JPH0970288A - New alkaline phosphatase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a high-purity new alkaline phosphatase having good stability and useful for detection, etc., of ligand in a specimen by culturing a bacterial strain belonging to the genus Bacillus and having a new alkaline phosphatase- producing ability. SOLUTION: This new alkaline phosphatase is obtained by culturing a bacterial strain belonging to the genus Bacillus and having a new alkaline phosphatase-producing ability [e.g. Bacillus badius TE 3592 (FERM BP-5329)] and collecting a product from the cultured products. The alkaline phosphatase catalyzes a reaction hydrolyzing orthophosphoric acid monoester and producing an alcohol and orthophosphoric acid and acts on p-nitrophenylphosphoric acid, 4-methylumbelliferylphosphoric acid, NADP, DL-α-glycerophosphoric acid, phenylphosphoric acid, phosphoethanolamine and glucose-6-phosphoric acid and is activated by Mg<++> or Co<++> and is stable for 30min by treatment at pH7.5 and 60 deg.C and has no sugar chain and has 140000 to 150000 molecular weight (by gel permeation method).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel alkaline phosphatase having high purity and good stability, a method for producing the same, a method for detecting a ligand in a sample using the enzyme as a label, and a reagent therefor.

[0002]

2. Description of the Related Art There are many known methods for detecting biological substances, and when a specific trace amount of a component is specifically measured in the presence of various components, biochemical affinity is required. An analysis method utilizing sex is used. For components such as glucose and uric acid that are present in body fluids at concentrations of the order of 10 ⁻² mole / L or more, detection methods that utilize an enzymatic reaction using the component as a substrate are often used. When trying to measure higher molecular weight components or lower concentration components that do not become, the affinity of each component, for example, antigen-antibody, hormone-hormone receptor, nucleic acid-nucleic acid, etc. with higher affinity should be used. It is generally used. In detection methods utilizing these affinities, it is often necessary to label and detect one affinity component. As one of them, a method using a radioactive substance (RI) as a label is excellent in terms of detection sensitivity and has been used conventionally. However, since facilities and measuring devices that handle special radioactive substances are required, in recent years, one affinity component has been labeled with an enzyme and the enzyme activity of the label bound or not bound depending on the affinity can be measured. A method of quantifying the other affinity component by measuring is used.

The substrate used for detecting the labeling enzyme has been dramatically improved in detection sensitivity by changing from a colorimetric substrate to a fluorescent substrate and further to a luminescent substrate. Generally, a high-purity, high-stability, high turnover, easy-to-label functional group, low Km value for the substrate, low background, and a substrate suitable for detection are used as the conditions for the labeling enzyme. There are some examples. The types of enzymes used are alkaline phosphatase and β
-Galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, peroxidase, β-lactamase, glucoamylase, lysozyme, and the like. Among them, the major advantage of alkaline phosphatase is that the background is lower than other enzymes. And a substrate suitable for detection.

Alcal phosphatase substrates suitable for the detection of antigens, antibodies or nucleic acids include p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate and 4-methylumbelliferyl phosphate. , Dioxetane luminescent substrates (PPD, AMPPD, etc.) have been used conventionally. The amount of the biological substance can be measured by measuring visible light, fluorescence, and luminescence generated by the reaction between these substrates and alkaline phosphatase.

As an alkaline phosphatase having the above-mentioned properties most, the one derived from the calf small intestine is mentioned and is most widely used. Alkaline phosphatase derived from calf small intestine has a specific activity of 3,000 U / mg or more, and since it has a sugar chain, it can be labeled by the periodate method, and is thus considered to be superior to enzymes of other origins. There is.
However, on the other hand, it is also known that the stability is poor, and that the background is generated due to the sugar chain (Besman, M., Coleman, JE, J. Biol. Chem., 26).
0,1190 (1985), JP-A-60-180584). Escherichia coli-derived alkaline phosphatase has good stability and a highly pure sample can be easily obtained, but its specific activity is 60 U / mg.
It is not suitable as a labeling enzyme and is only used as a dephosphorylation enzyme in molecular biology (Reid,
RW, Wilson, IB in "The Enzymes", 3rd Ed.373 (197
1)). In order to improve these enzymes, there has been an attempt to substitute the amino acid of Escherichia coli alkaline phosphatase by site-directed mutagenesis to improve the specific activity (JP-A-4-349881). However, the mutant alkaline phosphatase obtained here only obtained a 3.9-fold increase in specific activity, which is not comparable to that derived from calf small intestine.

There is also an attempt to acquire alkaline phosphatase having a high specific activity from the natural world, and an enzyme (M, M, derived from alkalophilic Bacillus sp.).
Nomoto et al., Agric. Biol. Chem., 52 (7), 1643 (1988)) and Bacillus licheniformis.
Report on Enzymes Derived (Hulett, FM, J.Gen. Microbio
l., 132, 2387 (1986)). However, the former has a specific activity of 1650 U / mg, and it cannot be said that it is comparable to the specific activity of the calf small intestine-derived enzyme. The latter has a specific activity of 21.
Although the enzyme activity was measured at 55 ° C. even though it was 15.9 U / mg, the data expected to be 70% or less of that at practical 37 ° C. has been shown (Hulett, FM et.
al., Biochemistry, 10 (8), 1364 (1971)).

[0007]

[Problems to be Solved by the Invention]
There has been a demand for obtaining alkaline phosphatase from microorganisms, which is highly stable and has a specific activity similar to that of the calf intestine-derived enzyme.

[0008]

The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result,
The present invention has been completed by discovering an alkaline phosphatase having excellent thermostability and high specific activity from bacteria belonging to the genus llus).

That is, the present invention is an alkaline phosphatase having the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes In terms of thermal stability, "stable" means that the residual activity is 80% or more. 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION One embodiment of the present invention is alkaline phosphatase having the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum pH: pH 9-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.34 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
It acts on phosphoethanolamine and glucose-6-phosphate.

Another embodiment of the present invention is alkaline phosphatase having the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum pH: pH 9-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.26 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
It acts on phosphoethanolamine and glucose-6-phosphate.

Another embodiment of the present invention is alkaline phosphatase having the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 70 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum pH: pH 9.5-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.28 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
Acts on phosphoethanolamine, glucose-1-phosphate, glucose-6-phosphate

The present invention also provides a method for producing alkaline phosphatase, which comprises culturing in a medium a strain belonging to the genus Bacillus and having the above-mentioned physicochemical properties and capable of producing alkaline phosphatase, and collecting alkaline phosphatase from the culture. is there.

The present invention is a method for detecting a ligand in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

The present invention also provides (i) a specific binding substance for a ligand directly or indirectly labeled with alkaline phosphatase having the following physicochemical properties or (i)
A reagent for detecting a ligand in a biological sample containing i) a specific binding substance for a ligand, a ligand labeled with alkaline phosphatase having the following physicochemical properties, and (iii) a reagent for measuring alkaline phosphatase. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

Further, the present invention provides (i) a substance having a specific affinity for a ligand that binds an avidin compound or a biotin compound, and (ii) an alkali having the following physicochemical properties that binds a biotin compound or an avidin compound. A reagent for detecting a ligand in a biological sample containing a substance for measuring phosphatase and (iii) alkaline phosphatase. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

The present invention is a method for quantifying a ligand in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

The present invention is a method for sequencing a nucleic acid in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

The enzyme of the present invention may be of any origin such as animals, plants and microorganisms as long as it produces alkaline phosphatase having the above-mentioned physicochemical properties. Preferred is a bacterium belonging to the genus Bacillus capable of producing alkaline phosphatase having the above-mentioned properties, and a suitable example is Bacillus badius.
llus badius) TE3592 (FREM BP-532
9) and Bacillus badius T
E3593 (FERM BP-5330), Bacillus
Bacillus badius TE3597 (FERM
BP-5120). In addition, Bacillus badius TE3592 and Bacillus badius TE
3593 is a strain isolated from soil in Yogo-cho, Ika-gun, Shiga Prefecture, and Bacillus badius TE3597 is a strain isolated from soil in Takefu City, Fukui Prefecture, and its mycological properties are as follows.

(A) Bacillus badius TE3592 (a) Morphology (1) Bacterial form: Short bacillus (2) Cell size: 0.6 × 1.8 to 3.0 μm (3) Cell polymorphism : None (4) Motility: None (5) Presence / absence of spores: The spores were elliptical and observed in the center or end of the cells. No swelling of the spores was observed.

(B) Growth state in each medium (1) Meat broth agar plate medium: Gray yellow colonies are formed by culturing at 30 ° C. for 24 hours. The periphery of the colony has an asymmetric tooth shape (Erose) and a convex shape (Convex). The surface is smooth, glossy and translucent. (2) Broth liquid culture: Growth is normal and it becomes cloudy uniformly. It does not form a sink or fungal ring. (3) Meat broth gelatin stab culture: The growth is normal, and only the upper part grows in a filament (Filiform). Gelatin liquefaction is weak. (4) Litmus milk: No change in color. Milk does not solidify. (5) MacConkey agar: No growth. (6) Phenylethyl alcohol agar medium: It grows badly.

(C) Physiological properties (1) Gram stainability: negative (-) or indeterminate (2) reduction of nitrate :-( 3) denitrification reaction :-( 4) MR test :-( 5) VP test :-( 6) Indole production :-( 7) Hydrogen sulfide production :-( 8) Starch degradation :-( 9) Casein degradation: + (10) Gelatin degradation: + (11) Tyrosine degradation + (12) Degradation of Tween 80 :-( 13) Utilization of citric acid: koser's medium-Christensen's medium- (14) Use of inorganic nitrogen source (carbon source is dl-malic acid) Sodium nitrate-Ammonium sulfate + sodium glutamate + ( 15) Dye formation :-( 16) urease- (17) oxidase + (18) catalase + (19) β-galactosidase- (20) arginine dihydrolar -(21) Lysine carboxylase- (22) Ornithine carboxylase- (23) Tryptophan deaminase- (24) β-glucosidase- (25) Extracellular DNase :-( 26) Range of growth: Growth temperature 10 ° C-20 ° C + 25 ° C + 30 ° C + 37 ° C + 40 ° C + 50 ° C − Growth pH pH 4 − pH 7 + pH 9 + NaCl concentration 2% + 5% + (27) Attitude toward oxygen: Aerobic (28) OF test (Hugh Leifson) Method) :-( does not decompose sugar) (29) Generation of acid and gas from sugar Acid gas L-arabinose-D-xylose-D-glucose-D-mannose-D-fructose-D- Galactose-maltose-sucrose-lactose-trehalose-D-sorbitol - - D-mannitol - - Inositol - - Glycerin - - Starch - - L-rhamnose - - D-melibiose - - D-Amidakurin - -

(30) Utilization of organic compound D-glucose-L-arabinose-D-mannose-D-mannitol-N-acetyl-D-glucosamine-maltose-potassium gluconate + n-capric acid-adipic acid-dl- Malic acid + Phenyl acetate-

(B) Bacillus badius TE3593 (a) Morphology (1) Bacterial form: Short bacillus (2) Cell size: 0.4 × 1.3 to 2.8 μm (3) Cell polymorphism : None (4) Motility: None (5) Presence / absence of spores: The spores were elliptical and observed in the center or end of the cells. No swelling of the spores was observed.

(B) Growth state in each medium (1) Meat broth agar plate medium: Gray yellow colonies are formed by culturing at 30 ° C. for 24 hours. The periphery of the colony has an asymmetric tooth shape (Erose) and a convex shape (Convex). The surface is smooth, glossy and translucent. (2) Broth liquid culture: Growth is normal and it becomes cloudy uniformly. It does not form a sink or fungal ring. (3) Meat broth gelatin stab culture: The growth is normal, and only the upper part grows in a filament (Filiform). Gelatin liquefaction is weak. (4) Litmus milk: No change in color. Milk does not solidify. (5) MacConkey agar: No growth. (6) Phenylethyl alcohol agar medium: It grows badly.

(C) Physiological properties (1) Gram stainability: negative (-) or indeterminate (2) reduction of nitrate :-( 3) denitrification reaction :-( 4) MR test :-( 5) VP test :-( 6) Indole production :-( 7) Hydrogen sulfide production :-( 8) Starch degradation :-( 9) Casein degradation: + (10) Gelatin degradation: + (11) Tyrosine degradation + (12) Degradation of Tween 80 :-( 13) Utilization of citric acid: koser's medium-Christensen's medium- (14) Use of inorganic nitrogen source (carbon source is dl-malic acid) Sodium nitrate-Ammonium sulfate + sodium glutamate + ( 15) Dye formation :-( 16) urease- (17) oxidase + (18) catalase + (19) β-galactosidase- (20) arginine dihydrolar -(21) Lysine carboxylase- (22) Ornithine carboxylase- (23) Tryptophan deaminase- (24) β-glucosidase- (25) Extracellular DNase: + (26) Range of growth: Growth temperature 10 ° C-20 ° C + 25 ° C. + 30 ° C. + 37 ° C. + 40 ° C. + 50 ° C.− Growth pH pH4−pH7 + pH9 + NaCl concentration 2% + 5% +

(27) Attitude toward oxygen: aerobic (28) OF test (Hugh Leifson method) :-( without decomposing sugar) (29) Generation of acid and gas from sugar Acid gas L-arabinose --- D-xylose-D-glucose-D-mannose-D-fructose-D-galactose-maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol-inositol- -Glycerin-starch-L-rhamnose-D-melibiose-D-amidacrine-

(30) Utilization of organic compound D-glucose-L-arabinose-D-mannose-D-mannitol-N-acetyl-D-glucosamine-maltose-potassium gluconate + n-capric acid-adipic acid-dl- Malic acid + citric acid-phenyl acetate-

(C) Bacillus badius TE3597 (a) Morphology (1) Bacterial form: Short bacillus (2) Cell size: 1.0 × 3.3 to 4.0 μm (3) Cell polymorphism : None (4) Motility: None (5) Presence / absence of spores: The spores were elliptical and observed in the center or end of the cells. No swelling of the spores was observed.

(B) Growth state in each medium (1) Meat broth agar plate medium: Gray yellow colonies are formed by culturing at 30 ° C. for 24 hours. The periphery of the colony has an asymmetric tooth shape (Erose) and a convex shape (Convex). The surface is smooth, glossy and translucent. (2) Broth liquid culture: Growth is normal and it becomes cloudy uniformly. It does not form a sink or fungal ring. (3) Meat broth gelatin stab culture: The growth is normal, and only the upper part grows in a filament (Filiform). Gelatin liquefaction is weak. (4) Litmus milk: No change in color. Milk does not solidify. (5) MacConkey agar: No growth. (6) Phenylethyl alcohol agar medium: It grows badly.

(C) Physiological properties (1) Gram stainability: positive (+) (2) reduction of nitrate: negative (-) (3) denitrification reaction :-( 4) MR test :-( 5) VP Test :-( 6) Indole production :-( 7) Hydrogen sulfide production :-( 8) Starch degradation :-( 9) Casein degradation: + (10) Gelatin degradation: + (11) Tyrosine degradation + (12) Degradation of Tween 80 :-( 13) Utilization of citric acid: Koser's medium-Christensen's medium- (14) Inorganic nitrogen source (carbon source is dl-malic acid) Sodium nitrate-Ammonium sulfate + Sodium glutamate + (15) Dye formation :-( 16) urease- (17) oxidase + (18) catalase + (19) β-galactosidase- (20) arginine dihydrolase (21) Lysine carboxylase- (22) Ornithine carboxylase- (23) Tryptophan deaminase- (24) β-glucosidase- (25) Extracellular DNase :-( 26) Growth range: Growth temperature 10 ° C-20 ° C +25 ℃ +30 ℃ +37 ℃ +40 ℃ +50 ℃ -Growth pH pH4-pH7 + pH9 + NaCl concentration 2% + 5% +

(27) Attitude toward oxygen: aerobic (28) OF test (Hugh Leifson method) :-( does not decompose sugar) (29) Generation of acid and gas from sugar Acid gas L-arabinose --- D-xylose-D-glucose-D-mannose-D-fructose-D-galactose-maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol-inositol- -Glycerin-starch-L-rhamnose-D-melibiose-D-amidacrine-

(30) Utilization of organic compound D-glucose-L-arabinose-D-mannose-D-mannitol-N-acetyl-D-glucosamine + maltose-potassium gluconate-n-capric acid-adipic acid-dl- Malic acid + citric acid-phenyl acetate +

The experimental method for identifying the above-mentioned mycological properties is mainly described in Takeji Hasegawa, revised edition "Classification and Identification of Microorganisms".
Performed by the Society Press Center (1985). As a criterion for classification and identification, “Burgess Manual of Systematic Bacteriology” (1984)
Year).

Referring to the above-mentioned literature and mycological properties, the production of extracellular DNase differs, but all of them are aerobic rods with indeterminate Gram stainability and spore-forming ability, so that they belong to the genus Bacillus. Considered to belong to. In the genus Bacillus, the spores are oval and do not swell,
Considering that it does not generate acid from D-glucose and decomposes gelatin but does not decompose starch, it is considered that all of them belong to Bacillus badius, and Bacillus badius (Bacillus badius) respectively.
ius) TE3592, Bacillus b
adius) TE3593 and Bacillus badius (Baci
llus badius) TE3597. These bacteria are deposited under the microorganism deposit numbers FERM BP-5329, FERM BP-5330 and FERM BP-5120, respectively.

In producing the enzyme of the present invention, the above alkaline phosphatase-producing bacterium is cultured in a nutrient medium,
Alkaline phosphatase is harvested from the culture. As a medium used for culturing the alkaline phosphatase-producing bacterium, either a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of a carbon source, a nitrogen source, an inorganic substance and other necessary nutrients that can be assimilated by the strain used. As the carbon source, for example, glucose, glycerol and the like are used. Examples of the nitrogen source include nitrogen-containing natural products such as peptones, meat extracts, and yeast extracts, and inorganic nitrogen-containing compounds such as ammonium chloride and ammonium citrate. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. Further, it is desirable to lower the phosphate concentration in order to induce the production of alkaline phosphatase.

The culture medium is usually shake culture or aeration-agitation culture. The culture temperature is 20 to 40 ° C., preferably 25.
~ 37 ° C, culture pH 5-11, preferably 6 ~
It is better to control to 10. It can be carried out under conditions other than these provided that the strain to be used grows. Culture period is usually 1 to
It grows up in 7 days, and alkaline phosphatase is produced and accumulated inside and outside the cells.

As the method for purifying the enzyme of the present invention, a generally used purification method may be used. For example, the medium after removing the bacterial cells,
Salting out methods such as ammonium sulfate and sodium sulfate, metal coagulation methods such as magnesium chloride and calcium chloride, coagulation methods such as protamine and polyethyleneimine, and further DEAE (diethylaminoethyl) -cellulose, CM (carboxymethyl)-
It can be purified by an ion exchange chromatography method such as Sepharose. The crude enzyme solution and the purified enzyme solution obtained by these methods can be powdered by, for example, spray drying or freeze drying. Furthermore, it can be immobilized on a suitable carrier and used as an immobilized enzyme.

Next, a method for measuring the activity of alkaline phosphatase of the present invention will be described. First, the following reaction mixture is prepared in a cuvette and preheated at 37 ° C. for about 5 minutes. 3.00 ml 1 M diethanolamine buffer containing 0.1 mM CoCl ₂ and 0.5 mM MgCl ₂ , pH 9.8 0.05 ml 0.67 M p-nitrophenyl phosphate solution Next, 0.05 ml of enzyme solution was added and gently. After mixing,
405nm with a spectrophotometer controlled at 37 ° C against water
Record the change in absorbance for 3 to 4 minutes, and determine the change in absorbance per minute from the initial linear portion. In the blind test, instead of the enzyme solution, the enzyme diluent (0.05 mM Co
50 mM with Cl ₂ and 0.05 mM MgCl ₂
Tris-hydrochloric acid buffer, pH 7.5) was added, and the same operation as above was performed to determine the absorbance per minute. The amount of the enzyme that produces 1 micromol of p-nitrophenol per minute under the above conditions is defined as 1 unit (U).

The method for detecting a ligand in a sample of the present invention utilizes an affinity reaction between the ligand in the sample and a substance having a specific affinity for the ligand, and the binding by the reaction. It is a method of measuring the alkaline phosphatase activity bound to the substance, or the unbound alkaline phosphatase activity.

In the present invention, examples of the ligand in the sample include antigens, antibodies, hormones, hormone receptors and nucleic acids. The affinity reaction between the ligand and a substance having a specific affinity for the ligand includes an antigen-antibody reaction, a hormone-hormone receptor reaction, a nucleic acid hybridization reaction and the like.

The alkaline phosphatase used in the present invention may be of any origin as long as it has the above-mentioned physicochemical properties. Suitable examples include alkaline phosphatase of the genus Bacillus. For example Bacillus Badius (Bac
illus badius) TE3492, Bacillus badius (B
acillus badius) TE3493, Bacillus badius
The alkaline phosphatase of (Bacillus badius) TE3497 is exemplified.

Bacillus badius
The physicochemical properties of TE3492 alkaline phosphatase are as follows. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glucerophosphate, β-glycerophosphate, phenylphosphate, phosphoethanolamine, glucose-6-phosphate To work. 3. Km value: 0.34 mM (relative to p-nitrophenyl phosphate) 4. Optimum pH: pH 9-10 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum temperature: 60 ° C or higher 7. Activating and stabilizing agents: Mg ²⁺ and Co ²⁺ 8. Specific activity: at least 2,300 U / mg 9. It has no sugar chains. 10. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 11. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

Bacillus badius
The physicochemical properties of TE3493 alkaline phosphatase are as follows. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glucerophosphate, β-glycerophosphate, phenylphosphate, phosphoethanolamine, glucose-6-phosphate To work. 3. Km value: 0.26 mM (relative to p-nitrophenyl phosphate) 4. Optimum pH: pH 9-10 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum temperature: 60 ° C or higher 7. Activating and stabilizing agents: Mg ²⁺ and Co ²⁺ 8. Specific activity: at least 2,300 U / mg 9. It has no sugar chains. 10. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 11. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

Bacillus badius
The physicochemical properties of TE3497 alkaline phosphatase are as follows. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glucerophosphate, β-glycerophosphate, phenylphosphate, phosphoethanolamine, glucose-6-phosphate To work. 3. Km value: 0.28 mM (relative to p-nitrophenyl phosphate) 4. Optimal pH: pH 9.5-10 5. Stable pH: pH 6 to 11 (25 ° C, 16 hours) 6. Optimum temperature: 60 ° C or higher 7. Activating and stabilizing agents: Mg ²⁺ and Co ²⁺ 8. Specific activity: at least 2,300 U / mg 9. It has no sugar chains. 10. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 11. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)

In the present invention, examples of the substance labeled with alkaline phosphatase include high molecular substances such as proteins and nucleic acids as the antigen. It is also possible to use a peptide in which the epitope site of an antigen that has recently been used is designed. As the antibody, those usually used, for example, a polyclonal antibody obtained by immunizing goat, rabbit, guinea pig and the like, a monoclonal antibody obtained from hybridoma such as mouse ascites, and an antigen obtained by further treating these antibodies with protease A binding active fragment (Fab ′) or the like can be used. It is also possible to use a protein having an antigen-binding activity such as an Fv antibody and a single chain Fv antibody obtained by a gene recombination technique.

In the present invention, the alkaline phosphatase is preferably bound to either the ligand or a substance having a specific affinity for the ligand.

As a method for labeling the above-mentioned antigen or antibody with alkaline phosphatase, the glutaraldehyde method, maleimide method, carbidiimide method, pyridine-disulfide method or the like can be used, but the maleimide method is preferred. It is a method that does not reduce the activity of antigens, antibodies and enzymes as described above.

As the labeling enzyme to be introduced into one molecule of antibody or antigen, it is usually desirable to use an enzyme-labeled product in which one or more molecules, preferably two or more molecules, are bound.

As a specific example of the present invention, a substance in a sample having a specific affinity for a ligand such as an antigen or an antibody bound to alkaline phosphatase, such as an antibody or an antigen, is reacted to produce a reaction. After separating the reaction product from the unreacted product, the alkaline phosphatase activity bound to the reaction product or the alkaline phosphatase activity of the unreacted product is measured.

The immunological assay method using alicariphosphatase as a labeling substance can be used in the competitive method or the non-competitive heterogeneous method in the case of measuring both antibodies and antigens. In any method, not only the primary antibody but also the method using the secondary antibody can be labeled with alkaline phosphatase. Instead of the secondary antibody, Fc receptors such as protein A and protein G can be labeled with alkaline phosphatase and used.

Another specific example of the method of the present invention is a method for detecting a ligand in which a ligand or a substance having a specific affinity for the ligand is bound to a solid phase. As the solid phase, a conventionally known one may be used,
Examples thereof include polystyrene beads.

As another specific example, an avidin compound or a biotin compound binds to a substance having a specific affinity for a ligand, for example, an antibody or an antigen, and an alkaline phosphatase binds to the biotin compound or avidin. A substance that binds to and has a specific affinity for a ligand, such as an antigen or antibody and a ligand,
For example, a method for detecting a ligand in a sample is performed by performing an avidin compound-biotin compound binding reaction simultaneously with or after an affinity reaction with an antibody or an antigen, and measuring the alkaline phosphatase activity bound by the reaction or the residual alkaline phosphatase activity. is there. Here, the avidin compound is a glycoprotein that strongly binds to a biotin compound, and examples thereof include avidin and streptavidin. A biotin compound is a vitamin B complex 1
One example is biotin.

The binding constants of avidin compound and biotin compound, especially avidin and biotin, are very high at a level of 10 ¹⁵ M ⁻¹ , avidin compound and especially avidin have four biotin binding sites, The introduction has a great merit in the avidin compound-biotin compound system because, for example, the loss of the activity of the antibody or the like is small.

For example, in the solid phase sandwich immunoassay, the antibody immobilized on the solid phase is reacted with the sample, and further reacted with the antibody bound with the biotin compound, and then the biotin compound in the reaction product or unreacted product is formed. The biotin compound in the product is detected by an avidin compound labeled with alkaline phosphatase. Avidin and its related substance streptavidin are proteins with a molecular weight of about 50,000,
These can be bound to alkaline phosphatase using a glutaraldehyde method, a maleimide method, a carbodiimide method, a pyridine-disulfide method, or the like.

On the other hand, when an antibody binding to an avidin compound is used in place of the biotin compound, the antigen-antibody reaction is followed by detection with an avidin compound and a biotin compound labeled with alkaline phosphatase. Reagents for biotinylating alkaline phosphatase are commercially available, for example, biotin-N-hydroxy-succinimide ester,
Examples thereof include biotin-N-hydroxy-sulfosuccinimide ester, biotinoyl-ε-aminocaproic acid-N-hydroxy-succinimide ester and the like.

The ligand detection reagent in the sample of the present invention is
It contains a specific binding substance for an alkaline phosphatase-labeled ligand having the following physicochemical properties, or an alkaline phosphatase-labeled ligand having the following physicochemical properties and an alkaline phosphatase measurement reagent. Examples of the reagent for measuring alkaline phosphatase include chromogenic substrates, fluorescent substrates, luminescent substrates and the like.

As the color-developing substrate, p-nitrophenyl phosphate, 1-naphtholphthalein monophosphate (Japanese Patent Publication No.
13958), 5-bromo-4-chloro-3-.
Examples include indolyl phosphoric acid and a combination thereof with nitroblue tetrazolium. Further, as a fluorescent substrate, 4-methylumbelliferyl phosphate, phenalenone-6-phosphate and its analogs, benzphenalene-6-phosphate and its analogs (JP-A-62-190)
191). Further, examples of the luminescent substrate include 1,2-dioxetane compounds such as PPD and AMPPD or derivatives thereof and mixtures of these compounds and enhancers such as surfactants, fluorescent substances or proteins.

The concentration of these substrates of the present invention is 0.01-.
200 mmol / l, preferably ~ 50 mmol / l. The enzymatic reaction of the present invention is usually carried out at pH 7 to 11, but it is desirable to carry out the enzymatic reaction at pH 8 to 11 in consideration of the optimum pH. The buffer to be used includes Tris-hydrochloric acid buffer, phosphate buffer, diethanolamine hydrochloric acid buffer, triethanolamine hydrochloric acid buffer, bicarbonate buffer,
Examples thereof include N-methyl-D-glucamine hydrochloric acid buffer solution, barbital buffer solution, glycine sodium hydroxide buffer solution, 2-amino-2-methylpropanol hydrochloric acid buffer solution, and amino alcohol buffer solution. The concentration of these buffers is 5-200 mmol / l, preferably 20
˜50 mmol / l.

The activity of the enzyme bound to the reaction product or the enzyme bound to the unreacted product can be measured by measuring the alkaline phosphatase activity by the rate method. It can also be carried out by detecting the product after stopping the reaction. Examples of the stopping agent that can be used include alkaline solutions, enzyme inhibitors, chelating agents such as EDTA, and inorganic phosphoric acid. Further, after the reaction is stopped, the reaction can be carried out under strong alkaline conditions to further increase the sensitivity for substrates such as p-nitrophenylphosphoric acid and dioxetane compounds (JP-A-2-273199).

It is desirable to add a metal salt to the buffer used in the present invention in order to prevent the inactivation of alkaline phosphatase. Examples of metal salts that can be used include magnesium salts, cobalt salts, zinc salts, manganese salts, and calcium salts, with magnesium salts and cobalt salts being preferred. The preferred concentration of magnesium salt added is 0.0
It is 5 mmol / l to 10 mmol / l, and magnesium complex compounds such as magnesium acetate, magnesium chloride, magnesium citrate, magnesium sulfate, and magnesium ethylenediaminetetraacetate can be used. The preferable addition concentration of cobalt salt is 0.02 to 5 mmol /
1, and a cobalt complex compound such as cobalt acetate, cobalt chloride, cobalt citrate, cobalt sulfate, and ethylenediaminetetraacetate cobalt can be used. It is desirable, but not essential, to use a magnesium salt and a cobalt salt together.

Any detergent that does not significantly inhibit alkaline phosphatase activity can be used as the detergent useful in the practice of the present invention. Generally useful surfactants are nonionic surfactants, but amphoteric and ionic surfactants can also be used. Further, in the present invention, an organic solvent miscible with water may be used in combination. Examples thereof are methanol, ethanol, propanol, N, N-dimethylformamide, dimethylsulfoxide, acetonitrile, hexamethylenephosphoamide and the like.

Further, other compounds may be added to the reagent of the present invention in order to smoothly carry out the enzymatic reaction or to maintain the activity of its constituent components. Examples of such compounds include stabilizers and excipients. It is also effective to add an inactivated enzyme of alkaline phosphatase to reduce the background and non-specific reaction.

The present invention is not limited to the above-mentioned solid-phase sandwich immunoassay method, but also uses hormone-receptor affinity to measure hormones and their receptors, nucleic acid-nucleic acid affinity,
For example, it can be used for measurement using a DNA-DNA reaction or a DNA-RNA reaction.

As an example of detecting the specific nucleic acid of the present invention, the nucleic acid (D
(NA or RNA), and then a detection oligonucleotide labeled with alkaline phosphatase to allow nucleic acid hybridization between the nucleic acid bound to the capture oligonucleotide and the labeled detection oligonucleotide to perform unreacted detection. After separating the oligonucleotides, there is a method of detecting the nucleic acid in the sample by measuring the alkaline phosphatase activity of the conjugate hybridized with the nucleic acid.

The nucleic acid in the sample is DNA or RN.
A single-stranded or double-stranded nucleic acid such as A is exemplified. Examples of the sample include body fluids such as serum, urine and lymph, and materials such as various tissues.

Alkaline phosphatase and DNA of the present invention
Alternatively, the preparation of a complex with RNA can be performed by, for example, M.
The method described in C. Kurz, Nucleic Acids Res., 12 (8), 3435 (1984) can be used. For example, alkaline phosphatase and polyethyleneimine are crosslinked to prepare a conjugate, and then a DNA or RNA oligonucleotide is crosslinked with glutaraldehyde to obtain a labeled nucleic acid. In the case of synthesizing an oligonucleotide, reagents for directly introducing an amino group or a thiol group into the 5'end or an arbitrary chain via a spacer arm are commercially available. These reagents and alkaline phosphatase are glutaraldehyde method. Alkaline phosphatase can also be introduced into an oligonucleotide by binding by a maleimide method, a carbodiimide method, a succinimide ester method, or a pyridine-disulfide method.

DNA is exemplified as a sample, and when the extension reaction is performed using DNA as a template, biotin is incorporated into the DNA fragment by using a biotinylated primer or a biotinylated terminator. Next, the fragment is developed by electrophoresis, and then reacted with avidinated alkaline phosphatase or avidin, followed by biotinylated alkaline phosphatase to detect the fragment.

Furthermore, in the present invention, instead of the method of taking out nucleic acid from a sample and performing nucleic acid hybridization,
In situ for nucleic acid hybridization in cells
It can also be used for hybridization.

Another specific example of the present invention is a method for sequencing a nucleic acid in a sample, which comprises using alkaline phosphatase having the above-mentioned physicochemical properties as a label. A known method such as the dideoxy termination method or the Maxam-Gilbert method can be selected for the sequence determination. For example, when the dideoxy termination method is used, the following method is exemplified. First, a biotinylated primer is hybridized with a nucleic acid to be sequenced (single-stranded or alkali-modified double-stranded), and one dideoxyribonucleotide (eg, ddATP) is added to four deoxyribonucleotides (dNTPs) and a nucleic acid. It is added together with a synthase (for example, Klenow enzyme, T7 polymerase, etc.), and an extension reaction and a termination reaction are simultaneously performed. The other three dideoxyribonucleotides (d
dCTP, ddGTP, and ddTTP) are also reacted in the same manner, and the resulting four reaction solutions are subjected to electrophoresis on a sequence gel, respectively, and then reacted with avidinylated alkaline phosphatase or avidin, followed by biotinylated alkaline phosphatase. Then, various extension fragments are detected using a chromogenic substrate or the like, and the respective lanes are compared and compared to determine the nucleic acid sequence. The nucleic acid sequencing reagent using the alkaline phosphatase-labeled nucleic acid includes a DNA or RNA that directly or indirectly binds the enzyme and an alkaline phosphatase assay reagent.

[0071]

INDUSTRIAL APPLICABILITY According to the present invention, a novel alkaline phosphatase having high purity and good stability can be obtained. Further, the binding assay reagent using alkaline phosphatase specified in the present invention and the method for detecting a ligand in a sample using the same have high sensitivity and excellent long-term storage stability. Moreover, in detecting the target substance, good results with little background can be provided.

[0072]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 3.0% glycerol, 1.0% polypeptone, 0.1
% Yeast extract, 0.02% magnesium sulfate, 0.00
100 ml of a medium containing 2% monopotassium phosphate and 0.3% sodium chloride was transferred to a 500 ml Sakaguchi flask, and 1
It was autoclaved at 21 ° C for 15 minutes. As an inoculum, Bacillus badius TE3592 (FERM B
P-5329) was inoculated with one platinum loop and cultured at 30 ° C. for 20 hours to obtain a seed culture solution. Next, 6 L of the same medium was transferred to a 10 L jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and 100 ml of the seed culture solution was transferred to 30
The culture was performed at 0 rpm, an aeration rate of 2 l / min, and 30 ° C for 20 hours. The obtained culture solution was centrifuged to obtain a supernatant. This solution was purified to a specific activity of 2,300 U / mg by ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl sepharose chromatography, and gel filtration with Sephadex G-200.

The obtained alkaline phosphatase had the following characteristics. 1. The following reaction was catalyzed. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid

2. Substrate specificity

[Table 1]

3. Km value The Km value for p-nitrophenol was 0.34 mM. 4. Optimal pH 0.97M diethanolamine buffer (pH 8.0-1
The enzyme activity in 1.0) was measured. The result is as shown in FIG. 1, and the optimum pH was 9 to 10. 5. Stable pH Glycine Hydrochloric acid buffer (pH 2-3), Acetate buffer (pH
3-6), K-phosphate buffer (pH 6-8), Tris-hydrochloric acid buffer (pH 8-9), glycine NaOH buffer (p
It was stored in H9 to 10) at 25 ° C for 16 hours, and its residual activity was measured. As a result, as shown in FIG. 2, the stable pH was pH 6-9. 6. Optimum temperature The enzyme activity at each temperature was measured. The results are shown in FIG. 3, and the optimum temperature was 60 ° C. or higher. 7. Thermostability The enzyme of the present invention was treated with 1.0 mM MgCl ₂ and 0.1 m
50 mM Tris-HCl buffer containing M CoCl ₂ (pH
After incubating in 7.5) for 30 minutes, the residual enzyme activity was measured. The result is as shown in FIG.
Was stable until. 8. Activation and Stabilizers: Mg ⁺⁺ and Co ⁺⁺ were mandatory. 9. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 10. Sugar content: No sugar was detected.

Example 2 3.0% glycerol, 1.0% polypeptone, 0.1
% Yeast extract, 0.02% magnesium sulfate, 0.00
100 ml of a medium containing 2% monopotassium phosphate and 0.3% sodium chloride was transferred to a 500 ml Sakaguchi flask, and 1
It was autoclaved at 21 ° C for 15 minutes. As an inoculum, Bacillus badius TE3593 (FERM B
P-5330) was inoculated with one platinum loop and cultured at 30 ° C. for 20 hours to obtain a seed culture solution. Next, 6 L of the same medium was transferred to a 10 L jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and 100 ml of the seed culture solution was transferred to 30
The culture was performed at 0 rpm, an aeration rate of 2 l / min, and 30 ° C for 20 hours. The obtained culture solution was centrifuged to obtain a supernatant. This solution was purified to a specific activity of 2,790 U / mg by ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl sepharose chromatography, and gel filtration with Sephadex G-200.

The obtained alkaline phosphatase had the following characteristics. 1. The following reaction was catalyzed. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid

2. Substrate specificity

[Table 2]

3. Km value The Km value for p-nitrophenol was 0.26 mM. 4. Optimal pH 0.97M diethanolamine buffer (pH 8.0-1
The enzyme activity in 1.0) was measured. The results are shown in FIG. 5, and the optimum pH was 9-10. 5. Stable pH Glycine Hydrochloric acid buffer (pH 2-3), Acetate buffer (pH
3-6), K-phosphate buffer (pH 6-8), Tris-hydrochloric acid buffer (pH 8-9), glycine NaOH buffer (p
It was stored in H9-10) at 25 ° C for 16 hours, and its residual activity was measured. The results are shown in FIG. 6, and the stable pH was pH 6-9. 6. Optimum temperature The enzyme activity at each temperature was measured. The results are shown in FIG. 7, and the optimum temperature was 60 ° C. or higher. 7. Thermostability The enzyme of the present invention was treated with 1.0 mM MgCl ₂ and 0.1 m
50 mM Tris-HCl buffer containing M CoCl ₂ (pH
After incubating in 7.5) for 30 minutes, the residual enzyme activity was measured. The result is shown in FIG.
Was stable until. 8. Activating and stabilizing agents Mg ⁺⁺ and Co ⁺⁺ were essential. 9. Molecular weight 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 10. Sugar content No sugar was detected.

Example 3 3.0% glycerol, 1.0% polypeptone, 0.1
% Yeast extract, 0.02% magnesium sulfate, 0.00
100 ml of a medium containing 2% monopotassium phosphate and 0.3% sodium chloride was transferred to a 500 ml Sakaguchi flask, and 1
It was autoclaved at 21 ° C for 15 minutes. As an inoculum, Bacillus badius TE3597 (FERM B
P-5120) was inoculated with one platinum loop and cultured at 30 ° C. for 20 hours to obtain a seed culture solution. Next, 6 L of the same medium was transferred to a 10 L jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and 100 ml of the seed culture solution was transferred to 30
The culture was performed at 0 rpm, an aeration rate of 2 l / min, and 30 ° C for 20 hours. The obtained culture solution was centrifuged to obtain a supernatant. This solution was purified to a specific activity of 2,300 U / mg by ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl sepharose chromatography, and gel filtration with Sephadex G-200.

The obtained alkaline phosphatase had the following characteristics. 1. The following reaction was catalyzed. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid

2. Substrate specificity

[Table 3]

3. Km value The Km value for p-nitrophenol was 0.28 mM. 4. Optimal pH 0.97M diethanolamine buffer (pH 8.0-1
The enzyme activity in 1.0) was measured. The results are shown in FIG. 9, and the optimum pH was 9.5-10. 5. Stable pH Glycine Hydrochloric acid buffer (pH 2-3), Acetate buffer (pH
3-6), K-phosphate buffer (pH 6-8), Tris-hydrochloric acid buffer (pH 8-9), glycine NaOH buffer (p
It was stored in H9-10) at 25 ° C for 16 hours, and its residual activity was measured. The results are shown in FIG. 10, and the stable pH was pH 6-11. 6. Optimum temperature The enzyme activity at each temperature was measured. The result is shown in Figure 11.
And the optimum temperature was 60 ° C or higher. 7. Thermostability The enzyme of the present invention was treated with 1.0 mM MgCl ₂ and 0.1 m
50 mM Tris-HCl buffer containing M CoCl ₂ (pH
After incubating in 7.5) for 30 minutes, the residual enzyme activity was measured. The result is shown in FIG.
It was stable up to ℃. 8. Activating and stabilizing agents Mg ⁺⁺ and Co ⁺⁺ were essential. 9. Molecular weight 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 10. Sugar content No sugar was detected.

Comparative Example 1 The properties of the enzyme of the present invention and the conventionally known enzyme are compared in the following table.

[0085]

[Table 4]

Example 4 (1) Alkaline phosphatase labeled goat anti-human CRPI
Preparation of gG 50m containing 5mg of alkaline phosphatase of Example 3
25% in 2.5 ml M phosphate buffer, pH 7.2 (containing 1 mM MgCl ₂ and 0.1 mM CoCl ₂ ).
Add 35 μl of glutaraldehyde solution and add 50 at 25 ° C.
Incubated for minutes. Next, 0.5 ml of 50 mM phosphate buffer, pH 7.2 containing 2.5 mg of goat anti-human CRP IgG fraction (manufactured by Japan Biotest Institute) was added, and the mixture was further incubated at 25 ° C. for 75 minutes. Then 2
After adding 150 μl of M Tris / HCl, pH 8.7, the mixture was stirred at 4 ° C. for 30 minutes, 150 μl of an aqueous solution containing 150 mg of NaBH ₄ was added, and then incubated at 4 ° C. for 15 hours. The resulting mixture is SuperdexTM
High performance liquid chromatography using 200 (Pharmacia) (0.1M NaCl, 1 mM as eluent)
MgCl ₂ , 0.1 mM CoCl ₂ , 0.1% Na
50 mM Tris / HCl containing N ₃ (pH 8.0 was used) to obtain the first peak as an enzyme-labeled antibody.

(2) Calibration curve of human CRP Human polystyrene CRP was coated on one polystyrene bead coated with goat anti-human CRP IgG fraction (manufactured by Japan Biotest Institute).
1 ml of 0 to 1000 ng / ml was added and incubated at 30 ° C. for 1 hour. Next, the solid phase was washed three times with PBS, 1 ml of enzyme-labeled antibody diluted to 1 U / ml per alkaline phosphatase activity was added, and the mixture was incubated at 30 ° C. for 1 hour. After washing 3 times with PBS, 11 mM
p-Nitrophenyl phosphate, 1M diethanolamine buffer containing 5 mM MgCl ₂ , pH 9.8 was added and 3
React for 30 minutes at 7 ° C, 2 ml of 0.5N NaOH
Was added to stop the reaction, the absorbance at 405 nm was measured, and a calibration curve was prepared (FIG. 13).

Comparative Example 2 Calf Intestinal Derived Alkaline Phosphatase (CIAP) 5 m
50 mM phosphate buffer (pH 7.2) containing 2.5 g
150 μl of 25% glutaraldehyde solution was added to ml and the same procedure as in Example 1 was performed to obtain an enzyme-labeled antibody. Using the above enzyme-labeled antibody, a calibration curve for human CRP was prepared in the same manner as in Example 1 (Fig. 13). The ratio (S / N ratio) of specific color development (10 ng / ml human CRP) and the absorbance of the blank (0 ng / ml human CRP) was CI vs. alkaline phosphatase 12.5 of the present invention.
AP was 5.13, and non-specific adsorption was smaller with the alkaline phosphatase of the present invention.

Example 5 (1) Alkaline phosphatase labeled sheep anti-human CRP
Preparation of Fab ′ 0.1 M phosphate buffer containing 5 mg of sheep anti-human CRP F (ab ′) ₂ (binding site), pH
6.0 0.1 M phosphate buffer containing 0.1 M 2-mercaptoethylamine and 10 mM EDTA in 1 ml,
100 ml of pH 6.0 was added and incubated at 37 ° C. for 90 minutes. The mixture was desalted with 0.1 M phosphate buffer containing 5 mM EDTA, pH 6.0, and then 0.5 m
and concentrated to 1. On the other hand, 30 mM triethanolamine buffer containing 2.5 mg of alkaline phosphatase of Example 3, pH 7.6 (1 mM MgCl ₂ , 0.1 mM).
To 500 μl of CoCl ₂ ) was added 10 μl of dimethylformamide containing 0.1 mg N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, and the mixture was incubated at 30 ° C. for 30 minutes. The obtained liquid was 1 mM MgCl ₂ , 0.1 mM Co.
0.1 M Tris-HCl buffer containing Cl ₂ , pH 7.0
It was desalted with and concentrated to 0.5 ml. Maleimidated alkaline phosphatase was added to the prepared sheep anti-human CRP Fab ′, and the mixture was incubated at 4 ° C. for 20 hours. Then, 50 μl of 10 mM 2-mercaptoethylamine was added, and the mixture was incubated at 25 ° C. for 20 minutes. The obtained mixture was purified by Superdex ™ 200, and the first peak was obtained as an enzyme-labeled antibody.

(2) Calibration curve of human CRP To one polystyrene bead coated with goat anti-human CRP IgG fraction (manufactured by Japan Biotest Laboratory Co., Ltd.), 1 ml of human CRP of 0 to 1000 ng / ml was added, and
Incubated at 0 ° C for 1 hour. Next, the solid phase is 3 times with PBS
After washing twice, 1 U / m per alkaline phosphatase activity
1 ml of the enzyme-labeled antibody diluted to 1 was added, and the mixture was incubated at 30 ° C for 1 hour. After washing 3 times with PBS,
mM p-nitrophenyl phosphate, 5 mM MgCl ₂
1M diethanolamine buffer containing pH 9.8 was added and reacted at 37 ° C. for 30 minutes, 0.5N NaOH
The reaction was stopped by adding 2 ml, and the absorbance at 405 nm was measured to prepare a calibration curve (FIG. 14).

Comparative Example 3 30 mM triethanolamine buffer containing 2.5 mg of CIAP, pH 7.6 (1 mM MgCl ₂ , 0.1
500 μl containing mM ZnCl ₂ , 3M NaCl)
The same operation as in Example 5 was performed to obtain an enzyme-labeled antibody. Using the above enzyme-labeled antibody, a calibration curve for human CRP was prepared in the same manner as in Example 4 (Fig. 1).
4). Ratio of specific color development (10 ng / ml human CRP) to blank absorbance (0 ng / ml human CRP) (S /
(N ratio) was 54.8 for the alkaline phosphatase of the present invention of 55.2, and the alkaline phosphatase of the present invention had smaller nonspecific adsorption.

Example 6 (1) Preparation of streptavidin-labeled alkaline phosphatase 0.1 M phosphate buffer containing 4 mg of streptavidin, pH 7.5 600 μl of 0.1 mg S-acetylmercaptosuccinic hydride 10 μl of dimethylformamide Was added and incubated at 30 ° C. for 30 minutes. Then 0.1M EDTA, pH
7.0 20 μl, 0.1 M Tris / HCl, pH
7.0 120 μl, 1M Hydroxylamine hydrochloride 1
Add 20 μl and incubate at 30 ° C. for 5 minutes,
600μ after desalting with 0.1M phosphate buffer, pH 6.0
and concentrated to 1. To 100 μl of the maleimidated alkaline phosphatase solution prepared in Example 5, 100 μl of the prepared mercaptosuccinylated streptavidin was added, and 4
After incubating at 20 ° C. for 20 hours, the resulting mixture was Su
Purified with perdex ™ 200, the first peak was enzyme-labeled streptavidin.

Comparative Example 4 With respect to CIAP, an enzyme-labeled streptavidin was prepared in the same manner as in Example 6. According to a conventional method, 0-5 ng of biotinyl BSA was applied to Immobilon (Millipore), blocked with PBS containing 1% casein, and then 0.3 U / ml of enzyme-labeled streptavidin and CIAP-labeled streptavidin of the present invention at 30 ° C. Incubated for 1 hour. Next, 1 M diethanolamine buffer containing luminescent substrate PPD, pH 9.8 (5 mM MgCl 2
(Including ₂ ) and exposed to an X-ray film for detection. Both the enzyme-labeled product of the present invention and the CIAP-labeled product could detect 0.5 ng of biotinylated streptavidin. Next, the enzyme-labeled streptavidin and the CIAP-labeled streptavidin of the present invention were added to 1 mM.
50 mM Tris / HCl containing MgCl ₂ , pH
Alkaline phosphatase activity was compared by storing at 7.5 ° C for 7 days at 40 ° C. The results are shown in FIG. 15, and the enzyme-labeled streptavidin of the present invention was more stable (FIG. 1).
5).

Example 7 (1) Preparation of biotinylated alkaline phosphatase 30 mM containing 6 mg of the alkaline phosphatase of the present invention
Triethanolamine buffer, pH 7.5 (1 mM M
gCl ₂ , containing 0.1 mM CoCl ₂ ) 600 μl
20 μl of dimethylformamide containing 0.128 mg D-biotinin-ε-aminocapric acid-N-hydroxysuccinimide ester, and stirred at 25 ° C. for 3 hours, and then 0.1 M NaCl, 1 mM MgCl ₂ ,
50 containing 0.1 mM CoCl ₂ and 0.1% NaN ₃
It was dialyzed against mM Tris-HCl buffer.

Comparative Example 5 With respect to CIAP, a biotinylated alkaline phosphata was prepared in the same manner as in Example 7. According to a conventional method, 0 to 5 ng of biotinyl BSA was applied to Immobilon (Millipore), followed by blocking with PBS containing 1% casein,
1 μg / ml of biotinylated alkaline phosphatase according to the invention and biotinylated CIAP 0.3 U / ml and 30
Incubated at 0 ° C for 1 hour. Next, a 1 M diethanolamine buffer containing a luminescent substrate PPD, pH 9.8 (5
(including mM MgCl ₂ ) and exposed to an X-ray film for detection. Enzyme-labeled product and CIA of the present invention
50 pg of biotinylated streptavidin could be detected in both P-labeled forms. Next, the biotinylated alkaline phosphatase of the present invention and biotinylated CIAP were added to 50 mM Tris / HC containing 1 mM MgCl _2.
The thermal stability of the thermal alkaline phosphatase activity in pH 7.5 was compared. The results are shown in FIG. 16, and the biotinylated alkaline phosphatase of the present invention was more stable.

Example 8 (1) Alkaline phosphatase-labeled probe An oligonucleotide having the following sequence in which Uni-Link ^™ AminoModifier (manufactured by Clontech) was incorporated at the 5'end was synthesized by a usual method and purified. 5 '
-GTAAACGACGGCCAGTGAGCGCG
CGTAAT-3 ′ 50 μl of a disuccimidyl suberic acid solution (10 mg / ml-DMSO) was added to 10 μl of 0.1 M NaHCO ₃ containing 10 nmole of the above probe, stirred and reacted at 25 ° C. for 15 minutes, and then gelled on a Sephadex G-25 column. After filtration, the peak containing the first oligonucleotide was collected. The peak was concentrated to 100 μl, 40 μl of 0.1 M NaHCO ₃ containing 1.5 mg of the alkaline phosphatase of the present invention was added, and the mixture was reacted at 25 ° C. overnight. About 500 μl of 1.0 mM to the mixture
20 mM Tris / HCl containing MgCl ₂ , pH
After adding 7.0, high performance liquid chromatography using MonoQ (Pharmacia) (eluent A: 1.0 m
20 mM Tris / HCl containing M MgCl ₂ , pH
7.0, eluent B: 1.0 mM MgCl ₂ , 1M N
20 mM Tris / HCl containing aCl, pH 7.
Purified in 0).

Comparative Example 6 CIAP A CIAP-labeled probe was prepared by the method of Example 5. Enzyme-labeled probe and CI of the present invention
The AP-labeled probe was treated with PBS containing 1 mM MgCl ₂ at 70 ° C. for 2 hours to compare the alkaline phosphatase activity. As shown in FIG. 17, the results showed that the enzyme-labeled probe of the present invention was more stable.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the reaction pH and the relative activity of an enzyme produced by Bacillus badius TE3592.

FIG. 2 is a graph showing pH stability of an enzyme produced by Bacillus badius TE3592.

FIG. 3 is a graph showing the relationship between the reaction temperature and the relative activity of the enzyme produced by Bacillus badius TE3592.

FIG. 4 is a graph showing the thermostability of the enzyme produced by Bacillus badius TE3592.

FIG. 5 is a graph showing the relationship between the reaction pH and the relative activity of the enzyme produced by Bacillus badius TE3593.

FIG. 6 is a graph showing pH stability of an enzyme produced by Bacillus badius TE3593.

FIG. 7 is a graph showing the relationship between the reaction temperature and the relative activity of the enzyme produced by Bacillus badius TE3593.

FIG. 8 is a graph showing the thermostability of the enzyme produced by Bacillus badius TE3593.

FIG. 9 is a graph showing the relationship between the reaction pH and the relative activity of the enzyme produced by Bacillus badius TE3597.

FIG. 10 is a graph showing pH stability of an enzyme produced by Bacillus badius TE3597.

FIG. 11 is a graph showing the relationship between the reaction temperature and the relative activity of the enzyme produced by Bacillus badius TE3597.

FIG. 12 is a graph showing the thermostability of the enzyme produced by Bacillus badius TE3597.

FIG. 13 shows a comparison of calibration curves of human CRP using the enzyme-labeled and CIAP-labeled goat anti-human CRP IgG of the present invention.

FIG. 14 shows a comparison of calibration curves for human CRP using the enzyme-labeled and CIA-labeled sheep anti-human CRP Fab of the present invention.

FIG. 15 shows a comparison of the storage stability of the enzyme-labeled product of the present invention with streptavidin and the CIAP-labeled product.

FIG. 16: Enzyme-labeled product of the present invention with biotin and CIA
The comparison of the thermal stability of P label | marker is shown.

FIG. 17 shows the thermostability of enzymes and CIAP labeled probes of the invention.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyocho, Tsuruga-shi, Fukui Toyobo Co., Ltd.

Claims (26)

[Claims] 1. An alkaline phosphatase having the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 2. The alkaline phosphatase according to claim 1, which has the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum pH: pH 9-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.34 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
It acts on phosphoethanolamine and glucose-6-phosphate. 3. The alkaline phosphatase according to claim 1, which has the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 60 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6-9 (25 ° C, 16 hours) 6. Optimum pH: pH 9-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.26 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
It acts on phosphoethanolamine and glucose-6-phosphate. 4. The alkaline phosphatase according to claim 1, which has the following physicochemical properties. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: 70 ° C. or lower (pH 7.5, 30 minutes) 4. Optimum temperature: 60 ° C or higher 5. Stable pH: pH 6 to 11 (25 ° C, 16 hours) 6. Optimum pH: pH 9.5-10 7. Specific activity: at least 2,300 U / mg 8. It has no sugar chains. 9. Km value: 0.28 mM (relative to p-nitrophenyl phosphate) 10. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 11. Substrate specificity: p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, NADP, DL-α-glycerophosphate, β-glycerophosphate, phenyl phosphate,
Acts on phosphoethanolamine, glucose-1-phosphate, glucose-6-phosphate 5. A method for producing alkaline phosphatase, which comprises culturing a strain belonging to the genus Bacillus and having an alkaline phosphatase-producing ability having the following physicochemical properties in a medium, and collecting the alkaline phosphatase from the culture. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 6. A strain capable of producing alkaline phosphatase is Bacillus badius TE3592 (FERM).
BP-5329), TE3593 (FERMBP-5)
330) or TE3597 (FERM BP-512)
0) The method for producing alkaline phosphatase according to claim 5. 7. A method for detecting a ligand in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 8. A method for detecting a ligand in a sample utilizes an affinity reaction between a ligand in the sample and a substance having a specific affinity for the ligand, and the substance bound by the reaction. The method for detecting a ligand in a sample according to claim 7, which is a method of measuring an alkaline phosphatase activity bound to the enzyme or a method of measuring an alkaline phosphatase activity not bound to the enzyme. 9. A method which utilizes an affinity reaction between a ligand and a substance having a specific affinity for the ligand, wherein alkaline phosphatase directly or directly binds to the substance having a specific affinity for the ligand. The method for detecting a ligand in a sample according to claim 7, which binds indirectly. 10. The sample according to claim 7, which utilizes an affinity reaction between a ligand and a substance having a specific affinity for the ligand, and alkaline phosphatase directly or indirectly binds to the ligand. For the detection of ligands in blood. 11. A substance having a specific affinity for a ligand to which an avidin compound or a biotin compound is bound, and a bond between alkaline phosphatase and a biotin compound for binding a ligand and a ligand with a specific affinity. The ligand in the sample according to claim 7, which is subjected to an avidin compound-biotin compound binding reaction at the same time as or after an affinity reaction with a substance possessed, and the alkaline phosphatase activity bound by the reaction or the residual alkaline phosphatase activity is measured. Detection method. 12. The method for detecting a ligand in a sample according to claim 7, wherein the ligand is an antigen, an antibody, a hormone, a hormone receptor or a nucleic acid. 13. The ligand in the sample according to claim 7, which utilizes an affinity reaction between the ligand and a substance having a specific affinity for the ligand, and the affinity reaction is an antigen-antibody reaction. Detection method. 14. The sample according to claim 7, which utilizes an affinity reaction between a ligand and a substance having a specific affinity for the ligand, and the affinity reaction is a hormone-receptor reaction. Ligand detection method. 15. The sample according to claim 7, which utilizes an affinity reaction between a ligand and a substance having a specific affinity for the ligand, and the affinity reaction is a nucleic acid hybridization reaction. Ligand detection method. 16. A method utilizing an affinity reaction between a ligand and a substance having a specific affinity for the ligand, wherein either the ligand or the substance having a specific affinity for the ligand is used. The method for detecting a ligand in a sample according to claim 7, which is bound to a solid phase. 17. (i) A specific binding substance for a ligand labeled with alkaline phosphatase having the following physicochemical properties, or (ii) a ligand labeled with alkaline phosphatase having the following physicochemical properties, and (iii)
A ligand detection reagent in a biological sample containing an alkaline phosphatase measurement reagent. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 18. (i) A substance having a specific affinity for a ligand that binds an avidin compound or a biotin compound, and (ii) an alkaline phosphatase having the following physicochemical properties that binds a biotin compound or an avidin compound, and ( iii) A ligand detection reagent in a biological sample containing a substance that measures alkaline phosphatase. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 19. The reagent for measuring alkaline phosphatase is a 1,2-dioxetane compound or a derivative thereof, phenalenone-6-phosphate, benzphenalene-6-phosphate,
18. A methyl 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate, 1-naphtholphthalein phosphate, 5-bromo-4-chloro-3-indolyl phosphate or a derivative of these compounds. 18. A reagent for detecting a ligand in a biological sample according to item 18. 20. The method according to claim 17 or 1, wherein the ligand is an antigen, an antibody, a hormone, a hormone receptor or a nucleic acid.
8. A reagent for detecting a ligand in the sample according to 8. 21. Ligand detection in a sample according to claim 17 or 18, wherein the ligand or a substance having a specific affinity for the ligand has affinity reactivity, and the affinity reaction is an antigen-antibody reaction. reagent. 22. The sample according to claim 17, wherein the ligand and the substance having a specific affinity for the ligand are affinity-reactive, and the affinity reaction is a hormone-hormone receptor reaction. Ligand detection reagent. 23. The ligand and the substance having a specific affinity for the ligand are affinity-reactive, and the affinity reaction is a nucleic acid hybridization reaction.
A ligand detection reagent in the sample according to 7 or 18. 24. The reagent for detecting a ligand in a sample according to claim 17, wherein either the ligand or a substance having a specific affinity for the ligand is bound to a solid phase. 25. A method for quantifying a ligand in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE) 26. A method for sequencing a nucleic acid in a sample, which comprises using alkaline phosphatase having the following physicochemical properties as a label. 1. It catalyzes the next reaction. Orthophosphoric acid monoester + H ₂ O → Alcohol + Orthophosphoric acid 2. Activator and stabilizer: Mg ⁺⁺ and Co ⁺⁺ 3. Thermal stability: pH 7.5, at least 3 at 60 ° C treatment
Stable for 0 minutes 4. Specific activity: at least 2,300 U / mg 5. It has no sugar chains. 6. Molecular weight: 140,000-150,000 (gel filtration method) 65,000-67,000 (SDS-PAGE)