JPH09322769A - Improved enzymatic substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an enzymatic substrate improved in long-term preservability and useful for enzyme activity measurement and enzyme immunoassay, etc., by making chromium coexistent with 4-methyl umbelliferonephosphate as a substrate for hydrolase such as alkaline phosphatase. SOLUTION: First, 2-amino-2-methyl-1-propanol and sodium azide are weighed and put into a vessel. Secondly, purified water is added to them to prepare an aqueous solution followed by adding concentrated hydrochloric acid to the aqueous solution while monitoring the solution with a pH meter to adjust the aqueous solution to pH10, and then, further purified water is added to the resultant aqueous solution to regulate its volume. Thirdly, 4-methyl umbelliferonephosphate(4MU), a substrate for hydrolase such as alkaline phosphatase, is added to the resultant solution to make into an 1mM 4MU solution. Finally, a chromium solution is added to the 4MU solution so as to have 0.1-300ppm of chromium concentration, thus obtaining the objective enzymatic substrate improved in stability after preserved for a long period and prevented from quality deterioration.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an improved enzyme substrate, a method for producing the improved enzyme substrate, and a reagent kit for measuring an enzyme activity, which comprises the improved enzyme substrate. The present invention provides an excellent enzyme substrate, etc., which has improved stability and can be used in enzyme immunoassays and the like.

[0002]

2. Description of the Related Art In recent years, the contents of trace proteins and the like in biological samples such as serum and urine can be known by carrying out enzyme immunoassay using antibodies and antigens. That is, for example, in the method called the sandwich method, a solid-phased monoclonal antibody against a protein to be measured (solid-phased antibody) and a monoclonal antibody different from the monoclonal antibody against the protein to be measured An enzyme bound antibody (labeled antibody) is used to form a sandwich complex with (immobilized antibody-protein etc.-labeled antibody) that correlates with the amount of protein or the like to be measured. After separating the labeled antibody that does not form a sandwich complex, the amount of the enzyme in the sandwich complex is measured based on its activity to estimate the amount of the protein or the like to be measured.

[0003] Enzyme substrates used in enzymatic reactions are often provided as solutions or in the form of lyophilized powders. When actually performing an enzyme reaction, this solution is used without dilution, or it is used after adding water etc. to this powder as a solution, but the provided solution or the enzyme substrate in powder state deteriorates. If so, reproducibility or the like has a great influence on the measurement result of the enzyme activity. Therefore, from the side of providing the enzyme substrate, it is necessary to provide the enzyme substrate which is stable for a long time and whose reactivity with the enzyme does not change.

[0004] In the enzyme immunoassay described above, the enzyme is used as a label for measuring a target antigen or the like. In other cases, for example, the enzyme content in a body fluid is determined by measuring the enzyme content. A method of knowing the activity by measuring it is frequently practiced.

[0005]

SUMMARY OF THE INVENTION For example, JP-A-2-18
No. 8578 describes stabilization by placing an enzyme substrate under alkaline conditions. However, since various enzymes have an optimum pH for expressing the activity,
When the enzyme substrate thus stabilized under alkaline conditions is used as it is, when the pH does not match the optimum pH of the enzyme whose activity is to be measured, the enzyme activity itself to be measured becomes small. However, there arises a problem that the measurement accuracy is lowered. On the other hand, although it is possible to adjust the pH of the enzyme solution thus stabilized under alkaline conditions to the optimum pH of the enzyme, a step of pH adjustment is added prior to measuring the enzyme activity.

Further, for example, US Pat. No. 5,143,825 describes stabilizing an enzyme solution by adding a metal chelating agent such as EDTA or EGTA to the enzyme substrate solution. However, in the case of an enzyme such as alkaline phosphatase frequently used for immunoassays, which requires a metal to express its activity, the enzyme activity may be inhibited in the presence of a chelating agent, and therefore, prior to use. Therefore, operations such as removing the chelating agent will be added.

As described above, the stability can be improved by freeze-drying the enzyme substrate as compared with the case of storing it as a solution. However, even when such a freeze-drying treatment is performed, there is a demand for an enzyme substrate that can stably maintain the quality for a longer period of time. In addition, as a matter of course, the enzyme substrate freeze-dried or the like is often dissolved in an appropriate solution before use, and it is required to have good storage stability after being dissolved.

[0008]

Means for Solving the Problems As a result of earnest studies to provide a more stable enzyme substrate, the present inventor has found that coexistence of chromium causes it to be frequently used as a labeling substance in the field of immunoassay and the like. The inventors have found that a substrate for a hydrolase typified by alkaline phosphatase, such as 4-methylumbelliferone phosphate, can be stabilized, and completed the present invention. That is, the present invention is a composition comprising chromium and a substrate for a hydrolase such as alkaline phosphatase, such as 4-methylumbelliferone phosphate or a salt thereof.

The present invention also provides a reagent set for measuring enzyme activity, which comprises this composition, and more particularly, a reagent set for immunoassay, which comprises this composition and uses an enzyme such as alkaline phosphatase as a labeling substance.

Furthermore, the present invention uses this composition for the measurement of enzyme activity of alkaline phosphatase and the like, a substrate for a hydrolase typified by alkaline phosphatase by coexistence of chromium, such as 4-methylumbelliferone phosphate. Alternatively, it is a method of stabilizing the salt.

The present invention is also a method for producing a stabilized hydrolase substrate such as alkaline phosphatase, which is characterized by adding chromium. Hereinafter, the present invention will be described in detail.

[0012] Chromium has an effect of suppressing non-enzymatic hydrolysis of the enzyme substrate and deterioration of quality during storage. The coexistence of chromium is effective for stabilizing 4-methylumbelliferone phosphate or its salt, which is a substrate for hydrolases such as alkaline phosphatase, but this fact indicates that 4-methylumbelliferone phosphate or its salt is stable. It is considered that this is due to the fact that the substrate of hydrolase such as salt has the property of being easily hydrolyzed non-enzymatically in a solution.

Chromium is most effectively added prior to long-term storage of a substrate for a hydrolase such as 4-methylumbelliferone phosphate or its salt, but it is most effective to use the enzyme substrate. There is no particular limitation as long as it is added in advance. For example, it is particularly preferable to prepare a stock solution of 4-methylumbelliferone phosphate or a salt thereof or before freeze-drying treatment when freeze-drying 4-methylumbelliferone phosphate or a salt thereof, but to measure enzyme activity. Any early addition is sufficient.

The amount of chromium added to and coexisting with the enzyme substrate is not particularly limited, but is usually 0.1 to 300 p.
pm is good, and particularly preferably 1 to 100 ppm.

According to the knowledge of the present inventor, although it is clear from the later examples, coexistence of chromium does not change the reactivity of the hydrolase and its substrate, etc., and affects the measurement of enzyme activity. Never give. Therefore, using the substrate for hydrolase such as 4-methylumbelliferone phosphate or a salt thereof produced in this manner as a component of a reagent kit for enzyme activity measurement such as enzyme immunoassay Therefore, it is possible to provide a reagent kit having high quality as a whole.

[0016]

According to the present invention, it is possible to improve the stability of the enzyme substrate as compared with the conventional one. As a result, a substrate for a hydrolase such as 4-methylumbelliferone phosphate or its salt, which was conventionally provided as a lyophilized product in order to improve stability, is maintained in a solution state while achieving equivalent stability. It is also possible to provide. Therefore, according to the present invention, it is possible to omit a complicated operation such as freeze-drying, which is strictly controlled in each step. Of course, the present invention does not exclude freeze-drying or the like of a substrate solution of a hydrolase such as 4-methylumbelliferone phosphate or its salt produced by the present invention.

The present invention is particularly effective for measuring the enzyme activity of a hydrolase typified by alkaline phosphatase which is frequently used in enzyme immunoassays, and improves the long-term stability of reagents in the field of enzyme immunoassay. Is effective for.

According to the present invention, even a substrate in a solution state can be stably stored for a long period of time. This makes it possible to eliminate the inconvenience of preparing and using the enzyme substrate solution in the required amount when needed.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In order to explain the present invention in more detail, examples of alkaline phosphatase which is a typical hydrolase will be described below, but the present invention is not limited to these examples. .

Example 1 Effect of Chromium (Chromium Ion) on Hydrolysis Rate of 4MUP (Production of Enzyme Substrate) 44.46 g of 2-amino-2-methyl-1-propanol (produced by Nacalai Chemical Co., Ltd.) and horse mackerel 1 g of sodium iodide (manufactured by Nakarai Chemical Co., Ltd.) was weighed in a container, and purified water was added to the container to make about 900 ml.
Concentrated hydrochloric acid was added to this solution while monitoring it with a pH meter to adjust the pH to 10, and then purified water was further added to adjust the liquid volume to 1.
I made it to liters.

4-Methylumbelliferone phosphate (manufactured by JBL, USA, 4M below) was added to the solution prepared as described above.
256.2 mg was added to obtain a 1 M 4M UP solution (hereinafter, this solution is referred to as a base substrate).

A chromium solution (manufactured by Nakarai Kagaku Co., Ltd.) was added to the base substrate so that the final concentrations were 1, 10, and 100 ppm, to prepare a control solution.

(Measurement of Stability of Substrate) The solution prepared as described above was placed in an incubator at 4, 25, 35 and 45 ° C. and taken out at regular intervals, and a fluorescence spectrophotometer (Hitachi F2
000 type, 1 cm glass square cell was used). The measurement using a fluorescence spectrophotometer was conducted with an excitation wavelength of 363 nm (bandpass 5 nm) and a fluorescence wavelength of 447 nm (bandpass 5 n).
m) at room temperature to measure 4MU concentration produced by hydrolysis of 4MUP. 4MU of known concentration (diluent (0.14M phosphate buffer, 0.1M EDTA (Nacalai Chemical Co., Ltd., 2 sodium・ Dihydrate), 0.1%
It was calculated by comparing the relative fluorescence intensity with that obtained by measuring a dilution series prepared with sodium azide, pH 9.1).

The results are shown in FIGS. These results show that the control solution produced less 4MU than the base substrate, that is, 4MUP was stabilized by the coexistence of chromium.

Example 2 Enzyme immunoassay of TSH (Preparation of antibody-immobilized solid phase beads) Average diameter 1.4 mm, average length 1.5 mm obtained by water strand method
Of the ethylene-vinyl acetate copolymer (EVA) pellets (manufactured by Tosoh Corporation) were spheroidized according to the method described in Japanese Patent Application No. 61-38279 and heat-bonded to ferrite (manufactured by Tosoh Corporation). Further, it was polymer-coated with glycidyl methacrylate (GMA). The obtained polymer-coated beads were treated with a solution of caustic soda / methanol to open the epoxy groups on the surface layer to form a diol.

Mouse anti-human TSH (thyroid stimulating hormone) monoclonal antibody (antibody 1) was immobilized on the beads obtained as described above, as shown below. First, for 100,000 beads, Japanese Patent Application No. 61-38279
500 mg of N, N 'according to the method described in the publication.
An activation treatment was carried out by vigorously stirring 25 ml of dry acetone containing carbonyldiimidazole (CDI: manufactured by Tokyo Chemical Industry Co., Ltd.) under a nitrogen atmosphere at room temperature for 30 minutes. 2.5 mg / 2 after washing the activated beads
0 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.

After washing the beads, a phosphate buffer (pH 7.0) containing 1.0% bovine serum albumin (BSA) was added for blocking treatment.

As an enzyme used for labeling, alkaline phosphatase derived from bovine small intestine was used, and this was combined with an anti-human TSH monoclonal antibody (antibody 2) according to a conventional method. Using the prepared antibody-immobilized beads, enzyme immunoassay of human TSH was performed. First, 12 antibody-immobilized beads were placed in a plastic cup, and 50 μl of the labeled antibody (antibody 2) was added thereto to prepare a solution. This was set in a measuring device (AIA-1200, manufactured by Tosoh Corporation) having a magnet at the bottom, and 0 or 48 μ was used as an antigen solution.
The enzyme immunoreaction was started by adding 100 μl of ISH / ml TSH solution. In order to proceed the antigen-antibody reaction of TSH, the mixture was incubated 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). ).

After washing, 4MUP prepared in Example 1
The enzyme reaction was carried out by dispensing 100 μl of a solution containing
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. Dilution solution (0.
The enzyme reaction was stopped by adding 500 μl of 14 M phosphate buffer, 0.1 M EDTA, 0.1% sodium azide, pH 9.1, and the reaction solution in which the reaction was stopped was diluted with a diluent. The amount of fluorescence was measured by the same method as that used in Example 1.

The results are shown in FIGS. 5 and 6. As a result, almost the same fluorescence amount (4 MU amount) was obtained for the base substrate to which chromium was not added and the control solution to which chromium was added, and it can be seen that coexistence of chromium does not affect the enzyme activity.

Example 3 Measurement of Fluorescence Spectra of Chromium and 4MUP Composition Of the 4MUP solution prepared in Example 1, a base substrate and a control substrate containing 10 ppm of chromium were incubated at 45 ° C. for 96 hours and then subjected to fluorescence spectrophotometry. The spectrum was measured using a meter.

The measurement conditions are as follows: (1) For the base substrate, the excitation wavelength is changed from 220 to 450 nm and 447 nm.
Fluorescence measurement with (2) Excitation wavelength of 36 for the base substrate
3 nm, fluorescence measurement at 320-550 nm, (3) change excitation wavelength to 220-450 nm for control substrate, 447 nm fluorescence measurement, (2) make excitation wavelength for base substrate 363 nm, fluorescence measurement at 320-550 nm, Is.

As a result, the fluorescence spectra of the base substrate and the control substrate are different from each other, and the presence of chromium causes 4 MU.
It was suggested that a structural change occurred in P, which resulted in structural stabilization.

From the above, it can be seen that in the measurement of enzyme activity in enzyme immunoassay and the like, coexistence of chromium in 4MUP or the like can improve the stability of the enzyme activity without inhibiting the activity of the enzyme.

[Brief description of drawings]

FIG. 1 shows the result of Example 1 when the temperature was kept at 4 ° C. The vertical axis shows the concentration of 4 MU (nM)
The horizontal axis indicates the storage time (hours) at 4 ° C., and the white circles, black circles, black triangles and black squares in the figure represent the base substrate,
The control solution with 1 ppm chromium added, the control solution with 10 ppm chromium and the control solution with 100 ppm chromium are shown.

FIG. 2 shows the result of Example 1 when the temperature was kept at 25 ° C. The vertical axis represents the concentration of 4MU (n
M), the horizontal axis shows the storage time (hours) at 25 ° C., and the white circles, black circles, black triangles and black squares in the figure represent the base substrate, the control solution containing 1 ppm of chromium, and 10 pp, respectively.
Figure 3 shows a control solution with m chromium added and a control solution with 100 ppm chromium added.

FIG. 3 shows the results of Example 1 when the temperature was kept at 35 ° C. The vertical axis represents the concentration of 4MU (n
M), the horizontal axis shows the storage time (hours) at 35 ° C., and the white circles, black circles, black triangles and black squares in the figure respectively represent the base substrate, a control solution containing 1 ppm of chromium, and 10 pp.
Figure 3 shows a control solution with m chromium added and a control solution with 100 ppm chromium added.

FIG. 4 shows the result of Example 1 when the temperature was kept at 45 ° C. The vertical axis represents the concentration of 4MU (n
M), the horizontal axis indicates the storage time (hours) at 45 ° C., and the white circles, black circles, black triangles and black squares in the figure represent the base substrate, the control solution containing 1 ppm of chromium, and 10 pp, respectively.
Figure 3 shows a control solution with m chromium added and a control solution with 100 ppm chromium added.

FIG. 5 shows the results of Example 2 with 0 μIU / ml of T
It shows the result of measurement for the SH solution. The vertical axis shows the concentration of 4MU (nM), the horizontal axis shows the enzyme reaction time (minutes).
In the figure, white circles, black circles, black triangles, and black squares represent the base substrate, the control solution containing 1 ppm of chromium, the control solution containing 10 ppm of chromium, and 10
A control solution with 0 ppm chromium added is shown.

FIG. 6 shows the results of measurement of a TSH solution of 48 μIU / ml among the results of Example 2. The vertical axis shows the concentration of 4 MU (nM), the horizontal axis shows the enzyme reaction time (minutes), white circles, black circles, black triangles and black squares in the figure are the base substrate, the control solution containing 1 ppm of chromium, A control solution with 10 ppm chromium and a control solution with 100 ppm chromium are shown.

Claims (18)

[Claims] 1. A composition comprising chromium and a substrate for a hydrolase. 2. The composition according to claim 1, wherein the substrate is a substrate for alkaline phosphatase. 3. The composition according to claim 2, wherein the substrate is 4-methylumbelliferone phosphate or its salt. 4. The composition according to claim 1, wherein the chromium content is 0.1 to 300 ppm. 5. The composition according to claim 1, which is an aqueous solution. 6. The composition according to any one of claims 1 to 4, which is a dried product. 7. A reagent set for measuring enzyme activity, which comprises the composition according to any one of claims 1 to 6. 8. The method according to any one of claims 1 to 6 for measuring enzyme activity.
Use of the composition according to any of the sections. 9. An immunoassay reagent set comprising an enzyme as a labeling substance, which comprises the composition according to any one of claims 1 to 6. 10. The reagent set according to claim 8, wherein the immunoassay uses alkaline phosphatase as a labeling substance. 11. A method for stabilizing a substrate for a hydrolase, which comprises coexisting with chromium. 12. The stabilization method according to claim 11, wherein the substrate is a substrate for alkaline phosphatase. 13. The stabilizing method according to claim 12, wherein the substrate is 4-methylumbelliferone phosphate or a salt thereof. 14. The stabilizing method according to claim 11, wherein the coexisting amount of chromium is 0.1 to 300 ppm. 15. A method for producing a stabilized hydrolase substrate, which comprises adding chromium. 16. The method according to claim 15, wherein the substrate is a substrate for alkaline phosphatase. 17. The method according to claim 16, wherein the substrate is 4-methylumbelliferone phosphate or a salt thereof. 18. The amount of chromium added is 0.1 to 300 ppm.
The manufacturing method according to any one of claims 15 to 17, wherein: