] As a result of various studies based on the above objectives, the present inventors found that when a catecholamine acidic metabolite and an enzyme are combined by a Mannich reaction using formaldehyde,
Enzyme-labeled substances can be prepared in a free state without involving the hydroxyl, methoxyl, and carboxyl groups, which are important in the hapten structure of catecholamine acidic metabolites, in the binding process.
The present invention was completed based on the finding that the enzyme activity of the enzyme still remains sufficiently.

In other words, the present invention provides an enzyme-labeled catecholamine acidic metabolite represented by the general formula [I] [wherein R1 represents a hydrogen atom or a methyl group, and R2 represents a hydrogen atom or a hydroxyl group] It is something.

In addition, the present invention provides an enzyme-labeled catecholamine acidic metabolite represented by the general formula [1], which is represented by the general formula [■] [wherein R1 and R2 have the same meanings as above]. The present invention provides a method for producing an enzyme-labeled catecholamine acidic metabolite, which is characterized in that the catecholamine acidic metabolite is bonded by a Mannich reaction using an enzyme and formaldehyde.

In the present invention, the catecholamine acidic metabolite is represented by the above general formula [11], and specifically, HVA CR"=Mefl J&, R"=Hydrogen atom")
, VMA (R''=methyl group, R2=hydroxyl group).

Dihydroxyphenylacetic acid (DOPAC, R1=RZ
==hydrogen atom) and dihydroxymandelic acid (DOM
A, R1=hydrogen atom, RZ==hydroxyl group).

Furthermore, there are no particular restrictions on the enzymes that can be used to label catecholamine acidic metabolites, and any enzyme that can be used in general enzyme immunoassay may be selected.A typical example of an enzyme is alkaline phosphatase ( ALP), glucose oxidase (GOD), peroxidase (
horseradish) (HRP), β-galactosidase,
Examples include acetylcholinesterase.

Enzyme 4Fff! In the Mannich reaction, for example, a catecholamine acidic metabolite is used in an amount several tens to hundreds of times the molar amount of the enzyme, and both are treated with an excess amount of formaldehyde in an aqueous solvent at a pH of approximately 6 to 7. This can be carried out by reacting. pH of aqueous solution
i! INgI is, for example, a sodium hydrogen carbonate aqueous solution,
An acetate buffer or the like may be used. The reaction is completed in several tens of minutes to several tens of hours under heating conditions at room temperature or within a range where enzyme activity is stable.

After the reaction is completed, excess formaldehyde and catecholamine acidic metabolites are removed by dialysis against an aqueous sodium acetate solution, water, etc., and the product is prepared as an enzyme-labeled reagent for enzyme immunoassay either as it is or by lyophilization. be able to.

The enzyme label of the present invention can be applied to so-called competitive enzyme immunoassay in which the enzyme label and the catecholamine acidic metabolite in the measurement sample are competitively reacted with an antibody against the catecholamine acidic metabolite. For the operating method of competitive enzyme immunoassay, please refer to books and reviews such as "Enzyme Immunoassay" 2nd edition edited by Eiji Ishikawa et al., Igakushoin Co., Ltd., published December 15, 1980. good.

[Operation of the Invention The Mannich reaction between a cocatecholamine acidic metabolite and an enzyme is represented by the following reaction formula.

R″=hydrogen atom, methyl group R2=hydrogen atom, hydroxyl group Therefore, the hydroxyl group, methoxyl group, and carboxyl group, which are important functional groups in the hapten structure of catecholamine acidic metabolites, are not involved in this Mannich reaction. Enzyme labels can be prepared without using functional groups for binding to enzymes.

[Example] The following is a detailed explanation of the embodiments of the present invention, with an example of the preparation of the enzyme molecule of the present invention as an example, and an example of its application to enzyme immunoassay as an application example.

The VMA and HVA of the example hapten are each VMA5.9.
mg, and HVA 5.5 mg were used. The enzymes are ALP, G
10 μ of OD or HRP were used.

Hapten and enzyme in 0.3M sodium bicarbonate aqueous solution 1
oon, 20 μQ of 37% formalin was added, stirred, and reacted at 37° C. for 1 hour.

After the reaction was completed, the ALP and HRP reaction solution was dialyzed against 10''C distilled water, and the GOD reaction solution was dialyzed several times against 10111M sodium acetate aqueous solution.

Freeze-dried six enzyme labels, VMA-ALP.

HVA-ALP, VMA-GOD, HVA-G.

D, VMA-HRP and HVA-HRP were obtained.

Electrophoresis was performed to confirm the formation of ligated bodies and the residual enzyme activity. One layer of the label solution was placed on a cellulose acetate membrane. 1 mA/am for 15 minutes.

A membrane run in 0.07M Veronal 41 buffer (pH 8,6) was immersed in each substrate solution.

The substrate solution for ALP was O containing 16mM Pm=trophenyl phosphate and 111M magnesium chloride.
, 1M Tris buffer (PH8,0).

GOD substrate solution was 16.7mM glucose, 4mM
4-aminoantipyrine, 7mM phenol.

Phosphate buffered saline (3.3mM phosphate buffer, pi 47.3-52mM sodium chloride) containing 67x/ml HRP was used.

The substrate solution for HRP was 0.2%/mQ o-phenylenediamine and 5.6m% hydrogen peroxide.
A 1M citrate buffer (pH 5.0) was used.

The electropherograms thus obtained are shown in Figures 1-3. If the starch transfer point is the origin, VMA-enzyme and H
Each VA-enzyme migrated more toward the anode than the untreated enzyme. This is due to the acidic substances VMA and HV in enzymes.
It is concluded that this is due to the binding of A.6 Application Example■ Antigen Preparation 0.3 mmol of HVA or VMA as a hapten and 100 mmol of human serum albumin (H5A) as a carrier are mixed into 1 mm of a 0.3 M aqueous sodium bicarbonate solution. Dissolved. 37% formalin 0.2- was added to this, the pH was adjusted to 6.0 to 7.0 with a 3M aqueous sodium acetate solution, and the Mannich reaction was carried out.
VMA-
H8A and HVA-H5A were obtained.

■ Preparation of monoclonal antibodies A 1:1 emulsion of physiological saline solution of the antigen (1■/-) and complete Freund's adjuvant was mixed with Ba1b/C.
It was administered intraperitoneally to mice (male, 6 weeks old) several times every two weeks. For the final immunization, 50 μg of the antigen was intravenously injected, and the spleen was removed from the mouse.

Spleen cells and mouse myeloma cells P and U1 were fused using polyethylene glycol according to a conventional method.

The fused cells were cloned, a single clone was grown in mouse ascites, 1M1 water was collected, and anti-HVA was extracted by ammonium sulfate fractionation.
Alternatively, anti-VMA monoclonal antibodies were purified.

As a result, anti-VMA monoclonal antibody Vl (subclass/type, IgG, /
Anti-HVA monoclonal antibody H1 (subclass/type, IgG,
/to) was obtained.

Anti-VMA monoclonal antibody v1 and anti-HVA monoclonal antibody H1 were used in the following enzyme immunoassay.

■ Enzyme immunoassay (EIA) 0.25μg/- of monoclonal antibody PBS solution
0 μΩ was dispensed into each well of the microplate. After leaving the plate overnight at 4°C, it was added to PBS (T-
Each well was washed with PBS).

Enzyme labeled 0, 2 itg/mQ 254 and various concentrations of free MVMA, HVA solution or urine 25d were added simultaneously and incubated at 37°C for 1 hour to compete the enzyme labeled and hapten for binding with the adsorbed antibody. React, T-PBS
Washed with.

After adding 60 ml of the substrate solution used in the above example and reacting at 37°C for 1 hour, the absorbance of the reaction solution was measured. The measurement wavelength is 405nm for ALP, 492nn+ for GOD,
HRP was set to 450 nm.

With EIA, it was possible to measure the difference in the amount of enzyme bound due to competition between the hapten and the enzyme label in response to the hapten concentration, and the dose-response curves shown in Figures 4 to 6 could be drawn6. Effect] The enzyme-labeled catecholamine acidic metabolite of the present invention is
Since the hydroxyl group, methoxyl group, and carboxyl group, which are important in the hapten structure of catecholamine acidic metabolites, are not used as conjugates with enzymes, enzyme immunoassays specific to catecholamine acidic metabolites can be performed. Thus, according to the present invention, it is possible to establish an enzyme immunoassay that is useful as a primary screening for urinary catecholamine acidic metabolites, and to prepare an enzyme-labeled substance that is useful as an enzyme-labeled reagent for a kit for the measurement. can.

[Brief explanation of drawings]

FIGS. 1 to 3 are electropherograms of enzyme-labeled substances prepared in Examples of the present invention. Figures 4-6 are. This figure shows a dose-response curve obtained by enzyme immunoassay using the enzyme labeled product of the present invention in an application example. Patent applicant (677) Yamasa Soy Sauce Co., Ltd. *tt-@
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sllNka&-t4 to 004';0-w5 Procedural amendment W (spontaneous) May 21, 1985 1, Case description 1985 Patent Application No. 77826 2, Name of the invention Enzyme-labeled catecholamine acidic metabolite 3 , Relationship to the case of the person making the amendment Patent applicant postal code 288 Address 2-10 Shinseicho, Choshi City, Chiba Prefecture Column 5 of the detailed description of the invention in the specification, Contents of the amendment

Claims (1)

[Claims] 1) General formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] [In the formula, R^1 represents a hydrogen atom or a methyl group, and R^2 represents a hydrogen atom or a hydroxyl group. . ] Enzyme-labeled catecholamine acidic metabolite. 2) General formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] [In the formula, R^1 represents a hydrogen atom or a methyl group, and R^2 represents a hydrogen atom or a hydroxyl group. ] In producing the enzyme-labeled catecholamine acidic metabolite represented by the general formula [II] ▲Mathematical formulas, chemical formulas, tables, etc.▼[II] [In the formula, R^1 and R^2 are the same as above. significance. ] A method for producing an enzyme-labeled catecholamine acidic metabolite, which comprises bonding the catecholamine acidic metabolite represented by the following by a Mannich reaction using an enzyme and formaldehyde.