JP2797102B2 - Acid phosphatase activity reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Application Field of the Invention] The present invention relates to an acid phosphatase (hereinafter abbreviated as ACP) in a biological sample such as blood or urine. It relates to a reagent for activity measurement.

[Background of the Invention] ACP is widely distributed in tissues such as the prostate, liver, spleen, granulocytes, erythrocytes, and bones. Since blood levels increase due to obstruction, it is used as a diagnostic index for prostate disease.

Conventional methods for measuring ACP activity can be classified into the glycerophosphate method, the phenylphosphate method,
-Nitrophenylphosphoric acid method, α-naphthylphosphoric acid method, thymolphthalein monophosphoric acid method, bromophenol blue monophosphoric acid method, 2,6-dichloro-4-nitrophenylphosphoric acid method and the like have been reported. Among them, methods that are relatively unaffected by the coexisting substance in the sample and can be measured by the late assay include the α-naphthyl phosphate method, the bromophenol blue monophosphate method, and the 2,6-dichloro-4-nitro acid method. The phenylphosphoric acid method is exemplified.
However, the α-naphthyl phosphoric acid method is a method in which α-naphthol decomposed and released by ACP is colored by a diazo coupling reaction using a diazo reagent, and thus is easily affected by a reducing substance which is an inhibitor of this reaction. On the other hand, in the bromophenol blue monophosphoric acid method and the 2,6-dichloro-4-nitrophenylphosphoric acid method, bromophenol blue or 2,6-dichloro-4-nitrophenol which is decomposed and released by ACP is directly used. Because of the measurement, the effect of substances coexisting in the sample is minimal, but the stability of the substrate itself is poor, and chemical decomposition is likely to occur not only in the test solution but also when stored in a solid state. The substrate needs to be synthesized or purified immediately before use,
There was a problem that it could only be used for a very short time after the preparation of the test solution.

On the other hand, instead of preventing the decomposition of the substrate, a method using 2-chloro-4-nitrophenyl phosphate, which is more stable than the conventional substrate, has been developed (Japanese Patent Publication No. Sho 58-6480, JP-A-62-96099, JP-A-62-115298). This 2-chloro-4-nitrophenyl phosphoric acid has better stability in solution than conventional ones, and there is no problem in that respect. However, in this method, 2-chloro-4- is released as a result of the reaction with ACP.
Since the pKa of nitrophenol is around 5.4 (meaning that it dissociates only 50% in a solution of pH 5.4), ACP
In the range of 5 to 6, which is the optimum pH, the detection sensitivity was low, and there was a problem that it could not be used for measurement by the late assay.

In order to solve this problem, inclusion reagents such as cyclodextrin (hereinafter abbreviated as CD) and crown ether coexist in a reagent solution for measuring ACP activity containing 2-chloro-4-nitrophenylphosphate as a substrate. A method of increasing the detection sensitivity by shifting the apparent pKa of 2-chloro-4-nitrophenol released as a result of the reaction with ACP to a more acidic side is disclosed in JP-A-58-501357 and JP-A-62-157357. −115298
No. 6,086,045. However, in the test solution for ACP activity measurement prepared by this method, the detection sensitivity of released 2-chloro-4-nitrophenol is increased, but on the other hand, the substrate 2-chloro-4-nitrophenol is released. A phenomenon occurs in which the stability of phenylphosphoric acid in the test solution is significantly reduced. Therefore, AC using this method
In the reagent solution for P measurement, the measurement reagent solution is divided into two, that is, a substrate solution and an inclusion compound solution. Unless the so-called two-component method is used, it is difficult to apply to actual measurement, and further improvement is desired. Was.

[Object of the Invention] The present invention has been made in view of the above situation,
An object of the present invention is to provide a reagent for measuring ACP activity which can be used in a late assay and has excellent stability.

[Constitution of the Invention] The present invention relates to a compound of the general formula [I] (In the formula, ^one of R ¹ and R ⁴ represents a halogen atom,
The other represents a hydrogen atom or a methyl group. R ² and R ³ are each independently a hydrogen atom, a lower alkyl group, a hydroxyalkyl group, a sulfoalkyl group, a carboxyalkyl group,
It represents a sulfonylalkyl group, a thioalkyl group, a lower alkoxy group, a sulfonic acid group, a carboxyl group or a halogen atom. ) Or a derivative thereof, or a salt thereof, and at least two of 21 hydroxyl groups.
An acid comprising a carboxyalkyl group having 2 to 4 carbon atoms, a sulfoalkyl group having 2 to 4 carbon atoms, a mono- or dihydroxyalkyl group having 2 to 4 carbon atoms, or a β-CD derivative having a methyl group This is a reagent solution for measuring phosphatase activity.

That is, the present inventors have intensively studied to develop a reagent for measuring ACP activity which can be used in a late assay and has excellent stability. As a result, HNP or a derivative thereof, or a salt thereof (hereinafter referred to as HNP, etc.) Abbreviated) is a specific C according to the present invention.
When coexisting with the D derivative, 2-halo-4-nitrophenol (hereinafter, referred to as HCP) generated from HNP or the like by the action of ACP
Abbreviated as HN. ) Or its derivative can reduce the apparent pKa so that it can be dissociated almost 100% within the optimal pH range of ACP, and the stability in a solution such as HNP is different from α or β-CD. Compared with the case of coexistence, the improvement was remarkable. When α or β-CD was used, the test solution for ACP activity measurement was inevitably used in the form of a two-liquid method. The inventors have found that they can be used, and have completed the present invention.

In the HNP of the present invention represented by the general formula [I] or the derivative thereof, the halogen atom represented by R ¹ or R ⁴ includes, for example, chlorine, bromine, iodine, fluorine and the like. Examples of the lower alkyl group represented by R ² and R ³ include a linear or branched alkyl group having 1 to 5 carbon atoms such as a methyl group, an ethyl group, a propyl group, a butyl group and an amyl group. A hydroxyalkyl group, a sulfoalkyl group,
Examples of the alkyl group of a carboxyalkyl group, a sulfonylalkyl group, and a thioalkyl group include, for example, a methyl group, an ethyl group, a propyl group, a butyl group, and an amyl group.
And a lower alkoxy group such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group, etc., having 1 to 4 carbon atoms. And examples of the halogen atom include chlorine, bromine, iodine and fluorine. Further, the sulfonic acid group, the sulfonic acid group of the sulfoalkyl group, the carboxyl group, and the carboxyl group of the carboxylalkyl group may be in the form of a salt such as an alkali metal salt such as sodium, potassium or lithium or an ammonium salt. Good. The salts of the HNP or its derivative represented by the general formula [I] according to the present invention include, for example, alkali metal salts such as sodium, potassium and lithium;
Ammonium salts, for example, salts of organic bases such as triethanolamine salt, triethylamine salt, cyclohexylamine salt and the like can be mentioned.

Among the various salts of the HNP or its derivative according to the present invention represented by the general formula [I], for example, monopotassium salt, monoammonium salt, and mono (ethyl 2-hydroxyethyl) of 2-chloro-4-nitrophenylphosphate Ammonium) salt, mono (1,1-dimethyl-2-hydroxyethylammonium) salt, mono [1,1-bis (hydroxyethylmethyl) -2-hydroxyethylammonium] salt,
Mono (2-hydroxyethylammonium) salts and the like are particularly preferable as the substrate used in the present invention because they have particularly excellent stability during storage and solubility in water as compared with others.

The HNP of the present invention represented by the general formula [I] or a derivative thereof can be prepared by a known method for synthesizing a phosphoric ester, for example, Experimental Chemistry Course 19, Synthesis of Organic Compounds I, Third Edition, pp. 206-210 [Maruzen Can be easily synthesized according to the method described in “Issued by K.K. That is, for example, HN or a derivative thereof is dissolved in an appropriate solvent such as benzene or toluene, and reacted by adding phosphorus oxychloride in the presence of a dehydrochlorinating agent such as pyridine, and then hydrolyzed by adding water. The desired HNP or its derivative can be obtained by extraction or precipitation from the aqueous layer. Further, by subjecting this to a salt formation reaction with a desired base or salt, the salt of HNP or a derivative thereof according to the present invention can be easily obtained.

As the concentration in the test solution for measuring ACP activity such as HNP according to the present invention, about 3 to 10 mM is preferably used.

Specific CD derivatives used in the present invention include:
Of the 21 hydroxyl groups of β-CD, at least two or more are carboxyalkyl groups having 2 to 4 carbon atoms, sulfoalkyl groups having 2 to 4 carbon atoms, mono- or dihydroxyalkyl groups having 2 to 4 carbon atoms, or methyl. Β-CD derivatives substituted with a group.

In the β-CD derivative according to the present invention, the remaining hydroxyl group substituted with a substituent as described above is generally unsubstituted, but part or all of the remaining hydroxyl group is replaced with another group. This does not hinder, even if the substituent is a group that does not impair the effects of the present invention.

Specific examples of the β-CD7 derivative according to the present invention include, for example, carboxymethyl-β-CD, carboxyethyl-β-
CD, carboxymethylethyl-β-CD, sulfopropyl-β-CD, hydroxyethyl-β-CD, 2-hydroxypropyl-β-CD, 3-hydroxypropyl-β-CD,
2,3-dihydroxypropyl-β-CD, methyl-β-CD
And the like, but of course is not limited to these.

These β-CD derivatives used in the present invention are described, for example, in US Pat. No. 3,453,258; US Pat.
3,259; U.S. Pat. No. 3,459,731 can be easily synthesized by a general production method, and thus, it is sufficient to use the one synthesized in such a manner.

These β-CD derivatives according to the present invention may be used alone or in combination of two or more, and the amount thereof is preferably about equimolar to about 5 times the molar amount of HNP used as a substrate. Used.

Stability is further improved when phenol, at least one compound selected from the group consisting of salicylic acid derivatives is further coexistent with the measurement solution of the present invention containing HNP or the like and a specific β-CD derivative, and the stability is further improved. It is desirable. Compounds having such a stabilizing action (hereinafter referred to as "
Abbreviated as stabilizer. ) Is not particularly limited as long as the interaction related to inclusion with the specific β-CD derivative according to the present invention is stronger than HNP or the like but weaker than HN or the like and has a certain degree of water solubility. But without
For example, phenol, for example, p-phenolsulfonic acid,
2,3-dichlorophenol, 2,4-dichlorophenol, 2,
5-dichlorophenol, 2,6-dichlorophenol, 3,4
-Dichlorophenol, 3,5-dichlorophenol, 4-chloro-2-methylphenol, 4-chloro-3-methylphenol, 2-chloro-4,5-dimethylphenol, 4-methoxyphenol, 2,4,6 Phenol derivatives such as -trichlorophenol, 4-chloro-3,5-dimethylphenol, 3,5-dimethylphenol, 4,4'-thiodiphenol,
For example, 5-bromosalicylic acid, 5-chlorosalicylic acid, 5,
Salicylic acid derivatives such as 5'-thiodisalicylic acid are preferred.

These stabilizers may be used alone or in combination of two or more. The amount of the stabilizer is not particularly limited as long as precipitation or the like does not occur in the ACP activity measurement reagent solution. The molar amount is at least 0.5 times the molar amount of HNP or the like used, and preferably about 1 to 5 times the molar amount.

In general, the stabilizing effect of these stabilizers is superior to those of weak hydrophilicity as compared with the substrate such as HNP. This suggests that the mechanism by which these compounds prevent spontaneous degradation of HNP and the like may be due to a competitive reaction in the inclusion reaction with the coexisting β-CD derivative. That is, it is considered that the decomposition rate of HNP and the like is accelerated by being included by the β-CD derivative, but when the above-mentioned stabilizer is further co-present in this solution, β -CD derivatives are more likely than HNP, etc.
It is presumed that an inclusion compound is formed preferentially with these stabilizers, and as a result, the formation of an inclusion compound between β-CD and HNP is hindered, so that the stability is improved.

The method of measuring ACP activity using the test solution of the present invention per se may be performed according to a conventional method of measuring ACP activity known per se except that the test solution of the present invention is used as the test solution for measurement. That is, for example, according to the method described in Enzyme, 20 , pp. 248-256 (1975), 2-halo-4-nitrophenol or a derivative thereof released by the enzymatic reaction of ACP is colorimetrically measured to determine the ACP activity. What is necessary is just to measure.

In the case of prostate cancer diagnosis, L (+) tartaric acid is AC
Utilizing the fact that it has an action of suppressing the prostate fraction in P, measurement may be performed using this.In such a case, the measurement reagent of the present invention naturally uses the existing AC.
Needless to say, it can be used in the same manner as the reagent for measuring P activity.

Buffers and other reagents used in the measurement method of the present invention may be used according to those used in the measurement method known per se, and measurement conditions such as pH and measurement temperature at the time of measurement. It suffices to conduct the measurement according to the method of ACP activity measurement known per se.

The reagent solution for measuring ACP activity of the present invention can be used very effectively for measuring ACP activity in a biological sample such as blood or urine, and can be applied to any one of a one-point measurement method, a late assay method, and the like. The assay method using the reagent solution for measuring ACP activity of the present invention can of course be used for the application method, but it is also applicable to an automatic analyzer, and particularly, the reagent solution for measuring ACP activity of the present invention and L When a buffer solution containing (+) tartaric acid is used in combination, total ACP activity and prostate-derived extracellular ACP activity can be continuously measured in one channel, which is very advantageous.
It is needless to say that the ACP activity can be measured similarly by dividing the components of the test solution for measuring ACP activity of the present invention into two liquids and measuring the ACP activity as a two-liquid method.

As a method for preparing the test solution for measuring ACP activity of the present invention, if dissolved in a predetermined amount with distilled water or purified water, it is stored as a lyophilized product prepared to have a desired composition, and dissolved if necessary. Or HNP as a substrate
And a solution containing other necessary components are prepared and stored, and according to a method for preparing a reagent for a clinical test known per se, such as a method of preparing the solution by appropriately mixing it at the time of use. You can do this.

Hereinafter, the present invention will be described in more detail with reference to Experimental Examples and Examples, but the present invention is not limited thereto.

[Example] Experimental example 1.2 Measurement of molecular extinction coefficient of 2-chloro-4-nitrophenol (buffer solution) 5.9 g of succinic acid was dissolved in 800 ml of distilled water, and adjusted to pH 6.0 with a 2N potassium hydroxide solution. After that, the total volume was made up to 1000 ml to prepare a buffer solution.

(Operating method) The above buffer was mixed with the CD derivative of the present invention at 8 mM and 2- mM.
Chlor-4-nitrophenol was added to a concentration of 0.5 mM as a sample, and 2-chloro-4-nitrophenol was obtained by a conventional method.
The molecular extinction coefficient of nitrophenol was determined.

(Results) The obtained results are shown in Table 1.

Experimental Example 2. Investigation of substrate stability (substrate buffer) The buffer used in Experimental Example 1 contains 4 mM monoammonium 2-chloro-4-nitrophenylphosphate and 8 mM CD derivative according to the present invention. As described above to obtain a substrate solution.

(Operation method) The absorbance E _{0 at} 410 nm immediately after the preparation of the substrate buffer and the absorbance E ₃ at 410 nm after storing it at room temperature for 3 days were measured.

In addition, a substrate buffer solution without CD derivative was prepared, and in the same manner as above, immediately after preparing the substrate buffer solution without CD derivative.
410nm Absorbance E _B10 and its absorbance was measured E _B13 of 410nm after storage for 3 days at room temperature.

These values were substituted into the following equation to determine the decomposition rate D.
In the formula, ε _CD is the molecular extinction coefficient of 2-chloro-4-nitrophenol obtained when the predetermined CD derivative obtained in Experimental Example 1 is used, and ε is 2-chloro-4-methyl chloride when no CD derivative is added. −
The molecular extinction coefficients of 4-nitrophenol are shown respectively.

A = (E ₃ −E ₀ ) ÷ ε _CD A ₀ = (E _B13 −E _B10 ) ÷ ε D (%) = A ÷ A ₀ × 100 (Results) The obtained results are also shown in Table 1.

Comparative Example 1. Measurement of molecular extinction coefficient of 2-chloro-4-nitrophenol using unsubstituted CD In Experimental Example 1, α was used instead of the CD derivative according to the present invention.
The molecular extinction coefficient of 2-chloro-4-nitrophenol was determined in the same manner as in Experimental Example 1 except that -CD, β-CD or γ-CD was used.

(Results) The obtained results are also shown in Table 1.

Comparative Example 2. Investigation of substrate stability when unsubstituted CD was used In the substrate solution of Experimental Example 2, α-CD, β-CD or γ-CD was used instead of the CD derivative according to the present invention. Decomposition rate D was determined in the same manner as in Experimental Example 2 except for the above.

(Results) The obtained results are also shown in Table 1.

In the following table, 3HP-β-CD is 3-hydroxypropyl-β-CD, M-β-CD is methyl-β-CD,
2,3DHP-β-CD is 2,3-dihydroxypropyl-β-CD
, 2HE-β-CD is 2-hydroxyethyl-β-CD, S
P-β-CD indicates sulfopropyl-β-CD, respectively.

The degree of substitution represents the number of substituents introduced out of the 21 hydroxyl groups present in β-CD.

As is clear from the results, when the CD derivative according to the present invention was used as an inclusion compound, the molecular extinction coefficient of 2-chloro-4-nitrophenol in a solution was α- or β-CD.
Shows almost the same value as in the case of using, and it can be seen that the stability of the substrate in solution is clearly improved as compared with α- and β-CD.

Experimental Example 3 Investigation of the Additive Effect of Phenol, Phenol Derivatives and Salicylic Acid Derivatives
The stabilizing effect of the test solution for ACP activity measurement when phenol, a phenol derivative or a salicylic acid derivative was further added as a stabilizer was examined.

(Reagent) A buffer having the following composition was used as a reagent.

Succinic acid 50 mM 2-Chloro-4-nitrophenyl monoammonium phosphate 4 mM HP-β-CD (Substitution degree: 5.0) 8 mM Stabilizer 4 mM KOH Appropriate amount pH 6.0 (Operation method) Reagent with specified stabilizer the absorbance E _S three days 410nm after storage was measured by 410nm absorbance E _{S · B1} and room temperature immediately after the preparation of the.

Similarly, immediately after the preparation of the reagent solution without stabilizer added 410n
The absorbance E _T three days 410nm after storage was measured by absorbance E _{T · B1} and room temperature m.

Substituting each measurement value obtained in this way into the following equation,
The stabilization rate S was determined.

S (%) = indicates the _{(E S -E S · B1)} ÷ (E T -E T · B1) × 100 The results obtained in Tables 2-1 and 2-2.

As is clear from the results of Tables 2-1 and 2-2, it is found that the stability of the test solution for measuring ACP activity increases by adding the stabilizer according to the present invention.

Experimental Example 4. Examination of the optimal amount of stabilizer added (part 1) The results of Experimental Example 3 confirmed the stabilizing effect of the stabilizer according to the present invention. Was.

(Specimen) Fresh human serum was used as the specimen.

(Reagent) A buffer having the following composition was used as a reagent.

Succinic acid 50 mM 2-Chloro-4-nitrophenylmonoammonium phosphate 4 mM HP-β-CD (degree of substitution: 5.0) 8 mM Stabilizer Predetermined concentration KOH Appropriate amount pH 6.0 (Operation method) (1) Examination of stabilization rate Immediately after the preparation of the test solution to which each stabilizer of a predetermined concentration was added 41
0nm absorbance E _{S · B1} and at room temperature after storage for 3 days 410
The absorbance E _S of nm was measured.

Similarly, immediately after the preparation of the reagent solution without stabilizer added 410n
absorbance ET _{· B1} and 410 nm after storage at room temperature for 3 days
The absorbance E _T was measured.

Substituting each measurement value obtained in this way into the following equation,
The stabilization rate S was determined.

_{S (%) = (E S} -E S · B1) ÷ (E T -E T · B1) × 100 (2) and a study sample 200μl and reagent solution 2.8ml of impact on ACP activity measurement was mixed well 37 After leaving at 37 ° C. for 2 minutes, the absorbance increase rate ΔE per minute at 410 ° C. at 37 ° C. was determined.

The same operation was performed using distilled water instead of the sample, and ΔE _B1 was determined.

Using the obtained (ΔE−ΔE _B1 ) value, 1 μmol per minute
The total ACP activity value A ₁ (IU / l) was determined using the amount of the enzyme releasing 2-chloro-4-nitrophenol as 1 unit (IU).

In addition, the same operation was performed using a sample, a test solution containing no distilled water and no stabilizer, and the total ACP activity A ₂ (IU / l) was determined.

Substituting A ₁ and A ₂ thus obtained into the following equation,
The influence rate Eff (%) of the stabilizer on the measurement of ACP activity was determined.

Eff (%) = A ₁ ÷ A ₂ × 100 The obtained results are shown in Tables 3-1, 3-2 and 3-3.

Experimental Example 5. Examination of optimal amount of stabilizer added (Part 2) M-β-CD was used instead of HP-β-CD in the test solution of Experimental Example 4.
(Substitution degree: 14.0) Except for using, the same specimen as in Experimental Example 4 was used and examined by the same operation method.

 Table 4 shows the results.

As is clear from the results of Tables 3-1 to 3 and Table 4, the required amount of the stabilizer according to the present invention is 0.5 to HNP or the like.
It turns out that it is more than double mol.

In Experimental Examples 4 and 5, the molecular extinction coefficient of 2-chloro-4-nitrophenol released as a result of the reaction with ACP was not affected by the addition of the stabilizer.

Example 1. (Sample) 30 samples of fresh human serum were used as samples.

(Test solution) (1) Substrate buffer A buffer having the following composition was used as a substrate buffer.

Succinic acid 50 mM 2-Chloro-4-nitrophenyl monoammonium phosphate 4 mM M-β-CD (degree of substitution: 14.0) 8 mM KOH Appropriate amount pH 6.0 (2) Tartaric acid solution A tartaric acid solution having the following composition was used.

L (+) tartaric acid 100 mM NaOH Appropriate amount pH 6.0 (Operation method) Mix well 200 μl of sample and 2.8 ml of substrate buffer, 37 ℃
After standing in 2 minutes, at 37 ° C., it was determined the absorbance increase Delta] E _T per minute in the 410 nm.

Then, in order to determine the ACP activity outside the prostate, 700 μl of the tartaric acid solution was added thereto, mixed well, and allowed to stand at 37 ° C. for 1 minute. Then, at 37 ° C., the absorbance increase rate ΔE _S per minute at 410 nm was determined. I asked.

The same operation was performed using distilled water instead of the sample, and ΔET _{· B1}
And to determine the ΔE _{S · B1.}

The obtained (ΔE _T −ΔE _{T · B1} ) value and (ΔE _S −ΔE
_{S · B1} ) value and 1 μmol of 2-chloro-
1 unit (IU) of enzyme that releases 4-nitrophenol
Obtains the total ACP activity value A ₁ in each sample (IU / l) and prostate-derived extracellular ACP activity value A ₂ (IU / l) as, (A ₁ -A ₂₎ prostate-derived ACP activity value from values (IU / l) asked.

Comparative Example 1. Prostate derived A using p-nitrophenyl phosphate
Measurement of CP activity (sample) The same sample as in Example 1 was used.

(Test solution) (1) Substrate buffer A buffer having the following composition was used as a substrate buffer.

 Citric acid monohydrate 50 mM p-Nitrophenyl phosphate disodium hexahydrate 10 mM NaOH Suitable amount pH 6.0 (2) Tartaric acid solution A tartaric acid solution having the following composition was used.

 L (+) tartaric acid 200 mM NaOH suitable amount pH 6.0 (3) Alkaline solution 2% sodium hydroxide solution was used.

(Operation method) Add 10 ml of distilled water to 100 ml of substrate buffer, mix well, and mix
A substrate buffer for measuring CP activity was used. This 0.5 ml was preheated for 3 minutes in a thermostat at 37 ° C., and 100 μl of the sample was added thereto.
7 ° C. for 30 minutes After incubation alkaline solution 5.0ml added to determine the absorbance at 405nm E _T. The same operation was performed using distilled water instead of the sample, and ET _{· B1} was determined.

A tartaric acid solution (10 ml) was added to the substrate buffer (100 ml) and mixed well to prepare a substrate buffer for prostate-derived extracellular ACP activity measurement. this
Using the same sample and distilled water in the same manner as described above using 0.5 ml, E _S and E _{S · B1} were determined.

Using the obtained (E _T -E _{T · B 1} ) value and (E _S -E _{S · B} 1) value, the amount of enzyme that releases 1 μmol of p-nitrophenol per minute is defined as 1 unit (IU). Total ACP activity B ₁ (IU / l) and ACP activity B ₂ outside prostate (IU / l) in the sample
From the (B ₁ −B ₂ ) value, the ACP activity value from prostate (IU /
l) asked.

Table 5 shows the results obtained by Example 1 and Comparative Example 1.
These are shown together with 1 and 5-2.

As is clear from the results of Tables 5-1 and 5-2, the substrate used in Example 1 and the substrate used in Comparative Example 1 have different substrate affinities.
(The measured values obtained in Example 1 are generally about 15% higher than the values obtained in Comparative Example 1.) The correlation is very good.

Example 2 (Sample) 53 samples of fresh human serum were used as samples.

(Test solution) (1) Substrate buffer A buffer having the following composition was used as a substrate buffer.

Succinic acid 50 mM 2-Chloro-4-nitrophenyl monoammonium phosphate 4 mM M-β-CD (degree of substitution: 14.0) 8 mM 5-bromosalicylic acid 6 mM KOH Appropriate amount pH 6.0 (2) Tartaric acid solution The solution was used.

L (+) tartaric acid 100 mM NaOH Appropriate amount pH 6.0 (Operation method) Mix well 200 μl of sample and 2.8 ml of substrate buffer, 37 ℃
After standing in 2 minutes, at 37 ° C., it was determined the absorbance increase Delta] E _T per minute in the 410 nm.

Then, in order to determine the ACP activity outside the prostate, 700 μl of the tartaric acid solution was added thereto, mixed well, and allowed to stand at 37 ° C. for 1 minute. Then, at 37 ° C., the absorbance increase rate ΔE _S at 410 nm at 410 nm was determined. I asked.

The same operation was performed using distilled water instead of the sample.
To determine the _{T · B1} and ΔE _{S · B1.}

The obtained (ΔE _T −ΔE _{T · B1} ) value and (ΔE _S −ΔE
_S.B1 ) value and 1 μmol of 2-chloro-4 per minute
Assuming that the amount of the enzyme that releases nitrophenol is 1 unit (IU), the total ACP activity value A ₁ (IU / l) and the prostatic extra-ACP activity value A ₂ (IU / l) in each sample were determined, prostate-derived ACP activity value (IU / l) was determined from ₁ -A ₂₎ value.

Comparative Example 2. Prostate derived A using p-nitrophenyl phosphate
Measurement of CP activity (sample) The same sample as in Example 2 was used.

(Test solution) A commercially available kit for measuring acid phosphatase activity [acid phosphatase B-test Wako (Wako Pure Chemical Industries, Ltd.)
Manufactured by the Company).

(Operation method) The operation was performed according to the standard operation method described in the instruction manual attached to the kit. In addition, each ACP activity value (IU / l) refers to the amount of enzyme that releases 1 μmol of p-nitrophenol per minute.
It was determined as a unit (IU).

The results obtained in Example 2 and Comparative Example 2 are also shown in FIGS. 1 and 2.

FIG. 1 shows the correlation between the total ACP activity value (IU / l) obtained in Example 2 and the total ACP activity value (IU / l) obtained in Comparative Example 2, and the vertical axis (Y ) The activity values obtained in Example 1 and the abscissa (X) indicate the activity values obtained in Comparative Example 2, respectively. The results of statistical processing of the obtained activity values are shown below.

Average: Y = 31.48, X = 29.04.

Correlation coefficient: γ = 0.988.

Regression linear equation: Y = 1.02X + 1.93.

FIG. 2 shows the prostate-derived ACP activity value (I
U / l) and the prostate-derived ACP activity value obtained in Comparative Example 2 (IU /
1), the vertical axis (Y) shows the activity value obtained in Example 2, and the horizontal axis (X) shows the activity value obtained in Comparative Example 2. The results of statistical processing of the obtained activity values are shown below.

Average: Y = 28.72, X = 24.51.

Correlation coefficient: γ = 0.998.

Regression linear equation: Y = 1.11X + 1.58.

As is clear from the results shown in FIGS.
Although the substrate used in Comparative Example 2 and the substrate used in Comparative Example 2 have different substrate affinities, individual measured values are significantly different from each other (the measured values obtained in Example 2 are the values obtained in Comparative Example 2). Generally higher)), the correlation is very good.

[Effects of the Invention] As described above, the present invention provides a reagent for measuring acid phosphatase activity which can be used for a late assay, has good stability, and can be used as a reagent for a one-component method. It is a great invention that contributes to the industry.

[Brief description of the drawings]

FIG. 1 shows the correlation between the total acid phosphatase (hereinafter abbreviated as ACP) activity value (IU / l) obtained in Example 2 and the total ACP activity value (IU / l) obtained in Comparative Example 2. The vertical axis indicates the activity value obtained in Example 2, and the horizontal axis indicates the activity value obtained in Comparative Example 2. FIG. 2 shows the prostate-derived ACP activity value obtained in Example 2 (IU /
l) and prostate-derived ACP activity values obtained in Comparative Example 2 (IU / l)
The vertical axis indicates the activity value obtained in Example 2, and the horizontal axis indicates the activity value obtained in Comparative Example 2.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12Q 1/42 REGISTRY (STN) CA (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A compound of the general formula [I] (In the formula, ^one of R ¹ and R ⁴ represents a halogen atom,
The other represents a hydrogen atom or a methyl group. R ² and R ³ are each independently a hydrogen atom, a lower alkyl group, a hydroxyalkyl group, a sulfoalkyl group, a carboxyalkyl group,
It represents a sulfonylalkyl group, a thioalkyl group, a lower alkoxy group, a sulfonic acid group, a carboxyl group or a halogen atom. 2-halo-4-nitrophenylphosphoric acid or a derivative thereof, or a salt thereof, and at least two of the 21 hydroxyl groups are carboxyalkyl groups having 2 to 4 carbon atoms, 2 to 2 carbon atoms. A sulfoalkyl group of 4,
A reagent for measuring an acid phosphatase activity, comprising a β-cyclodextrin derivative which is a mono- or dihydroxyalkyl group having 2 to 4 carbon atoms or a methyl group. 2. The reagent for measuring acid phosphatase activity according to claim 1, wherein at least one compound selected from the group consisting of phenol, phenol derivatives and salicylic acid derivatives coexists. 3. A compound represented by the general formula [I]: The reagent solution for measuring acid phosphatase activity according to claim 1 or 2, which is