JPH0462026B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a stable coloring agent composition for a reagent for measuring peroxidant or peroxidase activity, and more specifically to a stable coloring agent composition for use in a measurement system based on oxidative color development using a peroxidant or peroxidase and a coloring agent. Concerning the law. The measurement of peroxides, particularly 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, the content of each body fluid component can be determined. Furthermore, the measurement of peroxidase activity is becoming increasingly important in enzyme immunoassay using peroxidase as a labeling substance. Generally, in enzyme immunoassay, by measuring the peroxidase activity of labeled antibodies or labeled antigens, biologically active substances or biological components, such as insulin, growth hormones such as thyroid-stimulating hormone, α-fetoprotein, carcinolin, etc. , embryonic, antigen, immunoglobulin E (IgE), ferritin, β ₂ −
It is possible to measure extremely small amounts of serum proteins such as microglobulin. By the way, as a method for quantifying peroxide substances, particularly hydrogen peroxide, methods using o-dianisidine or methods using 4-aminoantipyrine and phenol as coloring agents are known (Inspection and Technology).
Vol.9, No.11, 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 amount of valuable samples such as serum. Moreover, it also has the disadvantage that it is easily influenced by coexisting substances during measurement. On the other hand, as a method for measuring peroxidase activity, a method is known in which a peroxide substance is used as a substrate and o-dianisidine or pyrogallol and a phenylenediamine derivative are used as coloring agents. However, as described above, the reaction using o-dianisidine is nonspecific. Furthermore, 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, because of that,
Not very practical. phenylenediamine derivative,
For example, a measurement method using o-phenylenediamine is commonly used and has satisfactory sensitivity. However, o-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, it has some inconveniences, such as the need to handle it in a dark place in order to suppress non-specific reactions such as photo-oxidation. Conventionally, methods for stabilizing color formers include blocking light, adding EDTA, etc., but 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 Technology Vol. 9, No. 11, P-867-871 ('81)). In the process of investigating oxidation reactions in aqueous solutions, the present inventors discovered the specificities of oxidation reactions (e.g., the reactivity of oxygen radicals, the reactivity of oxygen ions, and the reactivity of dissolved molecular oxygen). etc.), we found that in the oxidative coloring system of o-phenylenediamine, oxalic acid or its salt is a peroxidase,
For example, it was found that not only did the oxidation reaction by peroxidase not be inhibited, but also stable coloration could be achieved, and the present invention was completed. That is, the present invention is a stable coloring agent composition for a peroxidant or peroxidase activity measuring reagent, which is characterized by containing phenylenediamine or a derivative thereof, oxalic acid or a salt thereof, and a water-soluble polymer compound. Examples of oxalic acid or its salts include oxalic acid and its alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as magnesium and calcium, and ammonium salts thereof. Further, the phenylene diamine or its derivative used in the present invention includes, for example, oo-phenylene diamine, m-phenylene diamine, p-phenylene diamine, m-phenylene diamine, p-
phenylenediamines and their sulfates, hydrochlorides or oxalates, as well as 4-amino-N,N-
Dimethylaniline, 4-amino-N,N-diethylaniline, 4-amino-N,N-dipropylaniline, 4-aminotoluidine, 4-amino-
N,N-diethyl-m-toluidine, 4-amino-N-ethyl-N-β-hydroxyethyl-m-
Examples include toluidine. Examples of peroxidases include peroxidase, lactoperoxidase, and cytochrome peroxidase. Further, as the water-soluble polymer substance, polyethylene glycol (hereinafter abbreviated as PEG), sucrose, polyvinyl alcohol, etc. can be used. In the color former composition of the present invention, oxalic acid or its salt is usually 0.1mM to 5M, preferably 1mM to 1M.
and phenylenediamine or its derivative is 1mM to 100mM. Further, the water-soluble polymer substance is contained in an amount of 0.001 to 10% by weight, preferably 1 to 2% by weight, based on the aqueous solution. Also, the pH is 4-9
It is preferable that it is in the range of . 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, for example, glucose, uric acid,
Body fluid components such as cholesterol and monoamines, growth hormones such as insulin and thyroid stimulating hormone,
Examples include minute amounts of serum proteins such as α-fetoprotein, carcinogenic antigen, immunoglobulin E (IgE), ferritin, and β ₂ -microglobulin. Measurement examples using the color former composition of the present invention and hydrogen peroxide include, for example, insulin, growth hormones such as thyroid stimulating hormone, α-phetoprotein, carcinoid embryonic antigen, immunoglobulin E (IgE), Examples include trace amounts of serum proteins such as ferritin and β ₂ -microglobulin. When measuring hydrogen peroxide or peroxidase activity, oxalic acid or its salt is usually used.
0.1mM to 5M, preferably 1mM to 1M, a phenylenediamine derivative of 1mM to 100mM, and further a water-soluble polymeric substance, such as PEG ⁺ 4000, of 1 to 1M.
Buffer and peroxidase containing 2% by weight are added to the sample in any order or simultaneously. PH is any PH in the range of 4-9. 2～60
When the enzymatic reaction is carried out in the dark or in the light at a reaction temperature of 2 to 40 DEG C., a colored substance is produced depending on the amount of hydrogen peroxide produced or the amount of peroxidase present. 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 the hydrogen peroxide or peroxidase activity in the sample is measured by comparing with this calibration curve. In a method using an enzyme-labeled antibody or enzyme-labeled antigen, the amount of antigen or antibody in a sample can be calculated by comparing it with a standard curve prepared using an amount of antigen or antibody whose concentration is known. In the present invention, the color reaction can be handled both in the dark and in the light, and can be measured with high sensitivity while minimizing non-specific reactions. In addition to phenylenediamine or a derivative thereof, oxalic acid or a salt thereof, and a water-soluble polymer substance, the color former composition of the present invention contains peroxidase if necessary, and further contains buffer solutions, standard solutions, etc. It can include the reagents used. By containing oxalic acid or a salt thereof, the color former composition of the present invention has improved stability against light and can suppress non-specific reactions. Furthermore, by including a water-soluble polymeric substance, the color former composition becomes easier to handle.
That is, although the color former composition can be freeze-dried without a water-soluble polymeric substance, the shape stability against vibration is significantly improved by adding a water-soluble polymeric substance. Furthermore, when measuring peroxidase and hydrogen peroxide using the color former composition of the present invention, oxalic acid or its salt and the water-soluble polymer substance have the characteristic that they do not inhibit the enzymatic 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 Examination of non-specific reactions O-phenylenediamine dihydrochloride was added to a phosphate-citrate buffer (PH
5.7), add it to 0.3%, and further,
PEG ⁺ 4000 was added to 1% and dissolved.
Furthermore, various organic reducing agents were added as additives, and after dissolving, 0.5 ml each was dispensed into test tubes (12 mmφ x 7.5 cm) to obtain a coloring solution. 1N after being left under various conditions
- After adding 2 ml of sulfuric acid, the absorbance at 492 nm was measured to evaluate non-specific reactions. The results are shown in Table-1.

[Table] Table 1 shows that in the system with PEG ⁺ 4000 added, reducing substances such as oxalic acid or its salts, ascorbic acid, hydroxyl/amine/sulfate, glucose, etc. reacted nonspecifically compared to the system without addition. I found out that I can control it. Example 2 Examination of enzymatic reactions by peroxidase in various reducing substance addition systems
5.7) to be 0.3%, and further,
PEG ⁺ 4000 was added to 1% and dissolved.
Furthermore, various concentrations of reducing substances were added and dissolved, and 0.5 ml each was dispensed into test tubes (12 mmφ x 7.5 cm) to prepare a coloring solution. Next, add peroxidase to 0.15 m
Dissolve in 0.1% BSA aqueous solution at U/ml,
It was made into an enzyme solution. 50μ of this enzyme solution was added to the coloring solution prepared above and reacted in the light at room temperature for 30 minutes. After the reaction was completed, 2 ml of 1N sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured to examine the inhibitory effect on enzyme reactions in the presence of various reducing substances. The results are shown in Table-2.

[Table] * - was not measured because it could not be dissolved.
As is clear from Table 2, it was found that even when oxalic acid or its salt was added to the enzyme reaction system, it did not inhibit the enzyme reaction at all. 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. Example 3 Freeze-drying of color former composition A color former composition shown in Table 3 was freeze-dried. For freeze-drying, a 30-ml colored vial was used, and after dispensing 6 ml each, the solution was immediately freeze-dried using a conventional method. Note that after the freeze-drying was completed, the atmosphere was replaced with nitrogen. The color tone in Table 3 was visually observed, the shape stability was determined by rotating the vial, and the ease with which the powder came out.
It displayed its fragility. In addition, when preparing the solution before freeze-drying, distilled water or 0.01M citric acid-phosphate buffer (PH: 5.0) was used.

【table】

[Table] Note 1: Shape stability is determined by rotating the vial horizontally on a roller while applying slight vibrations, and determining the number of revolutions until the powder first separates, which indicates the fragility of the shape. did. Measurement Example 1 Storage Stability Test of Freeze-Dried Product A storage stability test of the freeze-dried product prepared in Example 3 at 4°C was conducted. That is, after storing at 4°C for a specified period of time, citric acid-phosphate buffer (PH: 5.7) containing 0.02% hydrogen peroxide was added per vial.
ml was added and completely dissolved (however, No. 5 was dissolved by adding 40 ml). After dissolving, transfer to test tube (12mmφ x 7.5cm)
Dispense 0.5 ml each to make a coloring solution, leave it at room temperature in a bright place for 1 hour, add 2 ml of 1N sulfuric acid, measure the absorbance at 492 nm, and check the degree of blank color development to determine storage stability. The gender was evaluated. As a control, o-
Phenylene diamine dihydrochloride 3 mg/ml, ammonium oxalate 3.5 mM, and PEG ⁺ 4000 0.5 mg/ml were each weighed and dissolved in citric acid-phosphate buffer (PH: 5.7). The results are shown in Table-4. As is clear from Table 4, compared to the additive-free system, the organic reducing agent-added system showed less blank color development, and the blank color development hardly increased even after long-term storage.

[Table] Measurement Example 2 Insulin Analysis Test Utilizing the results of Examples 1 and 2, insulin was analyzed by Sand-Germany EIA. Manufacture of insolubilization test for insulin measurement 45 from anti-porcine insulin antiserum (guinea pig)
After fractionation using saturated ammonium sulfate, the antibody fraction was purified using a DEAE-Sepharose column to obtain an antibody fraction. This antibody fraction was diluted with 0.01M phosphate buffer (PH: 7.2; containing 0.15M NaCl) and 400 thoroughly washed roughened polystyrene balls were immersed in 50ml of a solution with a concentration of 1mg per ml. The mixture was left standing at room temperature for 8 hours. Thereafter, the polystyrene balls were separated from the immersion solution, washed three times with 0.01M phosphate buffer (PH: 7.2; containing 0.15M NaCl) containing 1% bovine serum albumin, and then stored in the same buffer. . Preparation of peroxidase-labeled antibody Using 10 mg of peroxidase (derived from horseradish) as the enzyme, 5
mg of anti-porcine insulin antibody was conjugated with peroxidase, purified by gel filtration using Sephadex G-200, concentrated in a collodion bag,
It was used as a peroxidase-labeled antibody. A method for attaching peroxidase to antibodies is described by P., Nakane et al.: The Journal of Histochemistry and
Cytochemistry, Volume 22, No. 12, P-1084
~1091 (1974). The obtained peroxidase-labeled antibody was added to 0.01M phosphate buffer (PH:
7.2; containing 0.15M NaCl) and diluted to approximately 2000 times before use. Insulin measurement method: Add buffer solution (0.5%) to a test tube (12mmφ x 7.5cm)
0.01 phosphate buffer containing bovine serum albumin <PH:
7.2; Contains 0.15M NaCl>) 0.4ml and 0.1ml of standard insulin solution (5-320μU/ml) prepared based on WHO number 66/304, mix well, and then proceed to Measurement Example-2. One insolubilizing reagent prepared in Section 1 was added and heated at 37°C for 1 hour.
After heating, the reaction solution in the test tube was removed by suction, and 1 ml of buffer solution was added for washing. Perform this operation 3
After repeating the test several times, add 250μ of the peroxidase-labeled antibody obtained in Measurement Example-2, and
Warmed for an hour. After heating, the reaction solution was removed by suction in the same manner as above, and after washing three times, a coloring solution was prepared from the freeze-dried coloring agent composition prepared in Example 3 according to the method shown in Measurement Example-1. The enzyme reaction was carried out under various conditions. After the enzymatic reaction is complete, add 2 ml of 1N sulfuric acid to stop the reaction,
Red and colored pigments were measured at 492 nm. A standard insulin product was measured and a calibration curve was created according to the measurement method described in ``Examination of changes in the calibration curve when enzyme reaction conditions are changed''. Note that the lyophilized product prepared in Example 3 was used as the coloring liquid. In addition, an enzyme reaction was performed on the lyophilized product with the addition of 70 mM ammonium oxalate and the lyophilized color former without any additive. The enzymatic reaction was carried out at room temperature in the light for 1 hour and at 4° C. overnight, and the former results are shown in FIG. 1 and the latter results are shown in FIG. 2. As is clear from these figures, on the low insulin concentration side, sensitivity is significantly improved and non-specific reactions are suppressed. Insulin standard products were measured according to the measurement method described in ``Study of changes over time after color development''. The color stability was examined to see how it changes depending on the presence or absence of the addition of various reducing substances, and the results are shown in Table 5. It also shows the change over time when the absorbance (492 nm) immediately after the reaction is stopped is taken as 100.

[Table] As is clear from Table 5, it was found that the color stability was not impaired even when a reducing substance, especially oxalic acid or its salt, was added to the enzyme reaction system. Measurement example 3 Quantification of hydrogen peroxide 0.3% o-phenylenediamine dihydrochloride 1%,
Add 50μ of 20U/ml peroxidase aqueous solution to 500μ of phosphate buffer (PH: 5.0) containing PEG ⁺ 4000 and 30mM sodium oxalate, and add 25μ of hydrogen peroxide solution (concentration: 0, 10, 20mg/ml).
After the enzymatic reaction was completed at 37℃ for 30 minutes, the reaction was stopped with 2 ml of 1N sulfuric acid, and the absorbance at 492 nm was measured.
A calibration curve was created. Note that the above buffer solution containing no sodium oxalate was used as a control for measurement. The results are shown in Figure 3. As is clear from this figure, non-specific reactions are suppressed and sensitivity is also improved.

[Brief explanation of the drawing]

Figure 1 shows a lyophilized color former (lyophilized product consisting of o-phenylenediamine dihydrochloride: 10 mg/ml, ammonium oxalate: 70 mM, PEG ⁺ 4000: 10 mg/ml) containing 0.02% hydrogen peroxide. This is a calibration curve for insulin obtained by dissolving in 20 ml of citric acid-phosphate buffer (PH: 5.7), preparing a coloring solution, and performing an enzyme reaction at room temperature in the light for 1 hour. As a control, a calibration curve for insulin obtained with a coloring solution prepared from a freeze-dried coloring agent that does not contain ammonium oxalate is shown. In Figure 2, the enzyme reaction was carried out overnight at 4°C using the same coloring solution as in Figure 1. Furthermore, Figure 3 shows 30mM as a coloring agent.
of sodium oxalate and 1% PEG ⁺ 4000. This is a calibration curve for hydrogen peroxide determination using o-phenylenediamine dihydrochloride and peroxidase as the enzyme. Note that the measurement was performed using a coloring agent that did not contain sodium oxalate as a control. In Figures 1 to 3, ○†g

Claims (1)

[Claims] 1. A stable coloring agent composition for a peroxidant or a peroxidase activity measuring reagent, which comprises phenylenediamine or a derivative thereof, oxalic acid or a salt thereof, and a water-soluble polymer compound.