JPH06303996A - Determination of alkaline phosphatase and reagent therefor - Google Patents

Abstract

PURPOSE:To prevent the lowering of sensitivity caused by long term storage of a reagent by using a tris buffer solution as a buffer solution in a dephosphorylation reaction. CONSTITUTION:The alkaline phosphatase determination reagent is prepared by using a tris buffer solution as a buffer solution in a quantity-dependent dephosphorylation reaction of nicotinamide adenine dinucleotide phosphate by alkaline phosphatase. Even after a long term storage, alkaline phosphatase can be determined in high sensitivity.

Description

Detailed Description of the Invention

[0001]

This invention relates to an alkaline phosphatase (hereinafter referred to as useful for analysis of biological components and various food components).
ALP) and a reagent for measurement. More specifically, the present invention provides a signal obtained by enzymatically cycling ALP bound to a substance to be detected using nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or a reduced form thereof (hereinafter abbreviated as NADH). The present invention relates to a method for highly sensitive and stable measurement by an amplification system and a measuring reagent therefor.

[0002]

2. Description of the Related Art NAD and its reduced form NADH
Is a coenzyme with the highest biological content, and is involved in the redox reaction of many dehydrogenases by reversibly changing through the exchange of hydrogen atoms. These NAD or N
ADH can be detected with high sensitivity by a signal amplification system using its enzymatic cycling, and this signal amplification system is also widely applied to ALP detection systems in the fields of immunoassay and tissue staining ( A. Johannson et al .: C
linica Chimica Acta, 148 , 119-124, 19
85; FJ Dhahir et al .: Clinical Chemistry, 38 (2),
227-232, 1992).

This ALP detection system has a detection sensitivity about 10 times higher than that of a detection system using horseradish peroxidase, which is a general labeling enzyme. Despite the colorimetric method, a fluorescent dye or a radioactive dye is used. Since detection sensitivity equivalent to that of detection systems using isotopes can be obtained, in the immunoassay field,
It is positioned as a very good method (Ishikawa,
Other editions; enzyme immunoassay, 3rd edition, pp. 58-60, published by Ikusho Shoin).

This ALP detection system uses nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) or its reduced form (hereinafter abbreviated as NADPH).
By the catalytic action of ALP, respectively.
By changing to ADH and detecting this NAD or NADH by its signal amplification system, the presence of ALP, that is, the presence of the substance to be detected labeled with ALP is to be measured with high sensitivity.

For example, when NADP is used as a substrate for ALP, NADP is dephosphorylated and NAD is produced by the catalytic action of ALP, as shown in the reaction formula (Formula 1) below. Acts as a coenzyme in the oxidation reaction of ethanol to acetaldehyde by ADH in the presence of alcohol dehydrogenase (hereinafter abbreviated as ADH) and its substrate ethanol.
Reduce to DH. Furthermore, this NADH is a tetrazolium dye and its reductase diaphorase (hereinafter referred to as DIA).
(Hereinafter, abbreviated as "), it is oxidized to NAD again, but at the same time, the tetrazolium dye is reduced to produce formazan, and a colored signal is obtained. Since NAD repeats redox under such conditions, the signal intensity gradually increases (A. Johannson et al., Clinica Chemica Act, supra).
a).

[0006]

[Chemical 1]

Also, when NADPH is used as a substrate for ALP, as shown in the following reaction formula (Formula 2),
Color is generated by the same principle as NADP, and its intensity is amplified. In this case, the enzyme involved in the oxidation reaction of NADH → NAD needs to be an enzyme that recognizes only NADH but not NADPH, and is usually NADH-dependent DIA.
(FJ Dhahir et al., Clinica Chemis
try).

[0008]

[Chemical 2]

As described above, in the method for measuring ALP using the signal amplification system by enzymatic cycling of NAD or NADH, the first step is NAD by ALP.
Dephosphorylation of P or NADPH is performed, followed by a second
As a step, a signal amplification reaction by enzymatic cycling of NAD or NADH is performed. In addition, each reaction can be performed using a measurement reagent prepared in advance. That is, NADP or NA that is a substrate for ALP
A first reaction reagent containing DPH as a main component and NAD or N
It is a second reaction reagent containing an enzyme involved in the enzymatic cycling and signal amplification of ADH and its substrate as components.
Of these, the buffer solution for the first reaction reagent is generally
Dieranolamine buffer (pH 9-10) has been used (A. Johannson et al., Clinica Chimica Acta; F .;
J. Dhahir et al., Clinica Chimistry, supra).

[0010]

However, in the case of the conventional ALP assay method, NADP or NADPH in the first reaction reagent is liable to change during storage, and when mixed with the second reaction reagent, ALP Regardless of the presence of the non-specific color development (hereinafter sometimes referred to as reagent blank). Therefore, the first reaction reagent of the ALP detection system had to be prepared each time immediately before use.

The present invention has been made in view of the above circumstances, solves the drawbacks of the conventional ALP measuring method, and detects ALP with high sensitivity even after storing a previously prepared measuring reagent for a long time. It is intended to provide an ALP measuring method and a measuring reagent that can be used.

[0012]

In order to solve the above problems, the present invention provides NADP or NADPH as A
In an ALP detection system using signal amplification accompanied by enzymatic cycling of NAD or NADH produced by dephosphorylation in a dose-dependent manner by LP, a Tris buffer is used as a buffer during the dephosphorylation reaction. A method for measuring alkaline phosphatase is provided.

The present invention also provides an ALP measuring reagent using the above signal amplification, wherein the buffer solution for the dephosphorylation reaction reagent is a Tris buffer.
The constitution and preferred embodiments of the present invention will be described in more detail below. In the assay method of the present invention, first, NADP or NAD by ALP is used as the first reaction.
The dephosphorylation reaction of PH is performed. The composition of the measurement reagent used in this first reaction is NADP or NADPH, an ALP activator (NADP or NADPH magnesium, etc.),
And Tris buffer, which is mixed with a sample containing a substance to be detected labeled with ALP, and N
ADP or NADPH to NAD or NAD respectively
Change to H.

The Tris buffer used as the reagent for the first reaction is, for example, Tris-hydrochloric acid buffer, Tris-acetic acid buffer, etc., and has pH 7-14, preferably pH 8-11. NADP or NADPH does not contain a reaction component for the enzyme involved in the signal amplification reaction, and the concentration in the reagent can be a concentration required for expression of ALP activity. As the ALP activator, magnesium salt, cobalt salt or the like can be used.

Next, as a second reaction, NAD or NA
A signal amplification reaction is performed by enzymatic cycling of DH. The composition of the measuring reagent used in this second reaction is dehydrogenase, a substrate for dehydrogenase, DIA, a tetrazolium dye, a surfactant, etc., and is mixed with the reaction solution obtained in the first reaction to give a tetrazolium dye. The change in absorbance of formazan caused by the reduction of is measured.

Among various reagent components used in the second reaction reagent, the dehydrogenase is NAD or ADH having high specificity for NADH and amino acid dehydrogenase (eg, alanine dehydrogenase, leucine dehydrogenase, etc.). To use. The concentration of these dehydrogenases in the reagent is 0.01 to 1000 units / ml, more preferably 0.1 to 100 units / ml.

The substrate when amino acid dehydrogenase is used as the dehydrogenase is an amino acid corresponding to its type, and the substrate when ADH is used is alcohols such as ethanol. The concentrations of these substrates in the reagent are appropriately determined depending on the activity and concentration of dehydrogenase. The DIA is not particularly limited in its origin, but is preferably highly stable, for example, derived from Bacillus stearothermophilus, and its concentration in the measurement reagent is 0.01 to 1000.
Unit / ml, more preferably 0.1-100 unit / ml
The degree.

As the tetrazolium dye, 2- (P
Nitrophenyl) -3- (P-iodophenyl) -5-
Phenyltetrazolium chloride (hereinafter abbreviated as INT), 3,3 '-(3,3'-dimethoxy-4,
4'-diphenylene) bis (2- (P-nitrophenyl) -5-phenyl-tetrazolium chloride), 2
-(4 ', 5'-Dimethyl-2'-thiazolyl-3,5
-Diphenyltetrazolium bromide) and the like, and the concentration thereof in the measuring reagent is 0.1 to 10 m.
M, more preferably about 0.5 to 2 mM.

As the surface active agent, a nonionic surface active agent or an ionic surface active agent can be used. Among them, a Triton type surface active agent is preferable, and a Triton X-100 is more preferable. Specifically, the ALP measurement of the present invention can be performed, for example, as follows.

First, 10 μl of a sample containing a substance to be detected labeled with ALP was added to NADP or NADP having the above concentration.
A total of 200 μl of H, Tris buffer and ALP activator were added, and the mixture was incubated at the optimum temperature for enzyme activity for several minutes,
Dephosphorylation of NADP or NADPH is performed (first reaction), and then a dehydrogenase and its substrate, DIA, necessary for the enzymatic cycling of NAD or NADH,
The reaction solution of the first reaction was added to 400 μl of a buffer solution containing a tetrazolium dye and a surfactant, and the mixture was reacted for several minutes (second reaction). 200 μl of 1M hydrochloric acid was added to stop the reaction, and then at 490 nm. The absorbance (hereinafter referred to as A490) is measured using a commercially available spectrophotometric system or the like.

In the ALP measuring method of the present invention, the reagent used for the first reaction and the reagent for the second reaction may be simultaneously mixed with the ALP sample. In any case, in the measuring method of the present invention, NADP or NA
Since DPH does not denature during storage of the reagent, ALP can be measured with high sensitivity even when the above-mentioned first reaction reagent, second reaction reagent or a mixed reagent thereof is prepared in advance and used after storage for a long time.

Hereinafter, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0023]

【Example】

Example 1 (1) Preparation of measurement reagent A first reaction reagent and a second reaction reagent each having the following composition were prepared. First reaction reagent: Tris-HCl buffer, pH 9.8 500 mM NADPH 1 mM magnesium chloride 1 mM Second reaction reagent: Sodium phosphate buffer (pH 6.5) 250 mM INT 0.5 mM ethanol 4% Triton X-100 0.1% DIA (from Bacillus stearothermophilus) 1 unit / ml ADH (from Zymomonas mobilis) 1 unit / ml Further, as a control for the first reaction reagent, instead of the Tris-HCl buffer, diethanolamine buffer (pH 9.8). Was used to prepare a reagent (Comparative Example 1).

(2) Test method The first reaction reagent and its control (Comparative Example 1) were incubated at 37 ° C., and each was subjected to daily (0, 1, 4, 16, 35).
200 μl of the sample was mixed with 20 μl of water or 1 unit / ml of ALP (bovine origin, purchased from Boehringer Mannheim) and reacted at 30 ° C. for 10 minutes. Then, the second reaction reagent 4 is added to this reaction solution.
00 μl was added and reacted at 30 ° C. for 10 minutes, 200 μl of 1M hydrochloric acid was added to stop the reaction, and A490 was measured.

(3) Results Change in absorbance when water is reacted with the first reaction reagent and its control sampled daily (reagent blank)
Is as shown in Table 1 and FIG. As is clear from Table 1 and FIG. 1, the first reaction reagent (Example 1) using Tris-hydrochloric acid buffer as a buffer solution had a reagent blank more than the control (Comparative Example 1) using diethanolamine buffer solution. It was few and extremely stable over time.

Next, A49 when ALP was reacted with the first reaction reagent sampled daily and its control
The values (ALP activity values) obtained by subtracting the above reagent blanks from 0 are shown in Table 2 and FIG. Further, the ratio of ALP activity value / reagent blank is shown in Table 3 and FIG. 3 as a comprehensive index as a measuring reagent. As is clear from these results, it was confirmed that the reagent used in the measuring method of the present invention has little deterioration during storage and has stable ALP detection sensitivity.

[0027]

[Table 1]

[0028]

[Table 2]

[0029]

[Table 3]

Example 2 (1) Preparation of measurement reagent A first reaction reagent having the following composition was prepared. First reaction reagent: Tris-HCl buffer, pH 9.8 500 mM NADP 1 mM Magnesium chloride 1 mM A control of the first reaction reagent and a second reaction reagent were prepared in the same composition as in Experimental Example 1.

(2) Test Method The first reaction reagent and its control were incubated at 4 ° C., sampled daily (0, 9, 41 days) and tested in the same manner as in the examples. (3) Results A4 when water was reacted with the first reaction reagent (Example 2) and its control (Comparative Example 2) sampled daily
90 (reagent blank) is shown in Table 4 and FIG.

Similarly, the value (ALP activity value) obtained by subtracting the above-mentioned reagent blank from A490 when ALP was reacted with the first reaction reagent and its control is shown in Table 5 and FIG.
The ratio of ALP activity value / reagent blank is shown in Table 6 and FIG.

[0033]

[Table 4]

[0034]

[Table 5]

[0035]

[Table 6]

As is clear from these results, the assay reagent used in the assay method of the present invention has little reagent blank even when stored at 4 ° C. for 41 days, has a stable ALP activity value over time, and has a long duration. It was confirmed to have high ALP detection sensitivity over a period of time.

[0037]

As described above in detail, the A of the present invention
By the LP measurement method, it becomes possible to detect ALP with high sensitivity even after storing a previously prepared measurement reagent for a long period of time.

[Brief description of drawings]

FIG. 1 shows the change with time of a reagent blank of each of a measurement reagent (●) and a conventional reagent (◯) used in the method of the present invention.

[Fig. 2] Reagent (●) and conventional reagent (○) used in the present invention
3 shows the daily change of ALP activity value of.

FIG. 3 Reagents used in this invention (●) and conventional reagents (○)
3 shows the change over time in the ratio of ALP activity value / reagent blank of.

FIG. 4 Reagents used in this invention (●) and conventional reagents (○)
3 shows the daily change of the reagent blank of.

FIG. 5: Reagents used in this invention (●) and conventional reagents (○)
3 shows the daily change of ALP activity value of.

FIG. 6 Reagents used in this invention (●) and conventional reagents (○)
3 shows the change over time in the ratio of ALP activity value / reagent blank of.

Front page continuation (72) Inventor Nao Suzuki, 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Ltd. Central Research Laboratory

Claims (2)

[Claims] 1. A signal amplification accompanying enzymatic cycling of nicotinamide adenine dinucleotide phosphate or its reduced form, which is produced by dephosphorylating nicotinamide adenine dinucleotide phosphate or its reduced form with alkaline phosphatase in a dose-dependent manner. A method for measuring alkaline phosphatase, which comprises using a Tris buffer as a buffer during a dephosphorylation reaction in an alkaline phosphatase detection system using. 2. A signal amplification associated with enzymatic cycling of nicotinamide adenine dinucleotide phosphate or its reduced form, which is produced by dephosphorylating nicotinamide adenine dinucleotide phosphate or its reduced form with alkaline phosphatase in a dose-dependent manner. A reagent for measuring alkaline phosphatase using, wherein the buffer solution for the dephosphorylation reaction reagent is Tris buffer.