JP4120024B2 - Improved enzyme substrate - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an improved enzyme substrate, a method for producing an improved enzyme substrate, and a reagent kit for measuring enzyme activity comprising the improved enzyme substrate. The present invention provides an excellent enzyme substrate that can be used and has improved stability.
[0002]
[Prior art]
In recent years, the content of trace proteins and the like in biological samples such as serum and urine can be known by carrying out enzyme immunoassay such as so-called sandwich method and competitive method using antibodies and antigens. For example, in a method called a sandwich method, a solid-phase monoclonal antibody (solid-phase antibody) against a protein or the like to be measured and a monoclonal antibody different from the monoclonal antibody against the protein or the like to be measured, Using the bound antibody (labeled antibody), a sandwich complex with (solid phase antibody-protein etc.-labeled antibody) correlated with the amount of protein to be measured is formed, and such a Santiago complex After the labeled antibody that does not form is separated, the amount of enzyme in the sandwich complex is measured based on its activity, and the amount of protein or the like to be finally measured is estimated.
[0003]
The enzyme substrate used for the enzyme reaction is often provided as a solution or in a powder state such as freeze-drying. When actually carrying out the enzyme reaction, the solution is used without diluting or diluting, or by adding water or the like to the lyophilized powder, but the solution or powdered enzyme substrate is deteriorated. In such a case, the influence on the measurement result of the enzyme activity is large, such as deterioration of reproducibility. Therefore, it is necessary to provide an enzyme substrate that is stable for a longer time and does not change its reactivity with the enzyme from the side of producing and providing the enzyme substrate.
[0004]
In the enzyme immunoassay described above, the enzyme is used as a label for measuring the target antigen or the like. In addition to this, for example, the enzyme content in a body fluid is measured for the enzyme activity. The method of knowing by doing is also frequently implemented.
[0005]
[Problems to be solved by the invention]
For example, JP-A-2-188578 describes that an enzyme substrate is stabilized by placing it under alkaline conditions. However, since various enzymes have an optimum pH at the time of activity expression, when an enzyme substrate stabilized in this way under alkaline conditions is used as it is for the measurement of enzyme activity, this pH is the optimum pH of the enzyme to be measured. If it does not match, the enzyme activity itself becomes small, resulting in a problem that the measurement accuracy is lowered. Of course, it is possible to adjust the pH of the enzyme solution thus stabilized under alkaline conditions to the optimum pH of the enzyme, but a step of pH adjustment is required prior to enzyme activity measurement.
[0006]
For example, US Pat. No. 5,143,825 discloses a method for stabilizing an enzyme solution by adding a metal chelating agent such as EDTA or EGTA to the enzyme substrate solution. This method stabilizes these metals by capturing the metal ions that cause non-enzymatic degradation of 4-methylumbelliferone phosphate or its salt. In this method, however, 4-methylumbelliferone phosphate or the like as an enzyme substrate that requires magnesium ions or zinc ions to express its activity, such as alkaline phosphatase frequently used in immunoassays, is used. When the salt is used, the ions are also captured by the chelating agent, and the enzyme activity may be inhibited. Therefore, an operation such as removing the chelating agent is required prior to use.
[0007]
In addition, it is possible to improve stability by freeze-drying the enzyme substrate or the like as compared with the case where it is stored as a solution. However, even in the case of performing such lyophilization treatment, it is convenient for the user if an enzyme substrate that can maintain the quality of the enzyme substrate more stably for a longer period of time can be provided. In other words, since the lyophilized enzyme substrate is dissolved in an appropriate solution prior to use, a large amount of stock solution can be prepared and stored even if the lyophilized substrate has good storage stability after dissolution. It is possible to keep it.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to provide a more stable enzyme substrate, the present inventor has found that alkalinity frequently used as a labeling substance in fields such as immunoassay by coexisting citric acid and / or succinic acid. The inventors have found that a phosphate ester that is a substrate for a hydrolase such as phosphatase can be stabilized, and have completed the present invention. That is, the present invention relates to an additive which is an organic acid or a salt thereof having a chelate stability constant (log K) for zinc in an aqueous solution of 20 to 25 ° C. and an ionic strength of 0.1, and a buffer. a improved enzyme substrate containing phosphoric acid ester and an alkaline phosphatase measuring substrate, and the enzyme activity measuring reagent set comprising such enzyme substrate, comprising such an enzyme substrate, an enzyme as a labeling substance This is a reagent set for immunoassay to be used.
[0009]
The present invention also provides an additive which is an organic acid or a salt thereof having a chelate stability constant (log K) of 8 to 14 for zinc in an aqueous solution at 20 to 25 ° C. and an ionic strength of 0.1 , a buffer, Is a method for producing a stabilized phosphate ester. The present invention will be described in detail below.
[0010]
An organic acid or salt thereof having a chelate stability constant (log K) for zinc in an aqueous solution of 20 to 25 ° C. and an ionic strength of 0.1 is 4 methylumbelliferone phosphoric acid or a salt thereof By capturing a metal ion or the like that non-enzymatically degrades a phosphate ester such as 4MUP) , the quality and the like are prevented from deteriorating during storage. Under such conditions, the stability constant (log K) of EDTA is about 16.5. By using an organic acid or a salt thereof having a metal ion scavenging ability weaker than that of EDTA, phosphate esters such as 4MUP are activated. Therefore, there is no inconvenience that these metal ions necessary for the expression of activity are captured even when used as a substrate for an enzyme that requires metal ions.
[0011]
Examples of suitable organic acids or salts thereof used in the present invention include, for example, citric acid and oxalic acid having a constant of about 11, succinic acid having a constant of 13, or salts of these sodium and potassium (including hydrates). Can be shown. Organic acids such as citric acid, oxalic acid and succinic acid, or salts thereof can be used alone or in combination of two or more.
[0012]
The organic acid or a salt thereof is preferably added prior to long-term storage such as 4MUP, but can be added at various other times. For example, immediately after preparation of a 4 MUP stock solution or immediately before lyophilization treatment when lyophilizing it, the effect of the present invention can be expected if added prior to measurement of enzyme activity.
[0013]
The amount of the organic acid or a salt thereof added to a phosphate ester such as 4MUP and coexisting is not particularly limited, but is preferably 0.1 to 1000 mM in total amount, and particularly preferably 1 to 100 mM in total amount.
[0014]
As is clear from the following examples, according to the knowledge of the present inventor, the presence of citric acid or succinic acid does not affect the measurement of enzyme activity, and the reactivity of the enzyme with 4MUP or the like does not change. Therefore, the phosphate ester such as 4MUP produced by adding an organic acid or its salt is stabilized and reproducible by using it as a component reagent of an enzyme activity measurement reagent kit such as an enzyme immunoassay. It is possible to provide a high-quality reagent kit that can be expected to be improved.
[0015]
【The invention's effect】
According to the present invention, it is possible to stabilize the enzyme substrate for a longer period of time compared to the conventional case. As a result, it is also possible to provide a phosphate ester such as 4MUP, which has been conventionally provided as a lyophilized product in order to improve stability, in a solution state while assuring equivalent stability. Therefore, according to the present invention, it is possible to omit complicated operations such as freeze-drying and severe control in each step, and even a 4 MUP in a solution state can be stored stably for a long period of time. This eliminates the problem of reproducibility without preparing and using the necessary amount of the enzyme substrate solution when necessary, and can eliminate the inconvenience of sequentially preparing the substrate. Of course, this does not exclude lyophilization of a phosphate ester solution such as 4MUP produced according to the present invention, and when the enzyme substrate provided by the present invention is lyophilized, the dried product is particularly preferred. Stability after dissolution can be improved.
[0016]
The present invention can remarkably improve the stability of phosphate esters such as 4MUP frequently used for enzyme activity measurement of alkaline phosphatase frequently used in enzyme immunoassay. Useful for stability improvement.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
In order to describe the present invention in more detail, examples relating to 4MPU will be described below, but the present invention is not limited to these examples.
[0018]
Example 1 Effect of citric acid and / or succinic acid (citric acid and / or succinic ion) on hydrolysis rate of 4MUP (production of enzyme substrate)
Weigh 44.46 g of 2-amino-2-monomethyl-1-propanol (manufactured by Nacalai Chemical Co., Ltd.) and 1 g of sodium azide (manufactured by Nacalai Chemical Co., Ltd.) into a container, add purified water to this, It was 900 m1. Concentrated hydrochloric acid was added while monitoring this solution with a pH meter to adjust to pH 10, and then purified water was added to make the volume 1 liter.
[0019]
To the solution prepared as described above, 256.2 mg of 4-methylumbelliferone phosphate (manufactured by JBL, USA, hereinafter referred to as 4MUP) was added to form a 1 mM 4MUP solution (hereinafter, this solution is referred to as a base substrate).
[0020]
Manganese solution (Nacalai Chemical Co., Ltd.), zinc solution (Nacalai Chemical Co., Ltd.), cadmium solution (Nacalai Chemical Co., Ltd.), aluminum solution (Nacalai Chemical Co., Ltd.), iron solution (base substrate) Nacalai Chemical Co., Ltd.), Cobalt Solution (Nacalai Chemical Co., Ltd.), Nickel Solution (Nacalai Chemical Co., Ltd.), Magnesium Solution (Nacalai Chemical Co., Ltd.) or Lead Solution (Nacalai Chemical Co., Ltd.) ) Was added to a final concentration of 100 ppm, and used as a control solution.
[0021]
(Measurement of substrate stability)
The solution prepared as described above is put into a 4, 25, 35, and 45 ° C. incubator, taken out at regular intervals, and diluted (0.14 M phosphate buffer, 0.1 M EDTA (Nacalai Chemical Co., Ltd.). Product), 0.1% sodium azide, pH 9.1), diluted 10 times, and then measured with a fluorescence spectrophotometer (Hitachi F2000 model, using 1 cm glass square cell). The measurement with a fluorescence spectrophotometer is carried out at an excitation wavelength of 363 nm (bandpass 5 nm) and a fluorescence wavelength of 447 nm (bandpass 5 nm) at room temperature, and 4MU (diluted) of a known concentration is used to measure 4MU concentration generated by hydrolysis of 4MUP. Solution (0.14M phosphate buffer solution, 0.1M EDTA (manufactured by Nacalai Chemical Co., Ltd., disodium dihydrate), 0.1% sodium azide, pH 9.1) was used to prepare a dilution series) Was calculated by comparing the relative fluorescence intensity with the measured value.
[0022]
The result at the time of hold | maintaining at 45 degreeC is shown to FIGS. From this result, compared to the base substrate, the control solution, particularly the control solution to which the iron solution was added, produced 4MU faster, that is, the coexistence of these metal ions lost the stability of 4MUP (hydrolyzed). ) In addition, as a result of incubation at 4, 25 and 35 ° C. (not shown), the amount of 4MU produced decreases as the incubation temperature decreases, and non-enzymatic degradation of 4MUP in the presence of the metal ions is temperature dependent. Was also shown.
[0023]
Example 2 Influence of Iron Ion Concentration on Hydrolysis of 4MUP A base substrate prepared in the same manner as in Example 1 and an iron solution (manufactured by Nacalai Chemical Co., Ltd.) on the base substrate to a final concentration of 1, 10 or 100 ppm The added control solution was kept at a constant temperature for a certain time in the same manner as in Example 1, and then the 4MU concentration was measured using a fluorescence spectrophotometer.
[0024]
The results are shown in FIGS. According to these figures, it can be seen that the production of 4MU, that is, the degree of non-enzymatic hydrolysis of 4MUP increases depending on the iron ion concentration.
[0025]
Example 3 Effect of succinic acid or citric acid on hydrolysis of 4MUP A base substrate prepared in the same manner as in Example 1, and an iron solution (manufactured by Nacalai Chemical Co., Ltd.) as the base substrate to a final concentration of 1 or 10 ppm. A substrate solution (hereinafter referred to as a succinic acid-added substrate or a citric acid-added substrate) in which succinic acid or citric acid is added to the control solution to a final concentration of 10 mM is constant as in Example 1. After keeping the temperature constant for a certain time, the 4MU concentration was measured using a fluorescence spectrophotometer.
[0026]
The results when kept at 45 ° C. are shown in FIG. From this result, it can be seen that although the production of 4MU is increased in the control solution compared to the base substrate, the increase is suppressed in the citrate-added substrate or succinate-added substrate.
[0027]
Example 4 Measurement of Enzymatic Activity of Alkaline Phosphatase Based on a base substrate prepared in the same manner as in Example 1, a control solution in which an iron solution (manufactured by Nacalai Chemical Co., Ltd.) was added to the base substrate to a final concentration of 1 ppm. A substrate solution in which succinic acid, citric acid or EDTA was added to the control solution to a final concentration of 10 mM (hereinafter referred to as succinic acid-added substrate, citric acid-added substrate, and EDTA-added substrate) was prepared.
[0028]
50 mM Tris-HCl buffer containing 0.1% bovine serum albumin (manufactured by Sigma) prepared so that the final concentration of commercially available alkaline phosphatase (manufactured by Sigma) is 14 g / ml with respect to 1.5 ml of each substrate solution. After adding 20 μl of the solution (pH 7.5), the 4MU concentration was measured in the same manner as in Example 1. The operation of this example was performed under a temperature condition of 37 ° C.
[0029]
The results are shown in FIG. From FIG. 9, it can be seen that the 4MU concentration is similarly increased for the base substrate, the control solution, the succinate-added substrate, and the citrate-added substrate, that is, the enzyme activity producing 4MU is not inhibited. On the other hand, with the EDTA-added substrate, it can be seen that the increase in 4MU is suppressed as a result of inhibition of the enzyme activity.
[0030]
From the results of this example and the results of Examples 1 to 3, it can be understood that 4 MUP and the like can be stably stored by adding succinic acid or the like without inhibiting the enzyme activity.
[0031]
Example 5 Enzyme immunoassay of TSH (Preparation of antibody-immobilized solid phase beads)
An ethylene-vinyl acetate copolymer (EVA) pellet (manufactured by Tosoh Corporation) having an average diameter of 1.4 mm and an average length of 1.5 mm obtained by the water strand method is described in JP-A-62-197425. And ferrite (manufactured by Tosoh Corporation) was heat-sealed, and polymer coating was further performed with glycidyl methacrylate (GMA). The obtained polymer-coated beads were treated with a caustic soda / methanol solution to ring-open the epoxy groups of the surface layer to form diols. A mouse anti-human TSH (thyroid stimulating hormone) monoclonal antibody (antibody 1) was immobilized on the beads obtained as described above. First, 25 ml of dry acetone containing 500 mg of N, N′-carbonyldiimidazole (CDI: manufactured by Tokyo Chemical Industry Co., Ltd.) was added to 100000 beads according to the method described in JP-A-63-15167 under a nitrogen atmosphere. The activation treatment was performed by vigorously stirring at room temperature for 30 minutes. After washing the activated beads, 2.5 mg / 20 ml of mouse anti-human TSH monoclonal antibody was added and shaken at room temperature for 4 hours to bind the antibody to the particles.
[0032]
After washing the beads, a phosphate buffer (pH 7.0) containing 1.0% bovine serum albumin (BSA) was added for blocking treatment.
[0033]
Bovine small intestine-derived alkaline phosphatase was used as an enzyme for labeling, and this was conjugated with an anti-human TSH monoclonal antibody (antibody 2) according to a conventional method.
[0034]
Using the prepared antibody-immobilized beads, an enzyme immunoassay for human TSH was performed. First, 12 antibody-immobilized beads were placed in a plastic cup, and 50 μl of labeled antibody (antibody 2) was added thereto. This was set in a measuring apparatus (AIA 1200, manufactured by Tosoh Corporation) having a magnet at the bottom, and 100 μl of a 0 or 48 μIU / ml TSH solution was added as an antigen solution to start an enzyme immunoreaction. In order to advance the antigen-antibody reaction of TSH, incubation was performed at 37 ° C. for 40 minutes while shaking the lower magnet at about 83 strokes / minute, and then the reaction vessel was washed with a washing solution (B / F separation). ).
[0035]
After washing, 100 μl of various substrate solutions prepared in Example 4 were dispensed to carry out enzyme reaction. The reaction was carried out by incubating at 37 ° C. for 3, 6 and 10 minutes while shaking the lower magnet at about 83 strokes / minute. The enzyme reaction was stopped after adding 500 μl of a diluent (0.14 M phosphate buffer, 0.1 M EDTA, 0.1% sodium azide, pH 9.1) as an enzyme reaction stop solution, and then the reaction was stopped. The obtained reaction solution was diluted with the diluent shown in Example 1, and the amount of fluorescence was measured in the same manner as in Example 1.
[0036]
The results are shown in FIGS. From these results, it can be seen that the base substrate, the citrate-added substrate and the succinate-added substrate give almost the same amount of fluorescence (4 MU amount), that is, (1) coexistence of citric acid and / or succinic acid inhibits enzyme activity. And (2) and the coexistence of citric acid and / or succinic acid does not inhibit the immune response.
[Brief description of the drawings]
FIG. 1 shows the results of a control solution containing a base substrate and a mancan, zinc and cadmium solution among the results of Example 1. FIG. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing manganese, the black triangle is the control solution containing zinc, and the black square is cadmium. The results of the control solutions containing are shown respectively.
FIG. 2 shows the results of a control solution containing a base substrate and an aluminum, iron, and cobalt solution among the results of Example 1. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing aluminum, the black triangle is the control solution containing iron, and the black square is cobalt. The results of the control solutions containing are shown respectively.
FIG. 3 shows the results of a control solution containing a base substrate and nickel, magnesium, and lead solutions among the results of Example 1. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing nickel, the black triangle is the control solution containing magnesium, and the black square is the lead The results of the control solutions containing are shown respectively.
FIG. 4 shows the results of storing a base substrate or a control solution containing iron at 4 ° C. among the results of Example 2. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the storage time (hours), the self-circle indicates the base substrate, the black circle indicates the control solution containing 1 ppm iron, and the black triangle indicates the control solution containing 10 ppm iron. The black squares show the results of the control solutions containing 100 ppm of iron, respectively.
FIG. 5 shows the results of storing a base substrate or a control solution containing iron at 25 ° C. among the results of Example 2. In the figure, the vertical axis shows the 4MU concentration, the horizontal axis shows the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing 1 ppm iron, and the black triangle is the control solution containing 10 ppm iron. The black squares show the results of the control solution containing 100 ppm of iron, respectively.
FIG. 6 shows the results of storage of a control solution containing a base substrate or iron at 35 ° C. among the results of Example 2. In the figure, the vertical axis shows the 4MU concentration, the horizontal axis shows the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing 1 ppm iron, and the 4 black triangle is the control solution containing 10 ppm iron. The black squares show the results of the control solutions containing 100 ppm of iron, respectively.
FIG. 7 shows the results of storing a base substrate or a control solution containing iron at 45 ° C. among the results of Example 2. In the figure, the vertical axis shows the 4MU concentration, the horizontal axis shows the storage time (hours), the white circle is the base substrate, the black circle is the control solution containing 1 ppm iron, and the black triangle is the control solution containing 10 ppm iron. The black squares show the results of the control solution containing 100 ppm of iron, respectively.
FIG. 8 shows the results of storage at 45 ° C. among the results of Example 3. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the storage time (hours), the white circle indicates the base substrate, the self triangle indicates the iron-containing control solution, the black circle indicates the succinic acid-added substrate, and the black triangle indicates citric acid. The black squares of the added substrate indicate the results of the EDTA added substrate, respectively.
FIG. 9 shows the results of Example 4. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the enzyme reaction time (seconds), the white circle is the base substrate, the black square is the control solution containing iron, the black triangle is the succinic acid-added substrate, and the white triangle is The black circles of the citrate-added substrate indicate the results of the EDTA-added substrate, respectively.
FIG. 10 shows the results of measuring TSH at a concentration of 0 μIU / ml among the results of Example 5. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the enzyme reaction time (minutes), the white circle is the base substrate, the white triangle is the control solution containing iron, the black circle is the succinic acid-added substrate, and the black triangle is the quencher. The black squares of the acid-added substrate indicate the results of the EDTA-added substrate, respectively.
FIG. 11 shows the result of measuring TSH at a concentration of 50 μIU / ml among the results of Example 5. In the figure, the vertical axis indicates the 4MU concentration, the horizontal axis indicates the enzyme reaction time (minutes), the white circle is the base substrate, the white triangle is the control solution containing iron, the black circle is the succinic acid-added substrate, and the black triangle is the quencher. The black squares of the acid-added substrate indicate the results of the EDTA-added substrate, respectively.

Claims (11)

An additive which is an organic acid or a salt thereof having a chelate stability constant (log K) for zinc in an aqueous solution of 20 to 25 ° C. and an ionic strength of 0.1, a buffer, and alkaline phosphatase measurement An improved enzyme substrate composition comprising a substrate phosphate ester. The enzyme substrate composition according to claim 1, wherein the additive which is an organic acid or a salt thereof is citric acid and / or succinic acid, or a salt thereof . The enzyme substrate according to claim 1 or 2, wherein the content of the additive which is an organic acid or a salt thereof is 0.1 to 1000 mM. The enzyme substrate composition according to claim 1, wherein the phosphate ester which is a substrate for measuring alkaline phosphatase is 4-methylumbelliferone phosphate or a salt thereof . The enzyme substrate composition according to any one of claims 1 to 4, which is an aqueous solution. A reagent set for measuring enzyme activity comprising the enzyme substrate composition according to any one of claims 1 to 5. A reagent set for immunoassay using the enzyme as a labeling substance, comprising the enzyme substrate composition according to any one of claims 1 to 6. Addition of an additive which is an organic acid or salt thereof having a chelate stability constant (log K) to zinc in an aqueous solution of 20 to 25 ° C. and an ionic strength of 0.1 and a buffer, and a stability A method for producing a composition comprising a modified phosphate ester. The method for producing a composition containing a stabilized phosphate according to claim 8, wherein the additive which is an organic acid or a salt thereof is citric acid and / or succinic acid, or a salt thereof. The manufacturing method of Claim 8 or 9 which adds the additive which is 0.1-1000 mM organic acid or its salt. The method according to claim 10, wherein the phosphate ester as a substrate for measuring alkaline phosphatase is 4-methylumbelliferone phosphate or a salt thereof.

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