JPH03503121A - Enzymatic quantification of theophylline - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Amount of one ophylline Background of the invention Theophylline is a bronchodilator used to treat patients with asthma and allergy symptoms. It is a drug and a respiratory stimulant. Theophylline is also used to treat congestive heart failure and acute pulmonary edema. It is also used to treat. The benefits and risks of using this drug are Directly related to serum concentration. For theophylline to be effective, its blood concentration must be It is necessary to maintain a level of approximately 10-20 mg/L. Lower than 10mg/L Low theophylline levels are therapeutically ineffective, and levels higher than 20 mg/L are This toxicity can cause brain damage and death. Te Also because ophylline is therapeutically advantageous only within a narrow concentration range, dietary, other prescription There are large differences in drug excretion between patients due to drug and physiological differences. , it is important to monitor patients using this drug.

Theophylline measurement was performed using 5hah, J Pharm Sci 63(8), 1283 (1974) in gas chromatography, and DesaBe, et al. al.

J Chrosat 336 (2), 285 (1984) is a gas chromatograph. This is done using a combination of Raffey and a mass selective detector. Thoi*p-5 on, et al, J Lab Cl1n Med 84(4), 58 4 (1974) and Na1sh, et al,, 8nn Cl1 n Biochem 16(5), 254 (1979) by Theophy Phosphorus is measured using high pressure liquid chromatography. 5ehack, etc. al., J Pharm 97.283 (1949) quantification of theophylline UV spectrophotometry was used. However, these methods require tedious extraction Therefore, most clinical approaches to theophylline quantification today are based on test samples. For the recognition of theophylline in the sample, an antibody-dependent immunological method is used. child Theophylline quantification systems such as Involves protein binding. After the reaction with the antibody occurs, the quantification of theophylline is It is performed in various ways according to the schedule. In other words, the evaluation in the assay is whether turbidity occurs or not. Turbidity measurement, nephelometric analysis, radioactivity measurement or can be performed by colorimetric analysis. An example of this system is given by Painter, etal ,, J C11o Lib Autos 3(3), 179 (1983) , Samoszuk, etal, , Therp Drug Mon 5(1), 113 (1983), Opheim, et al.

Cl1n'Chem 30(11), 1870 (1984), Bo eckx and Nunson+Therp Drug Non 7 (1 ), 95 (1985), Cook, et al, Re5CoIls in Chew Path & Pharm 13(3), 4 97 (1976), and Landesa+an, et al, C1 1n Chew 29.1238 (1983) reports.

The immunological system is described in Li et al., C11n Chew 27(1), 2 2 (1981), Davis and Marks, Therp Drug Mon 5(4), 479 (1983), Chang, et al. C11n Chew 28(2), 361 (1982), old ods +et if,, C11n Chew 30(7), 1174 (1984), Jolley, et al.

Cl1n Chew 27. 1575 (1983), Norris , et ml,, ^nal Cbesi53, 658 (1981 ), and Tyhich, et al, C11n Chew 27゜149 9 (1981) also reported.

Additionally, the enzyme amplification assay disclosed in U.S. Pat. No. 3,817,837 It is used. In this disclosure, an enzyme is chemically coupled to a ligand, and the resulting enzyme is The bound ligand is attached to the seven-button. A ligand can be a drug. ligand The entire reaction has specificity because it reacts specifically with the receptor. one On the other hand, enzyme activity is used as a marker for the reaction. Therefore, the yeast used The application of such an approach, which does not perform enzymatic recognition of drugs, The method of the present invention, which uses enzymes instead of antibodies or ligands for phosphorus recognition, is It varies depending on the

European Patent Application No. EP8630022 filed on January 15, 1986 6.7, and C11n Chew 25.1370 (1979), the substrate The activity of alkaline phosphatase, which acts to cleave the phosphate group from It has been reported that it can be inhibited by theophylline. This approach The enzymatic reaction of alkaline phosphatase is a reaction that cleaves phosphate bonds. however, this effect is interfered with by the presence of theophylline. This opening The theophylline test performed in this study also indicates that theophylline plays a role in the enzymatic reaction. It is a test that cannot be used or modified by enzymes. In contrast, the present invention uses or recognizes theophylline for the determination of theophylline in liquids. We teach that a test system can be realized using an enzyme that uses theophylline as a substrate. Ru.

In the prior art, no enzymes that utilize theophylline are available. However, it was not disclosed.

Moreover, since theophylline is a xanthine derivative, commercially available xanthine oxy Dase and xanthine dehydrogenase and related enzymes utilize theophylline Attempts have been made regarding the ability to do so, but those attempts have been unsuccessful. In humans, Te Ophiline is metabolized mainly into 1,3-dimethyluric acid and 1-methyluric acid and 3-dimethyluric acid. -Methylxanthine [Cornish, H,H,a nd Christman, ^, 8,.

J Biol Chew 228.315 (1957)] The enzyme for use was neither isolated nor presented. However, recently, three types of theophylline have been used. Or the recognition enzyme is GDS Technology Ine.

P, O, Box 4F3. Elkhart, Indiana 46515? Now available. These enzymes are: 1. Enzyme T-090,2, Enzyme T-060 and 3. Identified as Enzyexe T-040 It was.

The present invention provides for the use of the above enzymes in applications such as body fluids, food extracts, pharmaceutical compounds and pharmaceutical compositions. It was used for the first time in the development and implementation of a test to quantify theophylline in food using an enzyme. enzyme The tests enabled by the approach according to the invention using the method of the present invention can be performed quickly and easily.

The advantages of the enzymatic approach are that 1) the potency of the reagent or test composition is uniform; 2) The addition of the sample to the reagent or test composition can be done in one step. , 3) capable of processing the liquid system in conjunction with an instrument reader; and 4) Reagents or test compositions can be easily incorporated into the solid matrix.

Brief description of the drawing The figure shows theophylline activity before and after contact with theophylline, as described below in Example 3. The absorbance curve of the enzyme used is shown.

beautiful The present invention basically describes enzymes that utilize or recognize theophylline (or A theophylline quantitative system in which an electron carrier is optionally used (a substance containing The quantification of theophylline according to the present invention consists of the enzyme and any present in the sample. The signal generated by the reaction with theophylline is measured and the amount of signal generated is calculated. This is done by converting or correlating to the abundance of theophylline in the sample. Ru.

Here, the term theophylline-utilizing enzyme refers to enzymes that recognize or utilize theophylline; Alone or in conjunction with other reagents or means, visual, instrumental or other an enzyme that generates a signal that can be measured by a known method, or a substance containing such an enzyme. means.

The principle of the test method is based on the utilization of theophylline by the enzyme, i.e. the enzyme It recognizes filin as a substrate and converts it into different compounds or products. this The system is shown in the diagram below.

enzyme Theophylline → product As mentioned above, the method of the present invention disclosed in the main text and claims uses theophylline and This signal involves detection of the signal generated by contact with theophylline-utilizing enzymes. The signal generated indicating that a specific enzymatic reaction has taken place is listed in the text. Measured in various ways. This treatment is performed on electron carriers such as electron acceptors or electron carriers. Examples of asci vessels are oxygen, nicotine, etc., carried out with or without the addition of donors. amide adenine dinucleotide (HAD), dichlorophenol indophenol (DCPIF), phenazine methosulfate (PMS), methylene blue, Cytochrome, ferricyanide, etc. An example of a child donor is reduced nicotinamide Adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide Chidophosphate (NADP)! >, reduced flavin adenine dinucleotide (F ADH) etc.

In addition, regular use using the optimal pi and temperature range for the activity of theophylline-utilizing enzymes is recommended. It is also possible to process by enzymatic quantitative method. Such quantification may also be performed using a buffered ring. It is best to do it at the border.

In principle, theophylline-utilizing enzymes that can be used for the enzymatic determination of theophylline All variations are included within the scope of this invention.

Various methods that can be used to quantify theophylline in a test sample by the method of the present invention The test system is shown below.

(1) When measuring the decrease in theophylline concentration in a system, theophylline The basis is determined by measuring the decrease in absorbance at the wavelength 272n*, where The disappearance of quality theophylline can be measured. This system uses theophylline enzyme T-060. According to Example 1 using and Example 2 using theophylline enzyme T-090 shown. Changes in concentration may also be detected by measuring reflectance, which is the reciprocal of absorbance. stomach.

(2) When measuring changes in the oxidation-reduction potential of a system, there are two methods for this purpose: (a) a method of measuring changes in an enzyme or enzyme complex system, e.g. For example, as in Example 3, changes in enzyme characteristic values were caused at wavelengths of 410 nm and 550 nm. or (b) e.g. ferricyanide. Electrification of changes in electron carriers added to the system, such as the reduction of from to ferrocyanide Use a method of scientific measurement.

Example 4 shows that the reaction occurs in the presence of theophylline enzyme T-090 and ferricyanide It shows the change in absorbance that occurs at 410 o when do changes to ferrocyanide. This change can also be measured by spectrophotometric or electrochemical methods.

(3) When measuring the appearance of products related to the enzymatic reaction of theophylline, this reaction The products produced in the reaction vary according to the enzymes involved. @Theophylline The enzyme is oxidized theophylline, dehydrogenated theophylline or demethylated theophylline. Can react. Example 5 shows the formaldehyde when using theophylline enzyme T-040. Example 6, which shows the formation of aldehyde and its measurement, shows that theophylline enzyme T-060 1 shows the formation of hydrogen peroxide and its measurement when using .

The products are believed to be formed by the following reactions or processes.

(i> Enzyme oxidizes theophylline using oxygen as an electron acceptor to produce theophylline (1,3 dimethylxanthine) to 1,3 dimethyluric acid, this is This is shown in the diagram below.

enzyme Theophylline 10x10HzO→ 1.3 dimethyluric acid hydrogen peroxide Hydrogen peroxide produced in this system can be measured using titration, potentiometry, and polarography methods. , can be quantified by colorimetric and enzymatic methods.

Determination of hydrogen peroxide by an enzymatic method is preferable because the enzymatic method has reliable characteristics. This is a different method, and at the same time the hydrogen peroxide in the above reaction can be easily colored. For example, ^nal Biochem 105.389. (1980) The peroxidase method described in . Uses theophylline enzyme T-060 However, Example 6 shows that hydrogen peroxide formation is proportional to the theophylline concentration in the sample. prove that Or, based on the above general reaction formula, for example, gas chromatograph The oxygen consumption rate is determined by the Raffy method and depolarization. You may measure (Yellow Spring In5 trusents, Use an oxygen electrode (available from Yellow Spring, 0)1) Depolarization methods are known and described in U.S. Pat. No. 3,838,011 and J Appl Ph. ysiol 18.1247 (1963).

(ii) Enzymes accept substances other than oxygen, such as ferricyanide, NAD, and cytochromes. The body oxidizes theophylline to produce 1.3 dimethyluric acid and reduced receptors. In this case, this is shown by the following equation.

enzyme Theophylline + oxidation receptor → 1.3 dimethyluric acid deca-reduction receptor The 1,3-dimethyluric acid produced in this system can be measured or quantified in several ways. Ru. Example 7 shows how the absorption at 29Zn+s was measured using theophylline enzyme T-090. Demonstrate how to measure degree.

1.3 Dimethyluric acid has an absorbance maximum at this wavelength; here again, the increase in absorbance is It was found to be proportional to the theophylline concentration in the sample.

Furthermore, using this reaction formula, the oxidation/reduction state of the electron acceptor after use can be measured. The theophylline concentration of a sample can also be determined by Examples 4 and 8 used ferricyanide as an acceptor and transferred it to theophylline enzyme T-090. Therefore, a test method for reducing it to ferrocyanide is shown.

Ferricyanide shows maximum absorbance at a wavelength of 410n-, and at this wavelength ferrocyanide Nido does not show any absorbance, so in Example 4, the absorbance at a wavelength of 410 nsa was Measure the weight loss of ferricyanide by measuring the decrease. Again, It was found that the decrease in absorbance was proportional to the theophylline concentration in the sample. fruit Example 8 shows another method for measuring ferrocyanide, in this case ^van, M , & 5havit N,, ^nalyBioehem 6.549 (196 Using the method of 3), 4.7 diphenyl-1.107 enanthroline sulfone The formation of ferrocyanide is determined chemically using Color development using the Avon method The zero color development measured at 535 ns was proportional to the theophylline concentration in the sample.

Example 9 demonstrates the use of another electron acceptor, ferricytochrome C. This implementation In the example, ferricytochrome C is reduced to ferrocytochrome C. theophylline To measure the increase in absorbance at wavelength 550n+a in the presence of enzyme T-090 Thus, the formation of ferrocytochrome C is measured. Again, absorption at wavelength 550n- It was found that the change in degree was proportional to the theophylline concentration in the sample.

Nicotinamide adenine dinucleotide (HAD), 2,6-cyclophenol Ruindophenol (DCPIF), phenazine methosulfate (PMS), etc. Other known electron acceptors can be used in the same manner.

Since reflectance is inversely proportional to absorbance, in all the above examples the change in reflectance may be measured.

Spectrophotometry is a method to measure the changes caused by the movement of electrons in the above reactions. or not limited to reflectance methods.

potentiometric method to measure the transfer or change of electrons in oxidation-reduction reactions; It is known that fluorescence or electrochemical methods can be used. For example, Reed and ■awkrec1ge is ^nal Chew 59.2334 (1987) disclosed the electron transfer reaction of cytochrome C at a silver electrode; In the invention, it can be used to measure changes in cytochrome C.

Additionally, the electrochemical method reported in U.S. Patent No. 4,545,382 Ferrocene or ferrocene derivatives are used as child receptors. These receptions The container is used for the enzymatic theophylline measurement of the present invention, and the change is measured electrochemically. It is also proven and reported in ^nal Chew 36.343<1964) ferrisi by measuring current changes using platinum electrodes as described. The change from anide to ferrocyanide may be quantified.

(iii) Enzymes bind theophile by cleaving one or both methyl groups. When one methyl group is cleaved, 1-methylxane is demethylated. 0 when 1 mole of formaldehyde and 3-methylxanthine are produced. When both methyl groups are cleaved, xanthine and 2 moles of formaldehyde are The zero reaction produced is shown by the following equation.

enzyme Theophylline + NADPH + 02 → 1 methyl lexantin + formua J redehyde + NADP and/or 3 methylxanthine + formaldehyde + NADP and/or Xanthine + Formaldehyde + NADP In the above reaction, as an electron donor NADPH is used. However, NADFI or FADfl The use of other electron donors is also possible. Known and conventional chemical, enzymatic or electrical The reaction product formaldehyde can be measured by gas chemical methods. Example 5 This is a method for measuring formaldehyde using theophylline enzyme T-040. Show one.

As shown in the example, the formaldehyde produced is It is proportional to the filrin concentration.

The reaction product xanthine or methylxanthine may be measured using the following procedure. stomach.

xanthine xanthine oxidase (or methylxanthine +02 Uric acid (or methyluric acid) + H, O.

The hydrogen peroxide formed (82 oz.) can be measured by various methods described above, and uric acid or Methyluric acid, for example, by quantifying the increase in absorbance at a wavelength of 292n- or C11n It can be measured by the colorimetric method described in Chew 26.227 (1980). .

A decrease in NADPH or NADH is also ^nal Biochem 12.3 spectrophotometrically or fluorometrically by conventional methods such as those described in 57 (1965). Can be measured accurately. The decrease in FADH is J Biol Chew246, 237 1 (1971), by measuring the decrease in absorbance at a wavelength of 450 nm. It can be measured.

(1) Separation of other products produced by enzyme recognition of theophylline conventional or practical methods such as photometric, electrochemical or chromatographic methods. Measured by decrease in theophylline concentration as in Example 1.

Liquid test reaction disclosed in the text In addition to s), the test reagent compositions and devices of the invention contain conventional additions that favor the reaction. Agents and adjuvants such as buffers, suspending agents, thickeners, color enhancers 1 surfactant.

Additionally, in addition to the liquid test reagent systems described above, the compositions of the present invention may be supported on solid carriers or matrices. Such formulations may be mixed into the alcoholic beverage! ! ! (conf iguratio n) That is, the format is known in the industry as Drygeme I/Suri or solid state test device. The most common matrix is paper, but polymers, Other water-absorbing materials such as gels, gels, etc. can also be used. Basically, the reagent composition is Mix or impregnate with lix and dry. Test sample when using the device immerse in or come into contact with. Test compositions containing especially theophylline-utilizing enzymes The signal generated in the device by the reaction between the substance and theophylline is detected using a method known in the art. Detected by techniques, e.g. visual comparison with a color chart, spectrophotometric measurement of reflectance and quantify. Example 10 shows that theophylline enzyme T-090 and cytochrome C were mixed on P paper. Observe the discoloration with increasing theophylline concentration, showing the device impregnated with Semi-quantitative reading by clinical examination or quantitative reading by existing reflectance meter Ru. Alternatively, changes in electron transfer in a solid matrix can be measured electrochemically.

In summary, all the examples above and below demonstrate the fermentation of theophylline according to the present invention. According to elementary quantitative methods, both liquid-based and solid matrix test device forms We have proven that we can produce a test format that is easy to use and convenient.

Heaven 1” 1 The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

In all the examples given below, the enzyme activity is measured per minute at a temperature of 30°C. Defined as 1 pmol of theophylline used in

夾1j1- The analytical mixture consisted of 0.05M potassium phosphate wifr solution at pH 7.0 and The seeds were added to 2 ml of the 0 analysis mixture that contained the enzyme T-0600, 4u/xi. Samples 100.1 containing different concentrations of theophylline were added to separate cuvettes. Ta. The reaction was carried out in a cuvette with 101 light paths at 30°C in a Gllford spectrophotometer. went. Observing the decrease in optical density at 272 nm after 30 minutes, the sample It was proportional to the theophylline concentration in

-Female 1 272nm 40xti/L 0.06720xg/L 0.03310M g/L　　　　　　　　　　O,017夾JilJLL The analytical mixture is 1. l17.5 potassium phosphate buffer 50 umoles/z1 , theophylline enzyme T-0901, 8u/z1 and 25 nmol of cytochrome C It contained es/li'. This analytical mixture 0.5i+1 was added with the following concentrations: Sample 25.1 containing ophylline was added into a separate cuvette. Gll Reactions were carried out in a 10-pass cuvette at 30° C. in a Ford spectrophotometer. 2 After 0 min, recording the decrease in optical density at 272n- It was proportional to filin concentration.

-Ly 1 line'272nII) 4011F/L O,06520mg/L O,0 3110my/L 0.015 factor m In this example, as shown in Figure 1, theophylline enzyme T-090 was used. Curve A shows theophylline fermentation in potassium phosphate buffer 0.0514 at pH 7.5. 0 curve B showing the absorbance spectrum of elementary T-090 at a concentration of 1.8 u/ml is the same Absorbance spectra of the same enzyme in the presence of theophylline concentration 2i+y/L under one condition Toll is shown.

As shown, theophylline has an absorbance at wavelengths of 417.5 and 550 n@ increased. These changes in absorbance are determined by the concentration of theophylline in the sample. be used as a basis for detection.

111 rainbow In this example, theophylline enzyme T-090 and potassium ferricyanide as receptors were used. I used um. When potassium ferricyanide is added to theophylline, the enzyme is activated. In its presence, it converts to ferrocyanide. Ferrocyanide formation is at 410 nm This can be determined by measuring the decrease in optical density at .

The analytical mixture consisted of potassium phosphate M solution 50 μmoles/ml, theophylline Enzyme 8u/xi and potassium ferricyanide 1.43pmole/xj! of Contained 0 analytical mixture 0.35zz! contains theophylline at the following concentrations: Sample 50Ij! were added into separate cuvettes. [;1lford spectroscopy Reactions were carried out in a 10 mm optical path cuvette at 30° C. in a photometer. After 30 minutes, 4 Observation of a decrease in optical density at a wavelength of 10n indicates that theophylline in the sample It was proportional to the concentration of

Theophylline (410nm) 40zy/L O, 14130xg/L 0. 10920mg/L O,07610 Stomach y/L O,047 dog JLLΣ In this example, theophylline enzyme T-040 was used. NADPH In the presence of NADH or NADH, this enzyme demethylates theophylline to form xanthine. and/or produce 1- and/or 3-methylxanthine and formaldehyde. to be accomplished.

Formaldehyde was converted to Biochem J 55,41+1l- by Na5h. 421 (1953). . The amount of formaldehyde produced was proportional to the theophylline concentration in the sample.

The analysis mixture was 1 tl of Tris-HC of ptis, o solution 50 Holes/xi ', NADP old Ho1e/yi' and semicarbazide 10nmoles/il It contained. Analytical mixture in test tube2. Ozl, theophylline enzyme T-040 1 reaction mixture to which 2.5 units were added was shaken at 30°C for 15 minutes, then 2 oz. The reaction was terminated by adding 0.6 ml of zinc sulfate and 0.66 ml of saturated barium hydroxide. Centrifuge the tube at 8,000g for 10 minutes. Ta. Supernatant liquid 1.0zl! and the following additives: Na5h reagent (ammonium acetate) 0.4 11+1! was added and the tubes were incubated in a 60°C water bath for 30 minutes. . The absorbance at a wavelength of 415 nm was immediately measured.

-Fiin' ゛ 1.48140+yg/L O, 26920 +yg/L O, 14010 y/L 　O,075 Ejun Nine Shadows In Examples A et al., thiopherin enzyme T-060 was used. This is Teoff 1,3-dimethyluric acid and hydrogen peroxide are produced in the presence of dilin. hydrogen peroxide Cl in Chew 26.227 <1980 by Fossati et al. It was measured by the known modified Trinder method described in ).

The analytical mixture consisted of 50 psoles/zN of potassium phosphate, pH 7.5, 3.5 -Dichloro-2-hydroxybenzenesulfonate hydrochloride (DHBS) 5Lm o1e/i1.4-aminoantipyridine lumole/zf, horseradish peroxidase 5, Ou/ml and theophylline enzyme T-0600, 7u/ 0.7 ml of the 0 analytical mixture containing z1 contained theophylline at the following concentrations: 50μr of sample were added into separate cuvettes. C11ford spectroscopy Reactions were carried out in a 10 mm optical path cuvette at 37° C. in a photometer. After 20 minutes, 5 The increase in absorbance at 10 n- was measured.

- Soi 1 inch 510nm 40ig/L O, 17220zy/L O, 089 10zy/L 0,045 1" 1L In this example, theophylline enzyme T-090 and cytochrome C were used. Teoff In the presence of dilin, the reaction produces 1,3-dimethyluric acid. This product , 1,3-dimethyluric acid was measured at 292 na, which is the wavelength of maximum absorbance.

The analytical mixture was 9H7.5 potassium phosphate Mtll solution 50 Holes/d, Theophylline enzyme T-0901,8u/zIl and cytochrome c 25mmol 0.5 mj of 0 analytical mixture containing es/i1! to theophylline at the following concentrations: 25 samples were added into separate cuvettes. Gllford spectroscopy The reaction was carried out in a 10 m-path cuvette at 30° C. in a thermometer. 20 minutes later, 29 Measuring the increase in optical density at 2ns+, this is due to theophylline in the sample. It was proportional to the concentration of

- 1 fan' 292 nm40zg/L O, 16230z y/L O, 12520zy/L 　　　　　　　　　　　　　O,08710zy/L　　 O, 036 cause J㇉m In this example, theophylline enzyme T-090 and potassium ferricyanide as receptors were used. I used um. It produces potassium ferrocyanide in the presence of theophylline. to be accomplished. The generated ferrocyanide was treated with ^van and 5havit. +aIBiochem 8,549 (1963) 4,7-di Chemically measured using phenyl-1゜10-phenanthroline sulfonate Ru.

The analytical mixture consisted of potassium phosphate buffer 5°Holes/i+1, pH 7.5; Theophylline enzyme T-0901, 8u/i1 and potassium ferricyanide T, 43 To 0.35 ml of one analytical mixture containing pmoles/z1, the following concentration of theophyl 50 ul of sample containing dilin was added. Gllford spectrophotometer, 3 The reaction was carried out in a cuvette with a 10 mm optical path at 0'Cr for 30 minutes. From the above, minutes 50ul of analytical mixture was mixed with 35μl of deionized water and 15hf of color forming reagent. The color forming reagent is acetic acid 0. In IH, sodium acetate IM, citric acid 0.066M, Ferric chloride 0.00055N and 4,7-diphenyl-1,10-7 enanthro Absorbance at a wavelength of 535 nm was measured after 6 minutes containing Phosphorus 83.3 uIF.

The absorbance was proportional to the theophylline concentration.

- Fin' 535nm 40mg/L O, 31930zy/L O, 241 20 zy/L O, 17110 zy/L O, 08G519/L O, 045 milk In this example, we used theophylline enzyme T-090 and ferricytochrome C as a receptor. It was used. This produces 1,3-dimethyluric acid and ferrochloride in the presence of theophylline. Produces cytochrome C. The production of ferrocytochrome C occurs at a wavelength of 550n- It is measured by the increase in absorbance.

The analytical mixture was prepared using 50 umoles/zL of potassium sulfate buffer in DB7.5. Filin enzyme T-0905L/ml and horse ferricitrochrome e 0.25 0.5x1 of the 0 analysis mixture containing 1 es/x1 was added with the following concentrations: 25ul of sample containing ophylline was added to a separate cuvette. Gllf Reactions were carried out in a cuvette with an Ions light path at 30° C. in an Ord spectrophotometer. 15 Once the reaction was complete after minutes, the increase in optical density was measured.

As in other examples, there is a direct relationship between absorbance and theophylline concentration in the sample. There is.

- 1 fan 550nm 40xg/L O, 45430xy/L O, 36 120zg/L O, 28410xy/L 0.11095x/L 0.11095x/L Follow up Add 0.05M solution containing theophylline enzyme T-09050u to a 10s+a square filter paper. Theophylline enzyme T-090 at various concentrations, such as phosphate buffer (p[I7.5) impregnated with a solution containing This solution contains ferricytochrome c lpmole/x It also included l. The filter paper was soaked in the above solution and air dried. Theophy in different concentrations When 50 μl of serum containing 5-40 my/'L of phosphorus is added to the filter paper, theophylline As the concentration of the dye increased, a deep pink shade appeared clearly.

Different theophylline concentrations can be assayed based on the shade of pink.

Engraving (no changes to the content) nano makeup Procedural amendment (formality) %formula% 2. Title of the invention Enzymatic determination of theophylline 3. Person making the correction Relationship to the incident: Patent applicant Name: G.D. Nissai Technology, Inc. 4. Agent: Yamada Building 5, 1-1-14 Shinjuku, Shinjuku-ku, Tokyo, amended order Date: April 16, 1991 6. Number of claims increased by amendment: None 7. Target of correction: Drawing translation 8. Contents of amendment (1) Supplement the engravings of the translations of the drawings as shown in the attached sheet.

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Claims (70)

[Claims] 1. Theophylline-utilizing enzymes and reagents that provide measurable signals in the presence of theophylline The amount of signal generated is measured, and this amount is calculated as the amount of theophylline in the sample. A method for measuring theophylline in a sample, characterized by correlating it with concentration. Law. 2. Selecting said sample from the group consisting of body fluids, food extracts and medical compositions. The method according to claim 1, characterized in that the method comprises: 3. A signal is generated when the theophylline present in the sample in contact with the enzyme is reduced. The method according to claim 1, characterized in that: 4. Confirm by observing the change in optical density of the sample at a wavelength of about 260 nm to about 280 nm. 4. The method according to claim 3, characterized in that: 5. The signal is the result of a change in the enzyme upon contact with theophylline present in the sample The method according to claim 1, characterized in that: 6. The increase in optical density of the sample at wavelengths ranging from about 411 nm to about 550 nm varies The method according to claim 5, characterized in that: 7. 2. A method according to claim 1, characterized in that an electron carrier is present. 8. A signal is generated when the theophylline present in the sample in contact with the enzyme is reduced. 8. The method according to claim 7, characterized in that: 9. Confirm by observing the change in optical density of the sample at a wavelength of about 260 nm to about 280 nm. 9. The method according to claim 8, characterized in that: 10. The signal is the result of a change in the enzyme that comes into contact with theophylline present in the sample. 8. The method according to claim 7, characterized in that: 11. The increase in optical density of the sample at wavelengths in the range of about 410 nm to about 550 nm is variable. 11. The method according to claim 10, characterized in that: 12. The method according to claim 10, characterized in that the change is measured electrochemically. Law. 13. 8. The method according to claim 7, wherein the electron carrier is an electron donor. 14. Reduced nicotinamide adenine dinucleotide, reduced nicotinamide Denine dinucleotide phosphate and reduced flavin adenine dinucleotide 14. The electron donor is selected from the group consisting of: Method described. 15. 8. The method according to claim 7, wherein the electron carrier is an electron acceptor. 16. 16. A method according to claim 15, characterized in that the electron acceptor is oxygen. 17. Cytochrome, ferricyanide, dichlorophenolindophenol, dichlorophenol Cotinamide adenine dinucleotide, phenazine methosulfate and methylene Claim characterized in that the electron acceptor is selected from the group consisting of The method according to item 15. 18. The interaction between theophylline-utilizing enzyme and theophylline present in the sample 2. The reaction product of claim 1, wherein the reaction product produced by the reaction product generates a measurable signal. How to put it on. 19. Claim 18, characterized in that the reaction product is 1,3-dimethyluric acid. Method described. 20. Measures the increase in optical density of the sample at wavelengths ranging from about 285 nm to about 305 nm. 20. The method according to claim 19, wherein 1,3-dimethyluric acid is determined by Method. 21. Specifically, hydrogen peroxide is generated as a reaction product and is also a measurable signal. 19. The method according to claim 18, wherein: 22. peroxidation in a system consisting essentially of a color-forming reagent that can change color in the presence of hydrogen peroxide. 22. The method according to claim 21, characterized in that hydrogen is measured. 23. A claim characterized in that a change in the electron carrier results in a measurable signal. The method described in 7. 24. Electron carrier is nicotinamide adenine dinucleotide and can be measured 8. A method according to claim 7, characterized in that the signal HADH is generated. 25. The electron carrier is ferricytochrome c, and ferrocytochrome c is measured 8. The method according to claim 7, characterized in that it is generated as an enable signal. 26. The electron carrier is a fluorophore that generates potassium ferrocyanide as a measurable signal. 8. The method according to claim 7, characterized in that it is potassium ericyanide. 27. Flavin adenine nuclei produce a measurable signal when the electron carrier is reduced 8. The method according to claim 7, characterized in that it is leotide. 28. 2,6 dichlorophenol that produces a measurable signal when the electron carrier is reduced 8. The method according to claim 7, characterized in that the indophenol is phenol. 29. Theophylline-utilizing enzyme is a glutamic acid consisting of xanthine and xanthine derivatives. characterized in that theophylline can be converted into a reaction product selected from among 19. The method according to claim 18. 30. In the presence of xanthine oxidase, appropriate hydrogen oxide is generated and the reaction product is 19. The method according to claim 18, characterized in that measuring. 31. Measurement of reaction products in the presence of xanthine dehydrogenase and electron acceptor 19. The method according to claim 18, characterized in that: 32. Formaldehyde is produced as a reaction product and is a measurable signal 19. The method according to claim 18, characterized in that: 33. Theophylline-utilizing enzymes capable of generating measurable signals in the presence of theophylline and means for measuring and correlating the signal to the amount of theophylline present in the sample. A test composition for measuring theophylline in a sample, characterized by comprising: . 34. A signal occurs when the theophylline present in the sample in contact with the enzyme is reduced. The test composition according to claim 33, characterized in that: 35. Observing the change in optical density of the sample at a wavelength of about 260 nm to about 280 nm 35. The test composition according to claim 34, wherein a signal is measured. 36. The signal is the result of a change in the enzyme that comes into contact with theophylline present in the sample. The test composition according to claim 33, characterized in that: 37. The increase in optical density of the sample at wavelengths in the range of about 410 nm to about 550 nm is variable. 37. The test composition according to claim 36, which is 38. The test composition according to claim 33, characterized in that an electron carrier is present. . 39. A signal occurs when the theophylline present in the sample in contact with the enzyme is reduced. The test composition according to claim 38, characterized in that: 40. Observe the change in optical density of the sample at wavelengths ranging from about 260 nm to about 280 nm. 40. The test composition according to claim 39, wherein the test composition is used to detect and measure a signal. 41. The signal is the result of a change in the enzyme that comes into contact with theophylline present in the sample. The test composition according to claim 38, characterized in that: 42. The increase in optical density of the sample at wavelengths in the range of about 410 nm to about 550 nm is variable. 42. The test composition according to claim 41, which is 43. The method for testing according to claim 41, characterized in that the change is measured electrochemically. Composition. 44. Test according to claim 38, characterized in that the electron carrier is an electron donor. Composition for use. 45. Reduced nicotinamide adenine dinucleotide, reduced nicotinamide Denine dinucleotide phosphate and reduced flavin adenine dinucleotide 45. The electron donor is selected from the group consisting of: Test composition as described. 46. The test according to claim 38, characterized in that the electron carrier is an electron acceptor. Composition for use. 47. 47. The test composition according to claim 46, wherein the electron acceptor is oxygen. thing. 48. Cytochrome, ferricyanide, dichlorophenolindophenol, dichlorophenol Cotinamide adenine dinucleotide, phenazine methosulfate and methylene A claim characterized in that an electron acceptor is selected from a groove consisting of 47. The test composition according to item 46. 49. The means includes the presence of a reaction product between a theophylline-utilizing enzyme and theophylline. The test composition according to claim 33, which is a reagent that reacts with . 50. Test according to claim 49, characterized in that the reaction product is hydrogen peroxide. Composition for use. 51. The reagent is selected from the group consisting of xanthine and xanthine derivatives. The test composition according to claim 49, characterized in that it reacts with a reaction product obtained by . 52. Claim 51, wherein the reagent comprises xanthine oxidase. The test composition described in . 53. The reagent further includes peroxidase and a redox indicator. 51. The test composition according to claim 50, characterized in that: 54. 50. The method according to claim 49, wherein the reagent reacts with formaldehyde. Composition for testing. 55. 50. The method according to claim 49, wherein the reagent reacts with 1,3-dimethyluric acid. The test composition described above. 56. A group consisting of spectrophotometry, electrochemical methods, fluorescence methods, reflectance methods and polarization methods. 3. A claim characterized in that the signal is measured using means selected from a group of 34. The test composition according to 33. 57. Theophylline-utilizing enzymes capable of generating measurable signals in the presence of theophylline comprising a solid matrix mixed with a residue of a composition consisting essentially of A testing device for measuring theophylline in a sample, characterized by: 58. 58. The composition further comprises an electron carrier. equipment for inspection. 59. Test according to claim 58, characterized in that the electron carrier is an electron donor. equipment. 60. Reduced nicotinamide adenine dinucleotide, reduced nicotinamide Denine dinucleotide phosphate and reduced flavin adenine dinucleotide Claim 59, characterized in that the electron donor is selected from the group consisting of Test equipment as described. 61. The test according to claim 58, characterized in that the electron carrier is an electron acceptor. equipment. 62. 62. The testing device according to claim 61, wherein the electron acceptor is oxygen. . 63. Cytochrome, ferricyanide, dichlorophenolindophenol, dichlorophenol Cotinamide adenine dinucleotide, phenazine methosulfate and methylene Claim characterized in that the electron acceptor is selected from the group consisting of 62. The testing device according to item 61. 64. A reaction product generated when theophylline-utilizing enzyme acts on existing theophylline. 58. The testing device according to claim 57, wherein the composition reacts with the product. 65. 58. The test device according to claim 57, which measures changes in enzymes. 66. 58. A method according to claim 57, characterized in that the reduction in theophylline present is determined. Inspection equipment included. 67. Testing device according to claim 57, characterized in that the solid matrix is paper. Device. 68. 58. A method according to claim 57, characterized in that the solid matrix is a polymer. Inspection equipment. 69. Testing device according to claim 58, characterized in that the solid matrix is paper. Device. 70. 59. A method according to claim 58, characterized in that the solid matrix is a polymer. Inspection equipment.