JPH01317399A - Method for stabilizing detection system - Google Patents

Abstract

PURPOSE:To suppress non-enzymic oxidation and measure a substance to be analyzed in high precision by stabilizing a detection system using phosphorusoxyacid, sulfamic acid, aromatic sulfonic acid, etc., as a reaction terminating agent for detection system utilizing oxidation reaction of a substrate by a peroxidase. CONSTITUTION:A mouse antibody, etc., against a substance to be measured is brought into contact with a sample for measuring to carry out antigen antibody reaction and then a peroxidase (e.g., Horseradishperoxidase) bound to antimouse immunoglobulin antibody is added thereto to react each other and unreacted material is separated and then phosphorusoxyacid, sulfamic acid, aromatic sulfonic acid, etc., used as a reaction terminating agent is added to a detection system utilizing oxidation reaction of a substrate by peroxidase colored by adding peroxide of enzymic substrate and o-phenylene diamine to terminate the reaction and then absorbance is measured to stably measure the substance to be measured in high precision without coloring by nonspecific absorption.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for analyzing biological components using peroxidase, and particularly to a method for stabilizing a detection system that utilizes the oxidation reaction of a substrate by peroxidase.

[Conventional technology and its problems]

In clinical tests and diagnostic analysis, qualitative analysis of various biological components,
For quantitative determination, measurement methods that utilize reactions of enzymes, particularly peroxidases, are widely used. Peroxidase is an enzyme that catalyzes oxidation reactions of various substances using hydrogen peroxide as a hydrogen acceptor, and can be used in detection systems based on optical changes based on oxidation reactions of chromogens, which are substrates.

For example, measurement of biological components to be analyzed such as cholesterol and glucose is carried out by applying the corresponding oxidizing enzymes such as cholesterol oxidase and glucose oxidase, and measuring the amount of hydrogen peroxide produced and the coloring reaction caused by the peroxidase. It can be quantified using In addition, in immunoassay methods that utilize antigen-antibody reactions, by using peroxidase as a label, the amount of activity or changes in activity can be detected by color reaction, and the target component in the sample can be measured. I can do it.

On the other hand, in order to accurately measure the target biological component, it is necessary to stop the oxidation reaction by peroxidase at an optimal point. Conventionally, this oxidation reaction has been stopped by adding an enzyme inhibitor or denaturant such as sodium azide or sodium fluoride, or an inorganic acid stopper such as hydrochloric acid or sulfuric acid. Using hydrochloric acid or sulfuric acid as a terminating agent, e.g. as a substrate. When -7 22-diamine is used, the absorbance at its maximum absorption wavelength increases by a factor of two or more, resulting in generally high sensitivity, which is advantageous for measurement. However, it is known that addition of an inorganic acid such as hydrochloric acid or sulfuric acid does not completely stop color development, and the absorbance increases over time due to a non-enzymatic oxidation reaction. It has also been reported that this increase can be suppressed by adding sodium sulfite to the acid (JournaI or Clnical C).
hemistry and C11nical Bio
chemistry, 23. Pd2-44.198
5). However, the addition of sodium sulfite also poses practical problems such as generation of sulfur dioxide gas.

[Purpose of the invention]

In light of the above circumstances, an object of the present invention is to provide a method for stabilizing a detection system that suppresses side reactions during oxidation of a substrate by peroxidase.

[Structure of the invention]

The object of the present invention described above is to provide a detection system that utilizes the oxidation reaction of a substrate by a peroxidase, in which at least one kind selected from phosphorus oxo acids, sulfamic acids, and aromatic sulfonic acid compounds is used as a reaction terminator. This is achieved by a method of stabilizing a detection system using a compound.

According to the present invention, the oxidation reaction of the substrate by the peracid enzyme is completely stopped, the increase in the detected value due to the non-enzymatic oxidation reaction as a side reaction is suppressed, and the detection system is stabilized. As a result, extremely precise and accurate measurement of the target biological component becomes possible.

The phosphorus oxo acids include phosphorus pentoxide, orthophosphoric acid, phosphorous acid, hypophosphorous acid, hypophosphorous acid, and condensed phosphorus oxo acids in which two or more phosphorus oxo acids are bonded, such as pyrophosphoric acid, polyphosphoric acid, and meth. Phosphoric acid and ultra phosphoric acid can be used. Preferred are phosphorus pentoxide, orthophosphoric acid, or their condensed combined phosphoric acids having an oxidation number of 5.

In particular, ori-phosphoric acid and pyrophosphoric acid are preferred.

The aromatic sulfonic acid compound may be any aromatic compound in which the aromatic ring is substituted with sulfonic acid. Examples of the aromatic ring include a benzene ring and a naphthalene ring. The aromatic ring may contain other substituents, such as t: aliphatic hydrocarbon residues such as alkyl groups and alkenyl groups, alicyclic compound residues such as cyclohexyl groups, pyridinyl groups, quinolyl groups, furaryl groups, thiazolyl groups, etc. It may be substituted with a heterocyclic residue, an aryl group such as a phenyl group or a naphthyl group, or a substituent such as a hydroxyl group, an alkoxy group, a carbonyl group, a carboxyl group, an amine group, an amide group, or a halogen group.

Typical aromatic sulfonic acid compounds useful in the present invention include benzenesulfonic acid derivatives, with benzenesulfonic acid, p-toluenesulfonic acid, and sulfosalicylic acid being particularly preferred.

The peroxidase according to the present invention may be derived from plants, animals, microorganisms, etc. Examples thereof include horseradish peroxidase, lactoperoxidase, glutathione peroxidase, myeloperoxidase, and cytochrome C. Examples include peroxidase.

Particularly preferred is horseradish peroxidase.

A peroxidant is required for the oxidation reaction of a substrate by peroxidase. The peroxide may be any peroxide, such as an organic peroxide, but hydrogen peroxide is preferred.

As the substrate for the peroxidase according to the present invention, substances that are oxidized to provide analytical means can be used, and chromogens that produce color pigments are common, but luminescent substances or fluorescent substances can be used. It may be a substance that generates a substance. Substrates generally used in detection systems that utilize an enzymatic reaction between peroxidase and hydrogen peroxide can be used. Examples include aromatic amine compounds and phenolic compounds. Representative examples include o-phenylenediamine, 3.3',5゜57-titramethylbenzidine, especially 0-7
Enylene diamine is preferred.

When the terminator according to the present invention is added in an appropriate amount to a solution in which the oxidation reaction by peroxidase is proceeding, it can lower the pH and completely deactivate the activity of peroxidase. For example, when o-phenylenediamine is used as a chromogen, the maximum absorption wavelength of the generated dye shifts, as is the case when hydrochloric acid or sulfuric acid is used as a stopper, and the absorbance increases accordingly, allowing highly sensitive measurements. becomes. Furthermore, it suppresses the generation of dye over time due to secondary non-enzymatic oxidation reactions, keeps the measured value constant, and stabilizes the detection system.

The amount of the terminator of the present invention added to the solution in which the enzymatic reaction is progressing may be any amount that can deactivate the activity of the enzyme, and the final optimal concentration in the reaction solution will vary somewhat depending on the type. , in the case of phosphorus oxoacid, 1 v/v (weight/
% or more, and more preferably 5 to 50 v/v%.

Further, in the case of sulfamic acid, the amount is preferably 1 v/v% or more, and more preferably 3 to 20 v/v%.

In the case of aromatic sulfonic acid compounds, the content is preferably 0.1 w/v% or more, more preferably 1 w/v% or more, and particularly preferably 5 to 50 v/v%.

The terminators according to the present invention may be used in combination of two or more, or may be used simultaneously with other known terminators such as hydrochloric acid, sulfuric acid, sodium sulfite, etc.

In that case, it is effective even if the concentration is below the above-mentioned optimum concentration. moreover,
Compounds that stabilize the detection system by adding them during the oxidation reaction of peroxidase, such as chelate compounds, oxalic acid,
Oxalate, aldehyde, bound phosphoric acid compounds, etc. may be used in combination to the extent that they do not inhibit the enzymatic reaction. These oxidation reaction stabilizers are added to the oxidation reaction solvent.

It may be added at any time up to immediately after the addition of the terminator, or it may be mixed with the terminator in advance.

Generally, an aqueous solvent is preferable as the solvent used for the oxidation reaction of peroxidase, but an organic solvent or a mixed solvent of an organic solvent and an aqueous solvent may be used as long as it does not extremely inhibit the enzymatic reaction. The pH of the solvent is desirably adjusted to an appropriate pH value for the oxidation reaction of peroxidase by adding a buffer, and the pH value is preferably 3 to 9. Further, the substrate concentration in the solvent is preferably 0.1 to 100 mM.

Next, an example of an embodiment of a detection system according to the present invention having each of the above-mentioned requirements will be shown.

For example, pH 3 containing 0.1-100mM chromogen
Prepare a buffer with a preferred pH in the range of ~9. When measuring peroxidase activity, a certain amount of hydrogen peroxide is further added, and when measuring the amount of hydrogen peroxide, a certain amount of peroxidase is further added to form a coloring reaction reagent solution. Further, the reagent solution may contain an appropriate amount of a stabilizer for the coloring reaction, such as a condensed phosphate. Mix the prepared reagent solution and sample 2.
A coloring reaction using an enzymatic reaction is performed at a temperature of ~50°C. After a certain period of time, a terminator is added to stop the reaction.

The chromogen is oxidized depending on the peroxidase activity or the amount of hydrogen peroxide in the sample, and the resulting dye is detected optically, for example, the absorbance at the maximum absorption wavelength is measured.

On the other hand, a calibration curve is prepared by performing a similar operation on a known amount of peroxidase or hydrogen peroxide, and the component to be analyzed in the sample is quantified by comparing the calibration curve with the measured values.

The invention applies to any enzyme immunoassay, such as homogeneous or heterogeneous assays, competitive or non-competitive assays, ELISA
It is applicable to any general measurement method, such as the method, sand winch method, etc.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example 1 0-phenylenediamine dihydrochloride at a concentration of 3 mg/mQ, 50 mM with a concentration of 0.02% hydrogen peroxide <
Citric acid-100n+lJ phosphate buffer (p14.9) was prepared and used as a coloring reaction reagent. Reagent solution Q, 0 in 5m12
．． 5 μQ of Horse Radison hyperoxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Tabo) at a concentration of 1 μg/+n12 was added, and the mixture was allowed to react in the dark at 37° C. for 30 minutes.

Next, 2 mQ of each terminator was added to stop the reaction, and the absorption spectrum was measured. As a comparative example, measurements were taken with sulfuric acid added and with pure water added instead of a terminating agent. Table 1 shows the maximum absorption wavelength and its absorbance.

Table 1 As shown in Table 1, the terminator of the present invention is 492n in the case of phosphorous oxoacid and sulfamic acid as well as in the case of sulfuric acid.
m, 502 nm in the case of aromatic sulfonic acid compounds,
The maximum absorption wavelength shifts to longer wavelengths, resulting in increased absorbance and improved sensitivity.

Example 2 As an antibody, monoclonal antibody 3872 against galactonrase isozyme ① described in JP-A-62-174100 was used.

After immobilizing 10 μg of monoclonal antibody 3872 on ABS beads at a concentration of 7 m (l), 1% bovine serum albumin (hereinafter referred to as BSA) in phosphate buffered saline (pH 7.4) was added.
(hereinafter referred to as PBS) was used for blocking. Furthermore, react with the positive sample, ascites fluid, at 21°C overnight and add PBS.
After washing with PSB, the plate was reacted with peroxidase-conjugated monoclonal antibody 3872 for 21'02 hours, and then washed again with PSB. Transfer the beads to a test tube and add a color reaction reagent solution, i.e., 50mM < citric acid-100mM phosphate buffer (pH 4) containing O-phenylenediamine dihydrochloride at a concentration of 3mg/mQ and hydrogen peroxide at a concentration of 0.02%.
-9) 0.5m12 was added. After reacting at 37°C for 30 minutes, stop solution 2IIIQ was added, and the mixture was left standing at room temperature in the dark.
Absorbance at 92 nm was measured over time.

Phosphorus oxoacid was used as the stop solution in the present invention, and sulfuric acid was used for comparison. The results are shown in Table 2.

The values in the table are the measured values at the first measurement (3 minutes), the second measurement (30 minutes), and the third measurement (150 minutes).
The difference between the measured value at the time of measurement and the first measured value is shown.

Table 2 It can be seen that when the terminating agent phosphorus oxoacid according to the present invention is used, the production of dye after the enzymatic reaction is stopped is suppressed.

Example 3 The same procedure as Example 2 was carried out except that sulfamic acid or a combination of sulfamic acid and birophosphoric acid was used as the terminator. The results are shown in Table 3.

Table 3 It can be seen that when sulfamic acid, which is the terminator of the present invention, is used, the production of dye after the enzymatic reaction is stopped is suppressed.

Example 4 According to Example 1, the effect of an aromatic sulfonic acid compound as a terminator was determined.

The experiment was also conducted using a coloring reagent solution in which 0.1 W/V% sodium tripolyphosphate was further added.

5 when aromatic sulfonic acid compound is used as a terminator
The absorbance at 00nn+ was measured. The results are shown in Table 4. It can be seen that when the aromatic sulfonic acid compound, which is the floating agent of the present invention, is used, the formation of dye after the enzymatic reaction is stopped is suppressed. Furthermore, it is more effective when sodium tripolyphosphate, which is a condensed phosphate, is used in combination.

〔Effect of the invention〕

According to the present invention, it is possible to completely stop the oxidation reaction of peroxidase, suppress non-enzymatic oxidation of the substrate after stopping, and maintain accurate values. It has become possible to measure the amount of hydrogen oxide as well as the corresponding substances to be analyzed with extremely high precision.

Claims (5)

[Claims] (1) In a detection system that utilizes the oxidation reaction of a substrate by peroxidase, at least one compound selected from phosphorus oxoacid, sulfamic acid, and aromatic sulfonic acid compounds should be used as a reaction terminator. Characteristic detection system stabilization method. (2) The method for stabilizing a detection system according to claim 1, wherein the phosphorus oxoacid is orthophosphoric acid or condensed phosphoric acid. (3) The method for stabilizing a detection system according to claim 1, wherein the aromatic sulfonic acid compound is a benzenesulfonic acid derivative. (4) The method for stabilizing a detection system according to claim 1 or 3, wherein the aromatic sulfonic acid compound is benzene, sulfonic acid, p-toluenesulfonic acid, or sulfosalicylic acid. (5) The method for stabilizing a detection system according to any one of claims 1 to 4, wherein the substrate of peroxidase is o-phenylenediamine.