JPS5962529A - Stable color-producing reagent composition - Google Patents

Abstract

PURPOSE:To provide the titled composition containing phenylenediamine or its derivative and an organic reducing agent, and useful for the analysis by the oxidative color development. CONSTITUTION:The objective stable color-producing reagent composition can be prepared by compounding (A) 1-100mM of phenylenediamine or its derivative (e.g. o-phenylenediamine, 4-amino-N,N-dimethylaniline, etc.), (B) 0.1mM-5M of an organic reducing agent (e.g. an oxalic acid compound, hydroxylamine sulfate, etc.), and if necessary, (C) a peroxidase (e.g. peroxidase, lactoperoxidase, cytochrome peroxidase, etc.), a buffer solution, a standard solution, etc. The contents of individual components of a body fluid can be determined by determining the evolved hydrogen peroxide using said composition. EFFECT:The stability to light can be improved and the non-specific reaction can be suppressed by the use of the organic reducing agent as a component of said composition. The composition is also excellent in the stability of color with time after the color development.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a stable color former composition, and more particularly to a method for stabilizing a color former used in a measurement system based on oxidative color development using a peroxide substance, a color former, and a peroxidase.

The measurement of perl'Nated substances, especially hydrogen peroxide, has recently become increasingly important in the field of clinical testing. Hydrogen peroxide is produced by an enzymatic reaction between body fluid components such as glucose, uric acid, cholesterol, and monoamines with glucose oxidase, uricase, cholesterol oxidase, and monoamine oxidase. By quantifying the generated hydrogen peroxide using a coloring agent and peroxidase, it is possible to know the nail content of each body fluid component.

In addition, the measurement of the activity of 1. hyperactive A. purple is becoming increasingly important in enzyme immunoassay using peroxidase as a labeling substance. In general, in enzyme immunoassay, the peroxidase activity of S-labeled antibodies or labeled antigens is measured to determine whether physiologically active substances such as carcinoids, embryonic enzymes, etc.
It is possible to measure trace amounts of serum proteins such as antigen/immunoglobulin G (FX), ferritin, β, and microglobulin.

As a method for quantifying peroxide substances, especially hydrogen peroxide, there is a method using 0-dianisidine as a coloring agent, or a method using 0-dianisidine as a coloring agent.
- A method using aminoandipirin and 7 enol is known (Inspection and Technology'l1rO1,9,/i l
1, P-867-871 (1981)). However, since the former has the property of reacting with reducing substances such as aldehydes, it has the disadvantage that its reaction is not specific to hydrogen peroxide. Furthermore, since the latter method does not have sufficient sensitivity, it requires a large number of valuable samples such as serum. or,
Therefore, it has the disadvantage that it is easily influenced by coexisting substances during measurement.

On the other hand, as a method for measuring the activity of peroxidase, a method is known in which a peroxide substance is used as a substrate and an 0-dianisidine or a pyroifo-phenylenediamine derivative is used as a coloring agent. However, in the method using SO-dianisidine, one reaction is nonspecific, as described above. In addition, the measurement method using pyrogallol uses ethyl ether, which tends to produce colored substances after enzymatic reaction, and has the inconvenience of requiring precision because the extraction operation is repeated. Also, it has great practicality. Measurement methods using phenylenediamine derivatives such as 10-phenylenediamine are commonly used)
It's perfect in terms of sensitivity. However, 0-phenylenediamine is susceptible to photo-oxidation, is unstable, and develops color due to non-specific reactions, so it must be handled with great care. Therefore, in order to suppress non-specific reactions such as photo-oxidation, it is inconvenient that it must be handled in a dark place. Conventional methods for stabilizing color formers include the method of blocking light.
Although methods such as adding iDTA are used, these methods cannot necessarily be said to be effective or advantageous. Furthermore, it is a well-known fact that adding a reducing substance to an oxidative coloring system interferes with the coloring reaction (Inspection and Ginan VoL9. All,
P-867-871 ('81)).

In the process of investigating oxidation reactions in aqueous solutions, the present inventors discovered that the oxidation degree of By intensively studying the reactivity of o-phenylenediamine, fl@:
It has been found that organic reducing agents such as hydroxylamine compounds and sulfur hydroxylamine not only do not inhibit the acetylation reaction caused by over-adsorption, for example, pero-V-sidase, but also can achieve stable coloration. was born.

That is, the present invention provides phenylenediamine or its derivative I
This is a stable color former composition characterized by containing a J-former and an organic reducing agent.

Among Arishiroito 31 base agents, oxalic acid compounds include butyric acid, t3
Alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as magnesium and calcium, and their ammonia conductors are, for example, 0
-phenylenethiamini, m-phenylenediamine, p
-phenylenediamines and their sulfates, hydrochlorides or (i&It'd salts 1 and also 4-amino-N,N-
Dimethylaniline, 4-amino-N, N-diethylaniline, 4-amino-NN-dipropylaniline, 4
-aminotriidine, 4-amino-N,N-diethyl-
m-) luidine, 4-amino-N-ethyl-N-β-hydroxyethyl-m-) luidine, and the like.

Examples of peroxidases include peroxidase, lactoperoxidase, and cytochrome peroxidase.

In the color former composition of the present invention) the organic reducing agent is usually 0.
1 m M to 5 M 1 preferably l m M to l
M is ant, phenylenediamine or its derivative is 1
mM to 100 mM. It is preferable that the field is in the range of 4 to 9.

The color former composition of the present invention may contain a peroxidant or a peroxidase, water, or a buffer.

Furthermore, other stabilizers, preservatives, etc. may be included if necessary.

Examples of measurements using the color former composition and peroxidase of the present invention include the following. When measuring the activity of hydrogen peroxide or peroxidase, an organic reducing agent is usually used at a concentration of 0.1 mλ to 5 Mz.
, Buffer containing SI<u11TIM~IM and phenylenediamine derivative at 1mM~lOOmM%@@, and peroxidase are added to the sample in any order or simultaneously. The pH is any pll in the range 4-9. When the enzymatic reaction is carried out in the dark or light at a reaction temperature of 2 to 60°C, preferably 2 to 40°C, a colored substance is produced depending on the amount of hydrogen peroxide produced or the amount of peroxidase present. is generated.

The absorbance at the wavelength of the maximum absorption value of the produced colored substance is measured. On the other hand, a calibration curve is created by similarly reacting the activity values of hydrogen peroxide or peroxidase with known concentrations, and compared with this calibration curve, the volume of peroxide or peroxidase activity in the sample is +11! I decide.

In methods using enzyme-labeled antibodies or enzyme-labeled antigens, a test F?
t('fl) to calculate the antigen or antibody in the sample.

In the present invention, the color reaction can be handled both in the dark and even in the light. In addition, the color former composition of the present invention contains peroxidase, but can also contain commonly used reagents such as buffer solutions and standard solutions.The color former composition of the present invention has stability against light by containing an organic reducing agent. is improved, making it possible to suppress non-specific reactions.

Furthermore, when measuring peroxidase and hydrogen peroxide using the color former composition of the present invention, the organic reducing agent has a characteristic that it does not inhibit the #tonic reaction.

Furthermore, the color former composition of the present invention is also excellent in changes over time after coloring.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Study of non-specific reactions O-phenylenediamine di-salt was further mixed with phosphoric acid-citric #J buffer solution (pj!, containing 0.02% hydrogen peroxide).
7) to a concentration of 0.3%. Furthermore, various additives were added, and after the whole child was dissolved, the absorbance at 492 nm was measured after 12 my+φ× in a test tube, and + was evaluated as a non-specific reaction.

The results are shown in Table-1.

From Table 1, it is clear that 1 has no additives.
It has been found that reducing substances such as adducts, ascorbic acid, hydroxyl amine sulfates, and glucose suppress non-specific varus J.

Detailed Example 2 Examination of enzyme reaction by peroxidase in a system in which 1 cyanogenic substance is added to each bucket.゜7)
It was dissolved to a concentration of 0.003%. Furthermore, 3-λ elemental substances of each concentration of yuzu were added and after completely dissolved, a test FA tube (12nφ
x 7°s cM) in 0°5 portions to prepare a coloring solution. Next, peroxidase. . The enzyme solution was dissolved in a 0.1% B% SA aqueous solution to a concentration of 15 mU/gal. 50 μl ft of this enzyme solution was added to the above-mentioned coloring solution and reacted in the light at room temperature for 30 minutes. After the reaction was completed, 2 times of X-N/sulfuric acid was added to stop one reaction, and the absorbance at 492 nm was measured.
'We investigated the inhibitory effect on enzyme reactions in the presence of quaternary substances.

The results are shown in Table-2.

As is clear from Table 2, even though the oxalic acid compound was added to the enzyme reaction thread, it did not inhibit the total elementary reaction. Ascorbic acid and the like greatly depend on the concentration, but at low concentrations, some color development was observed, but the enzyme reaction was significantly inhibited.

Δ11I Regular Example 1 Using the results of Insulin Analysis Test Examples-1 and -2, insulin was analyzed by Sandwich Method A.

(2) Production of non-bathed test mulch for insulin measurement Anti-pig insulin antiserum (guinea pig) was fractionated with 45% saturated ammonium sulfate and purified using a DICAE-Sepharose column to obtain an antibody fraction. This antibody fraction was dissolved in 0.01 M phosphate buffer (
Using p+ (near, 2: containing 0.15M NaO!), dissolved Kf was diluted to a concentration of 1 μ per 1 −,
400 thoroughly washed roughened polystyrene balls were immersed in 50 mt and subjected to III' JET at room temperature for 8 hours. Then, separate the polystyrene balls from the immersion liquid,
0, OL M phosphate 4 containing 1% bovine serum albumin
1 el liquid (14: 7.2 i 0.15M Na
p/containing) and then stored overnight in the same buffer.

■ Preparation of peroxidase-labeled antibody Using loq peroxidase (derived from horseradish) as the enzyme, peroxidase was conjugated to 51 anti-porcine insulin antibody by periodic acid cross-linking method, and purified by gel filtration with Sephadex G-200. Afterwards, it was concentrated in a collodion bag and used as a peroxidase-labeled antibody. Methods for binding peroxidase to antibodies are described in P1 Nakane et al.: The Journal of Histochemistry. And Cytochemistry, Volume 22, No. 12 1P-1
084-1091 (1974). The obtained peroxidase-labeled antibody was diluted with 1% immediately before use.
0.01 M NaCJ containing bovine serum albumin (p+I: 7.2 i 0.15 M NaCJ!
It was used after being diluted to approximately 2000 times with (containing).

■ Insulin measurement method Test bag (12 PIlφ x 7.5α) with buffer solution - (0.01 M!Jin vinegar containing 0.5% bovine serum albumin): 7.2; 0. Contains 15 M NaO!) o, a- and WHO number 66/304
Standard insulin solution (5-320
μu/m/)o. Add 1 ml and mix well, then add 1 insolubilizing reagent prepared in section ① of Measurement Example-1,
It was heated at 37'C for 1 hour. After heating, the reaction solution in the test tube was removed by suction, and buffer solution 1-1 was added for washing. After repeating this operation three times, 250μ of the peroxidase-labeled antibody obtained in section ① of Measurement Example-1 was added.
It was heated at 37°C for 2 hours. After heating, the reaction solution was removed by suction in the same manner as above, and after washing three times, only the balls were transferred to the coloring solution shown in Example 1, and each dye was measured at 492 mm.

■ Examination of liquefaction of the calibration curve when changing enzyme reaction conditions According to the measurement method in ■, standard insulin products were measured and a calibration curve was created. The enzyme reaction was performed with and without 30 mM sodium oxalate added to the coloring solution. In addition, the enzyme reaction was performed at room temperature in the light for 1 hour and at 4°C overnight, and the results of the former are shown in Figure 1.
The latter result is shown in Figure 2. As is clear from these figures, on the low insulin concentration side, sensitivity is significantly improved and non-specific reactions are suppressed.

■ Examination of changes over time after coloring Insulin standard product was measured according to the measurement method (2).

It should be noted that the color stability was examined to see how it changes depending on the presence or absence of the addition of each Maki-reducing substance, and the results are shown in Table 3. In addition, the absorbance immediately after stopping the reaction (49
It is shown by the change over time when znm) is set to 100.

Table 3 Examination of changes over time after color development As is clear from Table 3, color stability is not impaired even when reducing substances, especially oxalic acid compounds, are added to the e1γ-element reaction yarn. I understand.

Using the results of Analytical Test Practical Examples-1 and -2 in Measurement Example-219, FE was analyzed by sandwich method E-A.

(2) Preparation of insolubilizing reagent for 1FK measurement Anti-Human IH antiserum (rabbit) was fractionated with 45% saturated ammonium sulfate and purified with a DIGAE-Sepharose column to obtain an antibody fraction. Hereinafter, the preparation of the insolubilized test piece for IG measurement was performed using the same method as the production of a certain insolubilized test piece for insulin measurement in Measurement Example-1.

(2) Measurement of peroxidase-labeled antibody manufactured by 94 Co., Ltd. Synthesis was performed according to the method for preparing peroxidase-labeled antibody f11. Also, the same method was followed for usage.

■Measurement method for 1'H Buffer solution (0°5%
0.01 M phosphate buffer containing bovine serum albumin
7゜2j O, containing 15M Na0l>) 0.5- and a standard fE solution (12.5-1600U/ff1) prepared based on WHO number: '757502.
Add 20 μl of J, mix well, and then proceed to measurement example-2.
Add one insolubilizing reagent prepared in Section 0 of Section 0 and heat at 37'C for 1 hour. After heating, the reaction solution in the test tube is removed by suction, and the first buffer solution is added for washing. After repeating this operation three times, 250 Itl of the peroxidase-labeled antibody obtained in Section 2 of Measurement Example 2 was added, and the mixture was heated at 37°C for 2 hours. Below, ■ of measurement example-1
1 Red and colored pigments were measured at 492 nm in accordance with the method for measuring insulin in section ).

■ Enzyme reaction conditions? Detailed analysis of the changes in the calibration curve when changing the standard product was measured according to the 1XIll determination method described in (3), and a calibration curve was created. Note that the coloring liquid contains 1. OOIl
Enzyme reactions were performed with and without the addition of 1M of 7m rqt ammonium. The enzymatic reaction was carried out at room temperature, in the light, for 1 hour, and at 4°C overnight. The results of +iiJ are shown in Figure 3, and the results of the latter are shown in Figure 4. As is clear from these figures, engineering I/
It can be seen that sensitivity is significantly improved on the low B concentration side and non-specific reactions are suppressed.

Measurement example-3 Determination of hydrogen peroxide 0.3% 0-phenylenediamine di-salt dispersion and 60
Phosphate buffer (p
Add 50μl of 20U/l+l peroxidase aqueous solution to 500μl (5°0)! In addition, hydrogen peroxide solution (concentration 20, 10.20 mV/w=l) 25
After adding 1t1 and completing the enzyme reaction at 37°C for 130 minutes,
After stopping the reaction with IN sulfur 1i2-1/, the absorbance at 492 mm was measured and a calibration curve was created. In addition, oxalic acid) IJ
The above-mentioned loose solution containing no umum was used as a control for measurement.

The results are shown in FIG. As is clear from this figure, non-specific reactions are suppressed and sensitivity is also improved.

[Brief explanation of drawings]

Figure 1d O-3% 0-phenylenediamine, 2
Figure 2 is a calibration curve for insulin obtained by adding 30 M of sodium chloride to a coloring solution containing hydrochloride and carrying out an enzyme reaction at room temperature in the light for 1 hour. The enzymatic reaction was carried out overnight at 4°C using the same coloring solution. Figure 3 1d 0.3% 0-phenylenediamine. This is a calibration curve for IK obtained by adding 1100 II ammonium oxalate to a coloring solution containing dihydrochloride and carrying out an enzyme reaction at room temperature in the light for 1 hour. In Figure 4, the coloring solution is the same as in Figure 3, and enzyme anti-1+ is used.
The conditions were 4°C for one night. Moreover, the fifth
The figure shows a calibration curve for quantifying hydrogen diophthalmide using 0-phenylenediamine dihydrochloride containing 60FF1M sodium itate as a coloring agent and peroxidase as an enzyme. As a control, the measurement was conducted using a coloring agent that did not contain sodium chloride. In FIGS. 1 to 5, the symbol ■ indicates the case where a coloring liquid to which sodium oxalate was added is used, and the mark O indicates the case where an additive-free coloring liquid was used. Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] (1) The stable color forming agent composition according to claim 11iα, item 1, which is a stable color forming ammonium Jμ characterized by containing phenylenediamine or a derivative thereof and an organic reducing agent.