JPH0313880B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a reagent for measuring acid phosphatase activity. Measurement of acid phosphatase activity in body fluids has great clinical significance as it provides useful information for the diagnosis and follow-up of prostate cancer. (Prior art) Conventionally, acid phosphatase activity was determined by using a substrate in which p-nitrophenol was bound to the hydroxyl group of phosphoric acid, allowing acid phosphatase to act, and then colorimetrically measuring the liberated p-nitrophenol under alkaline conditions. , enzyme immunoassay (hereinafter abbreviated as EIA), radioimmunoassay (hereinafter abbreviated as RIA)
The method was used. However, in the method using p-nitrophenol, the optimum pH of the target enzyme acid phosphatase is
(pH around 5.0) and the coloring pH of p-nitrophenol (pH 9 or higher), which is a coloring group, are different. Therefore, in order to measure acid phosphatase activity, it is necessary to perform the enzymatic reaction and coloring separately. Therefore, it requires a large number of reagents and many operational steps, and has the disadvantage that rate assay method, which is said to be the most suitable method for determining enzyme activity, cannot be used. The EIA and RIA methods have drawbacks such as long measurement times and complicated operations. (Problems to be Solved by the Invention) An object of the present invention is to provide a reagent for measuring acid phosphatase activity that enables rate assay of acid phosphatase with excellent quantitative properties. (Means for Solving the Problems) In order to achieve the above object, the present inventors have conducted various intensive studies and found that by using a substrate represented by the general formula [], acid phosphatase activity in body fluids can be suppressed for a short time. We have discovered that it is possible to accurately and easily perform rate assays and have arrived at the present invention. That is, the present invention is a reagent for measuring acid phosphatase activity, which is characterized by containing a compound represented by the following general formula [] as a substrate. (In the formula, X represents a halogen atom, and Y represents a hydrogen atom or a halogen atom.) The substrate used in the present invention is a compound represented by the general formula [ ], that is, one hydroxyl group of phosphoric acid has at least 2 halogen atoms are substituted at the 2,6-position, and one nitro group is bonded to a phenyl group substituted at the 4-position. As a phenyl group in which at least two halogen atoms are substituted at the 2- and 6-1 positions and one nitro group is substituted at the 4-position, the cleaved aglycone exhibits an absorption spectrum different from that of the substrate, Moreover, it produces a compound that has a low pka and is sufficiently colored and dissociated in the measurement range of acid phosphatase, around pH 5.0. Specifically, the cleaved aglycone has the following general formula: It is a phenol derivative represented by (X and Y are the same as above). For example, 2,6-dichloro-4-nitrophenol, 2,6-dipromo-4-nitrophenol, 2,6-diiodo-4-nitrophenol,
Examples thereof include 2,3,6-trichloro-4-nitrophenol, 2,3,6-tribromo-4-nitrophenol, and 2,3,6-triiodo-4-nitrophenol. The method for synthesizing these substrates is, for example, by reacting halogen-substituted p-nitrophenol with phosphoryl chloride to form a phosphoric acid ester (Chemical Abstract 47 8032b), and then dehalogenating it (Bull.Chem.Soc.Japan 44 2743). , and then recrystallized as Na salt (J.Biol.Chem. 167
57), there is a method to obtain the target substrate. The pH of the reagent of the present invention is the optimum pH of acid phosphatase in body fluids. Any buffer solution may be used as long as it maintains a pH of 4.0 to 5.5. Examples include citric acid buffers and other organic acid buffers, such as acetic acid, succinic acid, and phthalic acid buffers. Although there are no particular limitations on the substrate concentration, it is preferably a concentration that exhibits the maximum enzyme activity of acid phosphatase. For example, it is 1 mM or more. If necessary, a surfactant, preservative, sodium chloride, cyclodextrin, stabilizer, etc. may be added to the reagent of the present invention. A method for measuring acid phosphatase activity using the reagent for measuring acid phosphatase activity of the present invention is to directly colorimetrically quantify the change in absorbance of the aglycone produced by reacting the reagent with the reagent using a spectrophotometer. be. (Effect of the invention) In the acid phosphatase activity measurement reagent of the present invention, by using the compound represented by the general formula [ ] as a substrate, acid phosphatase activity in body fluids can be accurately and easily rate assayed in a short time. . In particular, the enzymatic reaction and color reaction are reduced by 1% compared to substrates bound with nitrophenol.
It has the excellent effect of being able to be performed in one system. Furthermore, compared to a substrate bound to a monohalogenated nitrophenol such as 2-chloro-4-nitrophenol, it fully develops and dissociates in the measurement range of acid phosphatase, around pH 5.0, and has better quantitative performance. (Example) Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 and Comparative Example 1 The amount of acid phosphatase activity in the test liquid was measured using the following reagent and the following method. 1 Reagent A: 2-chloro-4-nitrophenyl phosphate 1.0mM citric acid buffer 0.1M (pH 4.5) Reagent B: 2,3,6-trichloro-4-nitrophenyl phosphate 1.0mM citric acid Buffer 0.1M (pH4.5) Reagent C: 2,6-dichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) 2 Measurement method Add the above to 50μ of the acid phosphatase-containing test solution 2 ml of reagent A was added and reacted at 37°C, and the absorbance was measured at 400 nm to determine the color development rate.
The reaction curve is shown in FIG. 1, and the calibration curve is shown in FIG. The color development rate was similarly determined using Reagent B or C instead of Reagent A. As is clear from Figures 1 and 2,
The reagents of the present invention using 2,3,6-trichloro-4-nitrophenyl phosphate (Reagent B) and 2,6-dichloro-4-nitrilophenyl phosphate (Reagent C) can be used accurately in a short time. and rate assay, the sensitivity of which is 2-chloro-4
- much higher than with nitrophenyl phosphate (reagent A). Example 2 and Comparative Example 2 The amount of acid phosphatase activity in the test liquid was measured using the following reagent and the following method. 1 Reagent A: 2,3,6-trichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH 4.5) Reagent B: 2,6-dichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) Reagent C: 2-chloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) Reagent D: 4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) 2 Measurement method a Test solution containing acid phosphatase (concentration
After adding 2 ml of the above reagents A, B, C, or D to 50μ (150 U/l) and heating at 37°C for 3 minutes, the change in absorbance was measured at a wavelength of 400 nm to determine the change in absorbance over 1 minute (0 D/min). . (For the blank, water was used instead of the acid phosphatase-containing test solution.) Table 1 shows the results.

[Table] b Test solution containing acid phosphatase (concentration
Add 2 ml of the above reagents A, B, C, or D to 50μ of 150U/l) and react at 37°C for 5 minutes, then add 0.1M sodium carbonate water to stop the reaction under alkaline conditions, and adjust the absorbance at a wavelength of 400nm. Measured (0D). (For the blank, use water instead of the acid phosphatase-containing test solution.) Second
The results are shown in the table.

[Table] As is clear from Table 1, reagents A and B of the present invention have sufficient sensitivity for measurement at PH4.5;
Sensitivity is low compared to . As is clear from Tables 1 and 2, Reagent D is not at a measurable level at all at PH4.5 and is only measurable under alkaline conditions. Reference Example 1 For various nitrophenols, the molar absorbance at PH5.0, a pH range suitable for measuring pKa and acid phosphatase activity, was measured. The results are shown in Table 3.

[Table] As is clear from the above results, the nitrophenol dissociated from the halonitrophenyl phosphate used in the present invention has a low pKa and a molar extinction coefficient near pH 5.0, which is suitable for measuring acid phosphatase activity. expensive. Therefore, the substrate used in the present invention fully develops and dissociates in the measurement range of acid phosphatase, around pH 5.0, and has excellent measurement sensitivity.

[Brief explanation of the drawing]

FIG. 1 shows the reaction curves of Example 1 of the present invention and Comparative Example 1. Figure 2 shows Example 1 of the present invention and Comparative Example 1.
The calibration curve is shown.

Claims (1)

[Scope of Claims] 1. A reagent for measuring acidic phosphatase activity, which contains a compound represented by the following general formula [] as a substrate. (In the formula, X represents a halogen atom, and Y represents a hydrogen atom or a halogen atom.)