JPS6296099A - Reagent for determination of acidic phosphatase activity - Google Patents

Abstract

PURPOSE:To provide the titled reagent containing a specific compound, capable of accurately, easily and quickly determining acidic phosphatase activity in body fluid and useful for clinical diagnosis, etc. CONSTITUTION:A halogen-substituted p-nitrophenol is made to react with phosphoryl chloride and the resultant phosphate is dehalogenated and converted to Na salt, etc., to obtain the compound of formula (X is halogen; n and m are 1-4). The objective reagent for the determination of acidic phosphatase activity can be produced by dissolving >=1mM of the above compound in a buffer solution of citric acid, etc., having a pH of 4.0-5.5 and, if necessary, adding a surfactant, an antiseptic, etc., to the solution.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) 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.

(Conventional technology) Conventionally, acid phosphatase activity was caused by p
- A method in which acid phosphatase is activated using a substrate bound to nitrophenol and p-nitrophenol released is colorimetrically measured under alkaline conditions, enzyme immunoassay (hereinafter abbreviated as EIA), and radioimmunoassay (hereinafter abbreviated as RIA). ) method was used.

However, in the method using p-nitrophenol, the optimum pH (pH 5,
0) and the coloring p of p-nitrophenol, which is a coloring group.
H (pH 9 or higher), it is necessary to perform the enzymatic reaction and coloring reaction separately in order to measure acid phosphatase activity.

For this reason, a large number of reagents and operational steps are required, and there is a drawback that the rate assay method, which is said to be the most appropriate method for determining enzyme activity, is not available.

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 7 osphatase activity, which enables rate assay of acid phosphatase with excellent quantitative properties.

(Means for Solving the Problems) In order to achieve the above-mentioned objective fc, the present inventors conducted various intensive studies and found that acid phosphatase activity in body fluids can be reduced in a short time by using a substrate represented by the general formula CD. We have discovered that rate assay can be performed accurately and easily, and have arrived at the present invention.

That is, one aspect of the present invention is a reagent for measuring acid phosphatase activity, which is characterized by containing a compound represented by the following general formula Ct) as a substrate.

(In the formula, X represents a halogen atom, and m is 1 to 4.
Indicates the number of ) The substrate used in the present invention is a compound represented by the general formula CD, in which one hydroxyl group of phosphoric acid is bonded to a halogen and a nitro group-substituted phenyl group. The cleaved aglycone of the halogen- and nitro-substituted phenyl group exhibits a different spectral absorption from that of the substrate. Specifically, the cleaved aglycone has the following general formula: For example, tlf, 2-chloro-4-ditrophenol, 2-
Promo-4-ditrophenol, 2-iodo-4-ditrophenol, 2,6-dichloro-4-ditrophenol, 2.6-dipromo-4-ditrophenol, 2.
6-diiodo-4-nitrophenol, 2,3.6-
1-dichloro-4-ditrophenol, 2.3.6
-) Lipcomo-4-nitrophenol, 2.3.6-
Driodo 4-nitropheno-/L/.

Examples include 2,4-dinitro-6-chlorophenol.

Methods for synthesizing these substrates include, for example, halogen-substituted (7)I
)-Chemical Abs by reacting ditrophenol with phosphoryl chloride to convert it into a phosphoric acid ester
tract 47 8032b), further dehalogenated Bull, Chem, Soc, Japan 44
2743) and then recrystallized as Na salt (J, Bi
ol, Chem, 167 57).

There is a method to obtain the target group W.

The pH of the reagent of the present invention may be any buffer solution that maintains the optimum pH of acid phosphatase in body fluids ($)H4.0 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 is no particular restriction on the degree of the group 'Ra, a degree showing the maximum enzyme activity of acid phosphatase is preferably suitable. 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.

Acid phosphatase activity of the present invention t! 11: A method for measuring acid phosphatase activity using a measurement reagent is to react the reagent with the reagent and measure the change in absorbance of the aglycon produced using a one-junction spectrophotometer. There is a method of colorimetric determination using

(Effect of the invention) In the acidic phosphamase activity measuring reagent of the present invention,
By using the compound represented by the general formula CI) as a substrate, acid phosphatase activity in 191 body fluids can be accurately and easily rate assayed in a short time. In particular, compared to substrates bound to nitrophenol, it has an excellent effect in that an enzymatic reaction and a coloring reaction can be carried out in one system.

(Example) The present invention will be explained in detail below using examples.

Example 1 Acid phosphatase activity in a test solution was measured using the following reagent and the following method.

1. Reagent 2-chloro-4-nitrophenyl phosphate 1.0771M citrate buffer 0.1M pH4,
5 2. Measurement method: Test solution containing acid phosphatase 50μ! Add the above reagent 2 to
zl k was added and reacted at 37°C, and the absorbance was measured at a wavelength of 400 nm to check the color development rate. The reaction curve is shown in FIG. 1, and the calibration curve is shown in FIG.

As is clear from FIGS. 1 and 2, the reagent of the present invention using a water-soluble substrate allows accurate and simple rate assays in a short period of time.

Example 2 The amount of acid phosphatase activity in the test solution was determined as fAIJ using the following reagent and the following method.

1. Reagent A, 2,3.6-dolichloro-4-nitrophenylrelic acid 1. (JmM citrate buffer U, I M pH4,5 B, 4-nitrophenyl phosphate 1.0mM citrate buffer 0.1 MpH4,5 2, measurement method a, acid phosphatase-containing test solution 50μ! and the above reagent A, 82 g? was added and heated at 37° C. for 3 minutes, and the absorbance change was measured at a wavelength of 400 nm to calculate the absorbance change tX for 1 minute (water was used instead of the acid phosphatase-containing test solution for the blank).

b. Add 82g of the above reagent A to 50μ of acid phosphatase-containing test solution. After reacting at 37°C for 5 minutes, 0.1
M sodium carbonate water was added, the reaction was stopped under alkaline conditions, and the absorbance at a wavelength of 400 nm was measured (water was used instead of the acid phosphatase-containing test solution for the blank).

Table 1 shows the results.

Reagent A of the present invention has sufficient measurable sensitivity at pH 4 and 5, but reagent Bit does not have a measurable level at all at pH 4 and 5, and can only be measured under alkaline conditions.

[Brief explanation of drawings]

FIG. 1 shows the reaction curve of Example 1 of the present invention. FIG. 2 shows the calibration curve of Example 1 of the present invention.

Claims (1)

[Scope of Claims] A reagent for measuring acid phosphatase activity, which contains a compound represented by the following general formula [I] as a substrate. ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, X represents a halogen atom, and n and m are 1 to 4
Indicates the number of )