JPH07501202A - Colorimetric determination of peroxidase and peroxide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] L4 to colorimetric determination of peroxidase and peroxide The present invention relates to the colorimetric determination of peroxidase and peroxide. peroxidase, peroxide, oxidizes directly or indirectly to produce stoichiometric amounts of peroxidation Compounds that produce hydrogen, as well as theories that directly or indirectly oxidize compounds A colorimetric determination of the amount of hydrogen peroxide produced by the catalyst was carried out.

Kobo! lL [ Polymorphonuclear leukocytes and macrophages are stimulated by some membrane stimulation. Generation of hydrogen peroxide (H2O1) when applied to muli).

as well as horseradish, which produces a reaction product that changes the color of the system from red to yellow. The reaction between I'I202 and phenol red in the presence of peroxidase has already been reported. Known [Pick et al., Journal of Immunolo 1c al Methods, Vol. 38.16 to 170 pages (1980)], previous The yellow of the above system becomes a purple mauve color (light bluish) when the pi (pi) is raised to 12.5. The absorbance of the purple mauve color system was measured at 610 nanometers. When compared with the absorbance of a buffered phenol red solution, H20□ is quantified.

Additionally, lipid peroxides (neutr) produced by neutrophils.

phil-generated 1ipid peroxides) to phenol A method for testing by oxidation of lured has also been described in the literature [Mo5len et al. Journs of Immunolo 1cal Methods, Vol. 98, pp. 71-78 (1987)], seeding in an extremely alkaline medium. Methods for measuring the degree of oxidation of molten fats and oils using various oxidizable dyes are also known [Deutsch et al. German Patent No. 2,630,052 and German Patent No. 2,543,543].

However, these methods require minimal oxidation of the dye used to obtain the desired color change. During or after oxidation, treatment with a strongly alkaline solution with a pH of 10 or higher is required.

”Suck to 11 The present invention provides peroxidase, hydrogen peroxide (LOi) and hydrogen peroxide. the presence of substances capable of producing or catalyzing the production of hydrogen peroxide; Provides a method for determining the presence or amount of In the method of the present invention, preferably about 5° Large absorbance peak in the visible range at wavelengths from nanometers to approximately 700 nanometers Surpone phthalein dye or phthalein dye with no strong alkali treatment oxidizes and disappears In addition to the sulfone phthalein dye or phthalein dye described above, this method uses Performed by forming a reaction system containing any one of the following test samples (a) An unknown amount of peroxidase and the disappearance of the absorbance peak of the dye mentioned above. A test sample containing a sufficient amount of H2O2 to generate H2O2, monitor the disappearance of the absorbance peak. Peroxidase can be quantified by monitoring.

(b) An unknown amount of a compound that can react with oxygen to produce H20□ and oxidase, and if necessary, causes the H20□ production reaction of the above compound. A test sample that also contains an appropriate amount of an enzyme or another catalyst, in which case the H2O2 produced is The absorbance peak of the dye disappears, and by monitoring the disappearance, it is determined that the compound is Quantitated.

(c) A test sample containing an unknown amount of peroxide and an appropriate amount of peroxidase, in this case The peroxidase causes the absorbance peak of the aforementioned dye to disappear, and the disappearance is monitored. Peroxide is quantified by the ring.

(d) an unknown amount of catalyst that reacts with the compound in the presence of oxygen to produce H2O, and a suitable A test sample containing an amount of peroxidase.

In this case, the amount of H2O2 produced disappears the absorbance peak of the aforementioned dye, Catalyst is quantified by monitoring the rate of disappearance.

Once the disappearance of the absorbance peak of the aforementioned dye has been monitored and its value determined, the Compare the extiBuishment value with a pre-prepared standard to determine the unknown quantity. The disappearance of the absorbance peak can be monitored instrumentally, Or it can be monitored visually by observing changes in color tone.

The method of the invention is particularly suitable for use in immunoassay methods or systems, such as for example with antibodies or immunoassay methods or systems. Periods that may be bound to the analyte (analyte of interest) When determining the presence or amount of oxidase, or e.g. The amount or production rate of the analyte or enzyme present in the test sample Peroxidation by dependent enzymatic oxidation (eozytaLic oxidation) Useful when determining the analyte or enzyme or other catalyst involved in the production of hydrogen. The dye used in the present invention may be present in solution in free form or may be It may be bound, absorbed or fixed to a wettable solid matrix by methods known to those skilled in the art.

＝'　Q　ll Figures 1 to 3 show liquid phase spectrophotometric measurements of peroxide-forming analytes in the present invention. Sei (1quid phase speedrophotometric a ssay) is a graph showing changes in absorbance of a dye.

Figures 4 and 5 are graphs showing the general principle of color change of the dyes used in the present invention. It is f.

FIG. 6 shows one of the non-ionized sulfonophthalein dyes as contemplated in the present invention. FIG. 2 is an explanatory diagram showing a general structure.

Figure 7 is an illustration of ionized sulfonephthalein as considered in the present invention. FIG. 2 is an explanatory diagram showing a general structure.

Figure 8 shows the general structure of a non-ionized phthalein dye as considered in the present invention. FIG.

Figures 9 to 13 show the peroxidase time course liquid phase assay (Iiq) of the present invention. Reaction mixture used for uicl phase kinetic assays) It is a graph showing the absorbance of.

Figures 14 to 17 are graphs showing the effect of pH when carrying out the method of the present invention. be.

11 animals 1 smoke The term H2O2 as used in the text and claims of this specification means hydrogen peroxide. "r%" and "parts" are weight% and parts by weight, respectively, unless otherwise specified. g is gram, +eg is milligram, μg is microgram, ng is nanogram. Yes, em means centimeter, -plow means millimeter, n- means nanometer. where A represents absorbance, l is liter, μl is microliter, ml is milliliter. liter and '8 is mole percent, which sets the number of moles of the specified component of the composition. Equal to the total number of moles of components multiplied by 100, where 8 is the volume percentage. and M is −afar, which is the number of grams of solute in one liter of solution. Equivalently, -H is -i11imolar and milliliter of solute in one liter of solution. equal to the number of moles, μH is -icromolar, the number of moles of solution in one liter of solution. is equal to the number of micromoles of the specified component, and μ-01 is the number of micromoles of the specified component. psi is the pressure in pounds per square inch. It is something that All temperatures are in °C unless otherwise specified.

In the present invention, certain sulfonophthalein dyes and phthalein dyes are It is gradually oxidized by H,0□ in the presence of a suitable catalyst such as a sidase enzyme; It was found that a series of color changes occur as a result of oxidation. This color change The oxidation is preferably performed using waves in the visible range of about 500 nanometers to about 700 nanometers. Oxidative disappearance of absorbance peaks of sulfonephthalein dyes and phthalein dyes at long temperatures Non-limiting specific examples of sulfonphthalein dyes considered in the present invention and Bromcresol green, bromcresol purple, bromcresol Examples include blue, bromthymol blue, thymol blue, and phenol red. Non-limiting examples of phenol red dyes include phenophthalein. Can be mentioned.

For example, bromcresol green and bromcresol purple are H2O2 is sufficient to cause complete oxidation and when there is an excess of 1120□ When the 6M formation is incomplete, yellow oxidation dye and bromocresol dye are used. non-oxidized which is blue for lean and purple for bromcresol purple The resulting color tone is obtained as a result of blending with dyes. Therefore, different known amounts of Perform oxidation of mucresol green or bromcresol purple as described above. However, observing the color tone after oxidation indicates the amount produced by enzymatic oxidation of the unknown sample. The amount of dye sufficient to react with H2O2 can be selected, or a known amount of Identify unknown analytes in samples by visually measuring the degree of dye color change It is possible to estimate the amount of In addition, the degree of color change of a known amount of dye can be measured using spectroscopic light. It is also possible to measure the degree of As will be clear to those skilled in the art, free peroxidase or peroxidase bound to an antibody or analyte, for example, in a reaction system. , H2O2 or H7O2 source, and also gradually oxidized by 1202. It can be combined with dyes such as those mentioned above which produce a range of color changes. Peruo The oxidation of the aforementioned dyes by oxidases is catalytic, so the resulting oxidation The rate of color change depends on the concentration of peroxidase or the stoichiometric amount of peroxidase. is a direct function of the concentration of any substance bound at . In addition, the amount of analyte enzymatic reactions that produce stoichiometrically relevant amounts of H20□ or other peroxides. peroxide or analyte, such as glucose or cholesterol, In the reaction system, peroxidase and dyes as mentioned above can also be combined, e.g. For example, glucose, cholesterol, etc. can be combined with an enzyme that reacts with H,0□ to produce H,0□. To Lo2 The degree of enzymatic oxidation and color change of the aforementioned dyes depends on the presence of the dye in the aqueous system. It is a measure of the amount of analyte present.

In particular, the amount of free or bound peroxidase, or the unknown amount of catalyst, may cause color changes. determined by monitoring the rate of oxidation, preferably by oxidation of the dye. By monitoring the rate of change in absorbance at a given wavelength at which absorbance loss occurs. Determined by ■The amount of 20□ or the amount of H2O2 producing analyte is as described above. The rate of change may be determined by monitoring visually or using an instrument. Like One of the new visual methods is to determine the volume of the sample, dispense reagents, or any chemically inert solid component to separate or perform other functions. a reagent containing peroxidase, a dye of the present invention, and a peroxide to be measured. or a sample of the analyte compound to be measured, if necessary. A reaction system is formed that also includes an enzyme that causes the H2O2 production reaction of the above compound. next Then, observe the reaction system and check the color tone. I20 of various known amounts for the investigated color tones If you compare the color tone obtained using 2 with the color tone of the standard chart, the peroxide or semi-quantitative visual or instrumental determination of the amount of peroxide-forming analyte. I can do it. When measuring peroxidase over time, use an instrument to measure absorbance. It is preferred to measure peroxide, glucose, cholesterol, or peracid. Semi-quantitative visual measurements of similar substances capable of producing compounds may also be performed.

Many phthalein and sulfone phthalein dyes (Figure 6 shows non-ionized sulfone phthalein dyes) Figure 7 shows an ionized sulfonophthalein dye; Figure 8 shows a thalein dye; indicates a non-ionized phthalein dye) catalyzed by peroxidase sensitive to oxidation by H20□ such as . These dyes preferably have the following properties: (a> All of the phthalein and sulfone phthalein dyes also contain small-sized acids. Non-oxidatively bleach+ehabl peak in the 500-700 nm range (green), which is large enough to mask the ~purple), which can be eliminated by oxidation, as shown in Figure 5. The compound has an absorbance peak at about 560n that can be oxidatively eliminated, but at about 430n The m peak indicates resistance to oxidative bleaching. However, even before oxidation, 43 The magnitude of the 0nm (yellow-orange) peak substantially exceeds the 560n-peak. Therefore, the result of peroxidase-catalyzed oxidation can be tracked instrumentally at 560 nm. However, with some difficulty, there is a visually observable color change (from orange close to deep red). (change to orange) occurs.

(b) pH within the range where the dye is in an ionized state and peroxidase is active The ionization pKa of the phenolic hydroxyl group (pH 5 to 8) Therefore, the shift from the acidic pi color, which is usually yellow, to the alkaline color, which is green to purple, is less than 8. , preferably 7 or less.

When used as a 9H indicator, bromcresol green has a pl(3,8-5 ．． 4, it ionizes and changes color, and bromcresol purple changes color at pH 5.2 to 6.8. Bromthymol blue changes color at pH 6.0 to 7.6, and phenol red The color changes at pi (6.8 to 8.2), and the thymol blue changes color at pH 8.0 to 9.2. Phenolphthalein changes color at pH 8.5-10. Also, phthalein and sulfonephthalein dyes at the lower end of the color shift range (e.g. bromcresol). can be oxidatively bleached at any pH above pll5.2) The oxidation rate was found to be maximum at 9)1, near the lower end of the color shift range.

However, the magnitude of the oxidatively bleachable absorbance peak between 500 and 700 n- is quite small at pll near the lower limit of pH where color change occurs, and color shift Above the upper limit pH of the range, l) increases rapidly as H increases. Therefore the assay The sensitivity is at pH near the lower limit where color shift begins (phenol ionization). Maximum speed and pit near or above the upper pH limit at which color shift occurs. Absorbance peak intensity before oxidation and absorbance peak intensity after oxidation (500-700nm> The maximum difference between (s + axi + sum discrimination) Determined by balance. Therefore, the pH of the buffer used to perform the assay is (1) Maximize the reaction rate p)l (pH close to the lower limit of the color shift range, many for instrument applications or assays of analytes expected to be present at low concentrations. (desirable), or (2) the magnitude of the difference between the initial and final absorbance values; pH that maximizes the apparent degree of color shift (close to the upper limit of the color shift range) pH1 is expected to be present in many semi-quantitative visual assays or at relatively high concentrations. , therefore, for applications that require a large dynamic range for quantification. (desired in the case of assays for analytes).

For general applications, it is best to strike a balance between speed of color change and degree of magnitude. The pH is within the pH-induced oxidation bioionization range. within 1/3 of the upper limit of 0 For example, in the case of phenol red, d7.5 to 7.8, Bromuk pH 5.2 to 5.6 for resol green, pH 5.2 to 5.6 for resol green, and pH 5.2 to 5.6 for resol green. For convenience, the pH is 6.5, and for bromothymol blue it is pll 7.4. Most of the assays described herein are performed at pH 7,4 (50+*M sodium phosphate). pH 6,0 (50 mM potassium 2-(tomorpholino)ethane) sulfonate (HES)] in buffer solution). Therefore, many reactions Although not necessarily carried out under optimal pII conditions for sensitivity, the The examples described include peroxides, substances capable of producing peroxides, and peroxides. This is sufficient to provide a detailed description of the present invention for assays relating to sidase enzyme activity. It is.

As will be clear to those skilled in the art, oxidation of bromcresol green, etc. The apparent color tone is caused by the subtractive nature of the shift. Therefore, rather than a true wavelength shift, the absorbance peak at a certain wavelength disappears. , another peak is unmasked, so the present invention is suitable for 0% to 100% oxidation dye Simply add dyes to make the range of analyte concentrations similar to the range of expected analyte concentrations. Can be modified to measure different ranges of analyte concentrations by varying the concentration. Wear.

The invention is illustrated by the following non-limiting examples.

Example 1 Glucose assay Spectrophotometric assay for quantifying glucose over time, molecular oxygen, glucose It was carried out using oxidase and bromcresol purple. Glucose Oki Sidase oxidizes glucose in solution to produce gluconic acid and hydrogen peroxide. It was used as a catalyst for The hydrogen peroxide is oxidized in the presence of peroxidase. Oxidized romcresol purple. This oxidation produces a dye of 5+39 nm. Since the absorbance at 589 nm disappeared, hydrogen peroxide was detected by measuring the absorbance at 589 nm. was quantified.

First, the following solution was prepared: (a) 50TaH sodium phosphate [*0.067mg in solution (pH 7,4>) /ml solution containing bromcresol purple, (b) 50mM sodium phosphate IB/ml of horseradish peroxidizer in 30% buffer (pH 7,4) ze(^-ano　[nLernati.

nal Enzyme Company), and (c) 50 end H Glucose oxy at 10 mg/infusion 1 in sodium phosphate M solution (pt(7,4)) solution containing dase.

The reaction was carried out using 1.51 of the bromocresol purple solution and the glucose oxy 20 μm of the horseradish peroxidase solution and 15 μm of the horseradish peroxidase solution. and 0.51 of water or 0.51 of water containing various amounts of D-glucose, 2-1 In a colorimetric container (spectrophotoe + tric cuvette) A mixture is prepared by mixing at a warm temperature (approximately 22°C), which is stirred vigorously and a 589 nm Absorbance was followed as a function of time. The amount of D-glucose is 0゜0~3.6mg (0 to 20 μm01e), Figures 1, 2, and 3 are each 0.0 ．． For reaction mixtures containing 25 and 0.5 μole/ml glucose, 5 890- is a graph showing absorbance as a function of time. It is clear from Figure 1 As shown, the absorbance of 589n- does not change unless peroxide is added, and the absorbance remains unchanged for 5 minutes. It maintains a value slightly below 3.0. In Figure 2, the reaction mixture is 0.5μ5o It is found that the absorbance decreases when le glucose is included. this curve The slope of is estimated to be 0.27. Figure 3 shows that the reaction mixture contains 1.0 μmole This shows that the absorbance decreases more rapidly when lucose is included. This song The slope of the line is estimated to be 2.8.

The three curves shown in Figures 1, 2 and 3, as well as other curves herein, , optical path length 1.0 cm, Beckman DLI-70 spectrophotometer (Becksa n Instruments) using an incandescent light source at a scanning speed of 15 nm/s. It was recorded in

As a result of further investigation of the reaction system of Example 1, it was found that system (i) was a glucoate with a molecular weight of 0.4μ or less. If the absorbance contains glucose, the rate of absorbance change is too slow to be reliable (ii) Glucose that cannot be used for quantitative determination and whose system has 1.0 μ5 ole or more 202, the rate of change in absorbance decreases because a saturating amount of The above procedure of Example 1 was found to be independent of glucose concentration. Although not, it is possible to optimize the combination of ■20□, glucose, and oxidation catalytic enzyme ( 1120 by reacting with molecular oxygen in the presence of oxidative enzyme) ．． can be used to produce reliable analyzes of different analytes and It should also be understood that these reactions are illustrative of the reactions caused by peroxides. Unless catalysts that interfere with oxidation such as bromcresol purple are used, Confidence of another analyte undergoing enzymatic or non-enzymatic oxidation to produce +1202 It can also be used to perform reliable analysis.

For example, the procedure described above plots the absorbance at 5890- as a function of glucose concentration. Glucose in the reaction mixture to obtain curve data indicating the slope of the curve to be plotted. Repeat using it while changing the amount in the range of 0.4μmole to 1.0μmole. can be done. In fact, the differential plotting the rate of change in absorbance at 589n+m A photometer may be used. In addition, by reducing the concentration of bromcresol purple, Obtain data when the glucose in the reaction mixture is 0.4 μmole or less, or Increase the concentration and obtain data when glucose is 1.0μ5ole or more can also be used to analyze peroxides or other analytes using similar methods. You can also get the data you need.

As will be discussed in more detail later, the , Bromcresol Green, Bromcresol Blue, Bromcresol Par Puru and other aforementioned dyes have large tonal changes in the light absorption spectrum of the dye. A common cause is that it results from the oxidative loss of absorbance by the dye at the wavelength peak of Several operations were performed to prove the theory.

In particular, the following solutions were prepared: (a) 50J sodium phosphate [0.04mg/ml in clear liquid (pH 6,8) A solution containing bromocresol green, and (b) 50J sodium phosphate 10B/ml horseradish peroxidizer in liquid (pl! 6.8) solution containing enzyme.

1.5 ml of the bromocresol green solution and 10 μm of 001M 202 After preparing the reaction mixture and recording the spectrophotometric curve, the horseradish 5 μm of Tsch peroxidase solution was added. The spectrophotometric curve of the reaction mixture Two more were recorded. The first curve was recorded 2 minutes after peroxidase addition; The second one was recorded 4 minutes later.

Figure 4 is a graph showing three curves recorded as described above from 325nm to 700nm. Curve A is the pre-oxidation curve recorded before the addition of peroxidase; IiB is the curve recorded 2 minutes after peroxidase addition, curve C is the peroxidase The curve was recorded 4 minutes after the addition of the enzyme. As is clear from Figure 4, Bromke Oxidation of resol green gradually eliminates the absorbance of 6121 (blue), but about This method does not affect the size or position of the absorbance peak at 400 nm (yellow). Under such oxidation conditions, the solution changes from blue through blue-green, green, and yellow-green to the final state. It is visually obvious that the color changes to a deep golden color. A single reaction can be performed using an instrument. A gradually disappearing peak is seen at 612n-, which can be traced in wavelength.

Bromcresol Purple and Bromthymol Blue and other fluorophores as mentioned above. Thalein dyes and sulfonephthalein dyes, when oxidized, produce bromocresol. A pattern qualitatively similar to that followed by lean, with maximum and minimum absorption. traces a pattern that differs only in the specific wavelength at which it occurs, the rate of change, and the magnitude of absorption. For example, the large absorbance peak of bromcresol purple is at 5890- The small absorbance peak is at 400 nm. 1.0μe+ole glucose A curve similar to that of FIG. 4 for the system described in Example 1 including It is clear that the situation would have been similar to that of The absorbance of the luminous intensity peak (5B9nm) was 3.5 nm before the addition of peroxidase. OO Slightly less than the unit, after 1 minute it is about 1,95.2 minutes after about 0.6.3 minutes after about It is also clear that after 0.3.5 minutes it will be about 0.16.

The curve shown when phenol red is oxidized was also examined. For that purpose, first, (a ) 50mM sodium phosphate buffer (0.067mg/m in pi(7,4) a solution containing phenol red and (b) 50 + 6M sodium phosphate ill. Contains 1-g/ml of horseradish peroxidase in solution (pH 7.4) A solution was prepared.

Next, 1.5+al of the phenol red solution was added to 50 μm of 0.1141'+ After preparing the reaction mixture and recording the spectrophotometric curve, the hose 15 μm of radish peroxidase solution was added. Peroxidase added After 15 minutes, the spectrophotometric curve of the reaction mixture was recorded once again.

Figure 5 shows the two curves (400-700n brass) recorded as described above. 6 Curve A is the pre-oxidation curve recorded before the addition of peroxidase, and curve B is the pre-oxidation curve recorded before the addition of peroxidase. Curve recorded 15 minutes after peroxidase addition. It is clear from Figure 5. Sea urchin, phenol red has a similar trajectory to that of bromcresol green. However, the "yellow" (actually orange) peak near 430n- before oxidation is 560n Quantitatively, it far exceeds the "red" peak that was lowered by oxidation near m. They follow different trajectories.

As shown in Figure 4, bromcresol green has two large absorption bands. . One at about 6251- and the other at about 405 nm. Also, Blomk Resol purple has one large absorption band at about 5890-, about 400ns+ has another large absorption band. The absorbance of 625n- and 589n- is different from other large absorbances. These dyes are peroxidants whose absorbance is much greater than their absorbance at wavelengths in the optical band. The absorbance of 625n- and 5890- when oxidized by H2O2 in the presence of disappears, resulting in a significant change in color tone and absorbance. Bromthymol blue Similarly, the absorption maximum was at 618 nm (disappeared due to oxidation) and 405 nm (disappeared due to oxidation). It acts as shown in (without).

Example 2 ゛　、の　Asse Ethanol on a P paper disk (Hhatman No. 1) with a diameter of 6.5+ue 5μm of a solution containing 7.5B/ml bromcresol green = cloudiness ÷ Impregnated and dried. Each dry disc was then coated with 60IIIM sodium phosphate. Mu&! 0.08% horseradish peroxidase in buffer solution (pH 7,4) was impregnated with 5 μm of an aqueous solution containing . Finally, add 0.1N to each disk. It was impregnated with 5 μm of sodium nanate aqueous solution and dried. Mark the dry disc. placed in the wells of a standard 96-well matrix microtiter plate; 0 color development by dropping 5μm of water or hydrogen peroxide aqueous solution up to 20mM onto each disk When f& has reached equilibrium (after about 20 minutes at room temperature of about 22 °C), dry the disk at room temperature. and observed the color. These discs contain dark royal blue (0mM peroxide). from blue-green, green and yellow-green (1-5mM peroxide) to yellow (5raHA Fa compound) Continuation up to a yellow color close to orange (10-2011M peroxide) A color change was visually observed. 50mM sodium phosphate buffer (pH 7) , 4> of a solution containing 7.5 mg/+sl of bromcresol green: Pipette into the wells of a microtiter plate and then add 5 μm 1111 1/all horseradish peroxidase and 5μm water or peroxide water Similar results were obtained when an aqueous solution was added, but in such an aqueous system, The yellow color tone, which is close to orange when the oxide concentration is high, could not be observed very well. disk The various colors produced above are proportional to the added peroxide (semi-quantitatively) and remain stable indefinitely. It appeared to be stable and did not continue to oxidize visibly at room temperature.

A qualitatively similar non-optimized assay was performed for cholesterol/cholesterolemia in plasma. Conducted on stealth. In this assay, cholesterol/cholesterol Ester-containing plasma is treated with cholesteryl ester esterase and cholesterol ointment. Oxidase (oxidizes cholesterol in the presence of ambient oxygen to form cholestamen) Applied to a porous polystyrene cylinder impregnated with hydrogen oxide (an enzyme that produces hydrogen oxide) and placed on similarly treated paper disks instead of water or hydrogen peroxide solution. . The degree of yellowness of the disc is directly proportional to the amount of cholesterol applied. It seemed to me.

Peroxidically oxidizable pigment dyes are A state in which the liquid phase analytes and reagents described herein are capable of reacting with each other. There are many methods known to those skilled in the art for affixing to a solid phase matrix such that the well known. Additionally, various potential analytes can be removed by chemical or enzymatic oxidation. Many methods of deriving peroxide from nitrates are also known to those skilled in the art, and are described herein. techniques can be used to determine the amount of peroxide produced by such methods. Wear.

Example 3 Bel Cise's Tusei Peroxidase time course liquid phase assay by first preparing the solution below. carried out; (a) 60 μM sodium phosphate buffer (0.0671 in pi(7,4) 111/I11 solution containing bromocresol purple, (b) 50 theory Hli 0 to IB/ml of horseradish spec in sodium chloride buffer (pH 7,4) (c) A solution containing 1005M H2O2 in water.

2-1 In a spectrophotometer container at room temperature (approximately 22°C), the bromocresol purple 1.5ml of solution and the above! ■, 0□ solution 50μm and 0-1mg/ml hose Mix with 10 μm of solution (b) containing raditsch peroxidase and mix the reaction. Mix it vigorously and monitor the absorbance of 5891- as a number over time. The reaction operation was carried out while controlling the temperature.

Figures 9, 10, 11, 12 and 13 are horseradish specs. oxidase at 1.0.0.5.0.2.0.1 and 0.05mg/respectively. Data showing the absorbance of 5890 mm as a function of time for reaction mixtures containing ml , and includes a tangent line representing the slope of the straight line portion of the curve. to each curve The calculated slopes are shown in Table I below.

Table 1 Conditions and concentrations of peroxidase tested (0-100 per 1°51 reaction volume) μg of horseradish peroxidase), the maximum rate of reaction is It is directly proportional to the amount of sidase enzyme added.

peroxide and chemical analytes that can oxidatively form peroxide (e.g. glucose). (e.g., cholesterol, xanthine, uric acid) over time or Catalyzing the oxidation of the substance with consumption of peroxide, which can be performed as an endpoint assay The present invention can be practiced as an assay for substances (i.e., peroxidase and its like) that If the dye concentration is sufficient to saturate the enzymatic reaction, the color change reaction will occur. The rate of reaction (disappearance of one of the absorbance peaks) depends on the peroxidant present. or similar active substances. Publicly available city Is the commercially available “peroxidase” enzyme derived from the horseradish root? However, there are also many other substances present in plant and animal tissues and microorganisms. Many natural sources are known, such as potatoes, white blood cells and various bacteria; The enzyme appears to be widely present in many other organisms. Also, many proteins in the pond Proteins and non-protein substances, such as heme, exhibit peroxidase activity, but usually Compared to general peroxidase enzymes, it is slower and has less substance specificity.

Use phenol red instead of bromcresol purple to change absorbance. Not 589nm (I repeated the above method while monitoring at 560n-) However, qualitatively similar results were obtained. Below is the data regarding phenol red. It is shown in Table 2.

Example 4 L±he Mausoleum Demonstrate the effect of DI on the rate of peroxide bleaching of sulfonephthalein dyes. First, the following solution was prepared.

(a) 0.067 in 50mM sodium citrate MrII solution (pH 5,6) mgl-1 bromcresol green, (a’) 10mg/m in 50mM sodium citrate buffer (pH 5,6) 1 of horseradish peroxidase, (b) 50-H sodium phosphate Bromocresol green of 0.067+eg/ugly 1 in buffer solution (pH 6,0), (b’’) 10-g/in 50ml 4 sodium phosphate buffer (pH 6,0) So1 horseradish peroxidase, (c) 50s+H phosphate sodium 0.067+sg/1 bromcresol glycol in lithium buffer (pH 6,5) Oh, (c’) 50mM sodium phosphate buffer (10e+g in pi(6,5) /+*1 horseradish peroxidase, (d) 50 tons of H phosphate sodium Bromocresol glycol at 0.067 mol/kg in thorium buffer (pH 7.4) Oh, (d’) 50a+M sodium phosphate [10-g/in soaking solution (pH 7,4) -1 horseradish peroxidase.

At room temperature (approximately 22 °C) in a spectrophotometer vessel of 15-1, Lean solution [(a), (b), (c) and (d) and tolJ+ 0.1M] H2O2 and each horseradish peroxidase solution [(a') of 5I+1 , (b'), (C') and (d')]. Four reactions were performed. These mixtures were mixed at high speed and the absorbance at 612 nm was monitored as a function of time. Figures 14, 15, 16 and 17 shows the absorbance at 612 nm as a function of time for the four reaction mixtures. It is a data graph.

Figures 14 to 17 show the higher wavelength absorbance peaks of sulfonephthalein dyes. shows a significant effect of pH on the initial absorbance value and rate of oxidative disappearance.

If the dye concentration is the same (0,067 mg/ml), the initial absorption at 612 nm is The luminous intensity is 1.2 at pH 5,6, 2.6 at 6.0, pI (6,5 and pH 7). , 4 is 2.8.

p) The 9H-dependent color change range of bromcresol green as I indicator dye is r3. 8 (yellow) to 5.4 (blue), but it appears greenish-blue even at pH 5 and 6, and above pH 6. Finally, it becomes royal blue.

On the other hand, the maximum rate of oxidation was the highest for 9H5,6 (6-70D/min), i [flH, tpH6, OF2.100/min, pH6.5TO, 4500/min, p H7.

4 was 0.2500/min. This result shows that the initial absorbance at higher pH and the maximum Clarify the balance between the maximum difference between the final absorbance and the greater rate at lower pH. Shown in white. As is clear from the foregoing data, the present invention can be used in various settings. It is possible to manipulate pi in order to optimize the conditions when programming and applying it for various purposes. It is possible.

It should be noted that the present invention can be modified in various ways without departing from its spirit and scope. limited only by 'Range'.

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Claims (26)

[Claims] 1. peroxidase, hydrogen peroxide, or hydrogen peroxide in the test sample. or the presence of a substance capable of catalyzing the formation of hydrogen peroxide. is a method of determining the amount, (a) (i) A sulfonate with an oxidatively quenchable absorbance peak in the visible region. thalein dye or phthalein dye; (ii) unknown amount of peroxidase, peracid; Hydrogen oxide, or hydrogen peroxide, or the production of hydrogen peroxide A reaction system containing a test sample suspected of containing a substance capable of catalyzing form, (b) comparing the disappearance of the absorbance peak with the number of unknown amounts present in the test sample; and monitoring the method. 2. The reaction system includes (i) an unknown amount of peroxidase, (ii) the dye, (iii) hydrogen peroxide in an amount sufficient to eliminate the absorbance peak of the dye; the test sample by monitoring the disappearance of the absorbance peak. 2. The method of claim 1, wherein the unknown amount of peroxidase present in the method is determined. 3. The reaction system includes (i) an unionized compound capable of reacting with oxygen to produce hydrogen peroxide; a test sample containing a known amount of the compound; (ii) said dye; and (iii) a peroxidant. The hydrogen peroxide produced disappears the absorbance peak of the dye. and detect unknown compounds present in the test sample by monitoring their disappearance. 2. The method of claim 1 for determining the amount. 4. The reaction system contains a sufficient amount of hydrogen peroxide production reaction of the compound. 4. The method of claim 3, comprising a catalyst. 5. The reaction system includes (i) a test sample containing an unknown amount of peroxide, and (ii) the dye. and (iii) peroxidase, wherein the peroxide acts as a dye. in the test sample by causing the absorbance peak to disappear and monitoring the disappearance. 2. The method of claim 1, wherein an unknown amount of peroxide is determined. 6. The reaction system (i) reacts with a compound in the presence of oxygen to produce hydrogen peroxide; (ii) said dye; and (iii) peroxidase. The amount of hydrogen peroxide produced extinguishes the absorbance heater of the dye and Determining the unknown amount of catalyst in the test sample by monitoring the loss A method according to claim 1, characterized in that: 7. 2. The method of claim 1, wherein the reaction system includes a sulfonephthalein dye. 8. 2. The method of claim 1, wherein the reaction system includes a phthalein dye. 9. The sulfone phthalein dye is used as bromocresol green or bromcresol dye. color purple, bromcresol blue, bromthymol blue, thymol blue 8. The method according to claim 7, wherein the method is selected from phenol red and phenol red. 10. 7. The sulfonephthalein dye is bromocresol green. The method described in. 11. 9. The method of claim 8, wherein the phthalein dye is phenolphthalein. . 12. The absorbance peak that can be eliminated by oxidation is between about 500 nm and about 700 nm. 2. The method according to claim 1. 13. The unknown amount is determined by the disappearance of the absorbance peak and the absorbance peak of the known standard sample. 2. The method according to claim 1, wherein the determination is made by comparison with the disappearance of . 14. The unknown amount is measured by monitoring the disappearance of the absorbance peak with an instrument. The method according to claim 1, wherein the method is determined by: 15. The unknown amount is determined by visually monitoring the disappearance of the absorbance peak. 2. The method according to claim 1, wherein: 16. Produces peroxidase, hydrogen peroxide, or hydrogen peroxide in the test sample the presence of substances capable of catalyzing the formation of hydrogen peroxide; or a reaction system for determining the amount of (i) that can be oxidatively lost in the visible region; a sulfonephthalein dye or phthalein dye having an absorbance peak of (ii) ) can produce unknown amounts of peroxidase, hydrogen peroxide, or hydrogen peroxide. or contain materials that can catalyze the production of hydrogen peroxide. contains a possible test sample, and the absorbance peak is present in the test sample. The reaction system disappears as a function of an unknown quantity. 17. (i) an unknown amount of a compound capable of reacting with oxygen to produce hydrogen peroxide; (ii) the dye and (iii) peroxidase. 17. The reaction system according to claim 16. 18. Contains a sufficient amount of catalyst to cause the compound to undergo a hydrogen peroxide production reaction The reaction system according to claim 17. 19. (i) a test sample containing an unknown amount of peroxide; (ii) the dye; (ii) 17. The reaction system according to claim 16, comprising: i) peroxidase. 20. (i) an unknown amount of catalyst that reacts with a compound in the presence of oxygen to produce hydrogen peroxide; a test sample containing a medium; (ii) the dye; and (iii) a peroxidase. 17. The reaction system according to claim 16, comprising: 21. 17. The reaction system according to claim 16, comprising a sulfonephthalein dye. 22. 17. The reaction system of claim 16, comprising a phthalein dye. 23. The sulfone phthalein dye is bromcresol green, bromcresol color purple, bromcresol blue, bromthymol blue, thymol blue 22. The reaction system according to claim 21, wherein the reaction system is selected from - and phenol red. 24. 23. The dye according to claim 22, wherein the phthalein dye is phenolphthalein. Compatible. 25. 2. The sulfonephthalein dye is bromocresol green. 3. The reaction system described in 3. 26. The oxidatively quenchable light-absorbing skin is in a range of about 500 nm to about 700 nm. Reaction system according to item 16.