SU1041568A1 - Reagent for detecting adenosine-5-triphosphate - Google Patents

Description

This invention relates to biochemistry and relates to a composition for the quantitative determination of adenosine-5-triphosphate (ATP) based on the immobilized bright luciferase. ATP is one of the most important metabolites, the quantitative determination of which is necessary for the purposes of medical diagnostics and for the analysis of environmental pollutants. A reagent is known based on a two-component enzyme system including phosphoglycerate kinase and glyceral dehydrophosphatase. The determination of ATP is carried out spectrophotometrically by the rate of accumulation of nicotinamide adenine dinucleotide (NAD). Sensitivity analysis does not exceed 1-50 microns. The closest to the present invention is a soluble luciferase-based reagent isolated from light-colored, containing luciferin, magnesium acetate, a buffer mixture of imidazole and acetic acid, the stabilizer is a mixture of sodium ethylenediaminetetraacetate (ED and acetylcysteine, bovine serum albumin, water in the following ratio Components, wt .: Luciferase, light coarse 0.002 Luciferin, 0.013 Magnesium acetate 0, OBS whey albumin, 0.3-3 Imidazole, 0.68 Acetic acid, 0.13 Ethylene diamine tetraacetate sodium 0.03 Acetylcysteine 9.24 Water, detail 2 .. Analysis It is based on recording the intensity of bioluminescence that occurs during enzymatic oxidation of luciferin by oxygen in the presence of ATP and magnesium salts. The intensity of the glow is proportional to the ATP concentration in the sample being analyzed. The dignity of the method is the ability to determine ATP in the presence of other metabolites (specificity), as well as high The sensitivity of the assay (10 m) is much higher than that achieved by other methods. The above composition allows a luminescence to be obtained which, having reached a maximum, remains constant for several minutes. The stability of luminescence in time provides high accuracy of determination and creates prerequisites for automating the analysis. The disadvantage of the composition is its low stability. In the lyophilized state with the reagent completely lost its enzymatic activity during the week. Therefore, in order to achieve the ultimate sensitivity of the determination, it is necessary to use a freshly prepared reagent solution and to rebuild the calibration graph before each measurement: ATP concentration — luminous intensity. This leads to jHe only to the complication of the method, but also to significant losses of the deficient enzyme. The purpose of the invention is to increase the stability of the reagent for the determination of ATP. The target is achieved by the fact that a reagent for the determination of adenosine 5-triphosphate, including luciferase, isolated from brightons, luciferin, magnesium salt, buffer, stabilizer, and water contains bright luciferase immobilized on a polysaccharide carrier. The mixture is a buffer Tris- (hydroxymethyl) -aminog. methane and acetic acid, as a stabilizer - a mixture of sodium ethylenediaminetetraacetate with dithiothreitol, c. As a salt, magnesium sulfate is magnesium, with the following ratio of components, May .: Immobilized Na.-, polysaccharide ohm-, .. the body of luciferaz OO7-0, b70 Luciferin 0.002-0.0.15 Magnesium sulphate -.r, 48-0.72 Acetic acid 0.02-0.04. Tris- (Oxymethyl), -aminomethane 0.08-0.16; Ethylene diamine tetraacetate sodium 0.03-0.06 Dithiothreitol 0.05-0.1 Water. Remaining Unlike the composition adopted as the prototype, the proposed reagent. contains luciferase svetov, immobilized covalently on an insoluble poly-saccharide carrier. The specific activity of the drug immobilized luciferase should be within 3 per 1 mg of the preparation of immobilized enzyme (E is the international unit of activity, 1 E is the amount of enzyme that catalyzes the conversion of 1 µmol of substrate per 1 minute). When the activity is less than S / S / mg, the sensitivity of the assay is reduced. A preparation with an activity of more than 10 U / mg should not be used. It is impractical because of the unproductive consumption of the enzyme in the preparation of the immobilized preparation. In addition, the use of an enzyme with an activity higher than 1 mg of immobilized enzyme does not lead to an increase in the detection sensitivity. As a buffer instead of an imidazole acetate, the proposed composition includes a tris- (oxymethyl) -aminome tan-acetate buffer, and instead of a stabilizing mixture of ethylene diamine tetraacetate (EDTA) and acetylcysteine a mixture of EDTA with dithiothreitol, an additional stabilizing effect is also introduced into the mixture of sulfate magnesium by increasing ionic strength. The reagent is obtained by adding immobilized luciferase in tris- (oxymethyl) -aminomethan-etatate buffer to luciferin, magnesium sulfate, EDTA, dithiothreitol in the same buffer to the centered suspension. . The reagent has increased stability. The period of his semi-incarnation. For 0-50 days. In the lyophilized state, the reagent is stored at 4 ° C without significant loss of enzymatic activity during the year. The stability of the obtained composition is several times higher than the stability of an individual preparation of immobilized luciferase. The observed effect is a result of co-presence in the reagent composition of the immobilized enzyme, a mixture of EDTA with ditertreyl, tris (oxymethyl) - aminomethane-ac buffer and magnesium sulfate. At the same time, the mixture of EDTA with dithiothreitolo does not have a noticeable stabilizing effect on the soluble enzyme. The limit of detection of ATP using the proposed reagent. Example 1: Preparation of a reagent for the determination of ATP on luciferase bases immobilized on sepharose, ultradex, ultragel, activated with cyan bromine. a. To 2 ml of a luciferase suspension immobilized covalently on ultragel by a known method with a concentration of 10 mg / ml and activity per 1 mg of the preparation of immobilized enzyme; in 0.01 M tris- (oxymethyl) -aminomethane-acetate buffer containing 1 mM EDTA and 0.5 mg / ml dithiothreitol, 4 ml of luciferin solution with a concentration of 0.5 mM and 2 ml of 50 mM magnesium sulfate solution in the same buffer are added. The reagent is obtained in the following ratio, wt%: Immobilized luciferase is light 0,867 luciferin 0,002 Magnesium sulphate 0, tris (Oxymethyl) amiomethane Ethylenedia amine tetraacetate sodium 0.03 Acetic acid 0.02 Dithiothreitol 0.05 Water. The rest is B. To h ml of a luciferase suspension immobilized covalently on ultradex, with a concentration of 10 mg / ml and an activity of 6-10 U per 1 mg of immobilized enzyme in 0.015 M tris- (oxymethyl) -aminomethane-acetate buffer containing 1.5 mM EDTA and 0.7 mg / ml dithiothreitol, k ml of luciferin solution with a concentration of 1.6 mm and 22 ml of a 67 mm solution of magnesium sulfate in the same buffer are added. A reagent is obtained having the following composition, in wt%: The immobilized luciferase is light 0.13 Luciferino, 006 Magnesium sulfate 0.60 tris (Oxymethyl) aminomethane 0.12 Ethylene diamine tetraacetate. Acetic acid - 0.03 Dithiothreitol 0.07 Water. The rest is. To a 20 ml suspension of luciferase, immobilized covalently on Sepharose, with a concentration of 10 mg / ml and an activity of 10 U per 1 mg of the immobilized enzyme, in 0.02 M tris - (oxymethyl) -aminomethane-acetate buffer containing 2 mM EDTA and

1 mg / ml dithiothreitol, a "ml of luciferin solution with a concentration of 3.9 mmol and 6 ml of a 0.3 M solution of magnesium sulfate in the same buffer is added. Get reagent having the following composition, masd:

Immobilized luciferase svetov 0,67 Luciferin 0,015

Magnesium sulfate 0.72 Tris- (Oxymethyl) -aminomethan0, 16

Ethylene diamine tetraacetate O, O Acetic acid O., 04 Dithiothreitol 0,, 1

WaterEverything

Example 2. Preparation of the agent for determining ATP based on luciferase immobilized on a cellophane film.

To 1.0 ml of 0.02 M trie- (oxymethyl) -aminomethane-acetate buffer containing 2 mM EDTA and 1 mg / ml dithiothreitol, add 0.2 ml of luciferin solution with a concentration of 1.5 mM and 0.2 ml 0, M solution of magnesium sulfate in the same buffer. A cellulose film is immersed in the resulting solution. immobilized on it by a known method of luciferase weighing 7 mg and an area of 25 mm with activity per 1 mg of immobilized enzymes. The following reagent composition is obtained, wt .: Immobilized luciferase light 0.5 Luciferin .006

Magnesium sulfate 0.69 Tris- (Oxymethyl) -aminomethane 0.16 Ethylene diamine tetraacetate sodium 0.06 Acetic acid 0.0 Dithiothreitol 0.1 Water Remaining

Changing the area of the pulp film taken makes it possible to vary the content of the enzyme in the fines described in the claims.

.Primerz. Reagent stability test.

The table indicates the values of the period of semi-inactivation of the reagent, including in the lyophilized state. For comparison, a similar indicator is given for a known reagent based on soluble luciferase and an individual luciferase preparation immobilized on sepharose. To estimate the half-inactivation period, the change in the level of the enzymatic activity of the sample is determined from the intensity of luminescence that occurs when a standard amount of ATP is added to the sample (example k). The numbers in the table below show that the stability of the proposed reagent is at least 25 times greater than the stability of the composition of the prototype and 2.5 times greater than the stability of the individual preparation of immobilized luciferase.

CO

150

-50

150 50 150 50

2

30 20

Example k. The technique will determine laziness ATP.

1, ml of reagent, prepared as described in Example 1 or 2, is injected into a 3 ml cylindrical cuvette with a diameter of 1 cm, which is then placed in the cuvette compartment of the luminometer and the background signal is recorded. Then, 0.5 ml of ATP solution with a known concentration, without interrupting the registration of the glow. The difference between the magnitude of the bioluminescence intensity signal in the presence and in the absence of ATP is a value proportional to the concentration of ATP. The experiments are repeated for ATP solutions with ATP concentrations.

s the same range in which measurements are intended to be made. The direct proportionality between the intensity of bioluminescence and the ATP concentration is observed in the range from 10M to 10% of ATP. Based on the data obtained, a graph of the dependence of the bioluminescence intensity on the ATP concentration is plotted. When working with samples with an ATP concentration, it is recommended to use the reagent described in example 1a or 2, 8 at a concentration of 10 - the reagent described in example 16, with an ATP concentration of 10g-10 m - the reagent described in example 1c. The concentration of ATP in an unknown sample is determined by the intensity of luminescence that occurs when adding 6.5 ml of unknown

ATP sample to 1, A. ml reagent, using a calibration graph, obtained as described above. The detection limit of the ATP concentration by this method is.

The proposed reagent for the determination of ATP can be used for scientific purposes to study the processes associated with the transformation of ATP, as well as for the purpose of medical diagnostics. With the help of this drug, by the level of creatine phosphokinase in the blood, it is possible to carry out an early diagnosis of myocardial infarction and a number of other diseases, a rapid analysis of the sensitivity of microorganisms to antibiotics. The determination of the level of ATP 8 reservoirs allows one to wake up about their biological productivity and level of contamination.

Claims (1)

1. REAGENT FOR THE DETERMINATION of 'Adenosine-5 ^{1 2 3} -TRIPHOSPHATE, including luciferase isolated from fireflies, luciferin, a magnesium salt, a buffer, a stabilizer and water, due to the fact that, in order to increase stability, it contains luciferase immobilized on a polysaccharide carrier, as a buffer - a mixture of tris (oxymethyl) -aminomethane and acetic acid, as a stabilizer - a mixture of sodium ethylene diamine tetraacetate with dithiothreitol, as magnesium salt of magnesium sulfate, in the following the ratio of components, wt.%: Luciferase immobilized on a polysaccharide carrier Luciferin Magnesium sulfate Acetic acid tris- (Oxymethyl) aminomethane Ethylene di sodium aminotetraacetate Dithiothreitol Water 2. The reagent according to which live luciferase immobilized on a polysaccharide 3 ⁴ -! '10 ⁵ ) U / mg. 0,067-0,670 0.002-0.015 0.48-0.72 0.02-0.04 0.08-0.16 1, 0.03-0.06 0.05-0.1 The rest is about t l and what it contains SU „„ 1041568 I I