Nicotinamide agent and preparation method thereof

Abstract

The present invention is reducing nicotine amide coenzyme as enzyme testing reagent for testing body fluid. The enzyme testing reagent of the present invention includes oxidizing nicotine amide coenzyme, corresponding enzyme and substrate. The oxidizing coenzyme is reduced into reducing nicotine amide coenzyme slowly to test the tested matter. The reagent includes alanine aminotransferase reagent, aspartic acid aminotransferase reagent, urea reagent, ammonia reagent, creatinine reagent, CO2 reagent, etc. Owing to constant creation of nicotine amide coenzyme, the present invention has greatly raised reagent stability and greatly lowered reagent cost.

Description

technical field

[0001] The invention relates to a reagent for measuring reduced nicotinamide coenzyme (NADH or NADPH) and a preparation method thereof. Background technique

[0002] Using reduced nicotinamide coenzyme (NADH or NADPH) to determine the analyte in the body fluid sample is a widely used method, and the oxidation degree of NADH or NADPH in the reagent is directly proportional to the concentration of the analyte in the sample. According to the different analytes in the sample, the corresponding enzyme reaction mechanism is adopted. After the enzyme reacts with the substrate, NADH or NADPH in the reagent is oxidized, and the absorbance of the reaction system changes. By measuring the absorbance change value of the reaction system, it can be calculated Come out the concentration of the analyte in the book.

[0003] For example, the determination of ammonia concentration in plasma samples, ammonia in plasma reacts with α-ketoglutarate under the action of glutamate d...

Examples

Embodiment 1

[0113] Embodiment 1: prepare alanine nitrogen base transferase reagent, be made up of the following components by the content of every liter,

[0114] Tris buffer 100mM pH7.15, 37°C,

[0115] L~alanine 500mM,

[0116] Lactate dehydrogenase 2.7ku / L,

[0117] α-ketoglutaric acid 15mM,

[0118] EDTA.Na 2 .2H 2 O 60mM

[0119] NAD 0.27mM

[0120] D~glucose 50mM.

[0121] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700U / L,

[0122] Dipotassium hydrogen phosphate 5mM

[0123] Sodium azide 1.5mM

[0124] According to the method of the above reagent solution, after the preparation of the above alanine aminotransferase reagent is completed, place it at 2-8°C for 1-3 days, and the reagent blank absorbance is ≥1.10, 340nm, 10mm optical path. When measuring the sample, use the rate method, the sample reagent ratio is 1:12, the temperature is 37°C, the delay time is 90 seconds, the measurement time is 180 seconds, and the number of readings is ≥ 6 points.

Embodiment 2

[0125] Embodiment 2: preparation aspartate aminotransferase reagent, is made up of the following components by the content of every liter,

[0126] Tris buffer 80mM, pH7.50, 37°C,

[0127] L~aspartic acid 240mM,

[0128] Malate dehydrogenase 600U / L

[0129] Lactate dehydrogenase 1000U / L

[0130] a~Ketoglutarate 12mM,

[0131] EDTA.Na 2 .2H 2 O 6mM

[0132] NAD 0.28mM

[0133] D~glucose 80mM.

[0134] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700U / L,

[0135] Dipotassium hydrogen phosphate 5mM

[0136] Sodium azide 1.5mM

[0137] According to the above method for preparing the reagent solution, after the preparation of the above aspartate aminotransferase reagent is completed, place it at 2-8°C for 1-5 days, and the reagent blank absorbance is ≥1.20, 340nm, 10mm optical path. When measuring the sample, use the rate method, the sample reagent ratio is 1:12, the temperature is 37°C, the delay time is 90 seconds, the measurement time is 180 seconds, and the numbe...

Embodiment 3

[0138] Embodiment 3: prepare urea reagent, be made up of following component by the content of every liter,

[0139] Tris buffer pH7.50, 37°C, 14.54g

[0140] Alpha-ketoglutarate 1.43g

[0141] Bovine Serum Acid 5.0g

[0142] Adenosine diphosphate potassium salt 0.65

[0143] Glutamate dehydrogenase (microbial source) 550u

[0144] Urease 12000u

[0145]NAD 0.192g

[0146] D~glucose 18.0g

[0147] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700u

[0148] Dipotassium hydrogen phosphate 0.87g

[0149] EDTA.Na 2 .2H 2 O 0.37g

[0150] Sodium azide 0.26g

[0151] According to the method for preparing the reagent solution above, after the preparation of the above urea reagent is completed, place it at 2-8°C for 1-7 days, and the reagent blank absorbance is ≥1.20, 340nm, 10mM optical path. When measuring the sample or calibrator, use the two-point method, the sample reagent ratio is 1:100, the temperature is 37°C, the delay time is 30 seconds, the measurement time i...

technical field

[0001] The invention relates to a reagent for measuring reduced nicotinamide coenzyme (NADH or NADPH) and a preparation method thereof. Background technique

[0002] Using reduced nicotinamide coenzyme (NADH or NADPH) to determine the analyte in the body fluid sample is a widely used method, and the oxidation degree of NADH or NADPH in the reagent is directly proportional to the concentration of the analyte in the sample. According to the different analytes in the sample, the corresponding enzyme reaction mechanism is adopted. After the enzyme reacts with the substrate, NADH or NADPH in the reagent is oxidized, and the absorbance of the reaction system changes. By measuring the absorbance change value of the reaction system, it can be calculated Come out the concentration of the analyte in the book.

[0003] For example, the determination of ammonia concentration in plasma samples, ammonia in plasma reacts with α-ketoglutarate under the action of glutamate d...

Examples

Embodiment 1

[0113] Embodiment 1: prepare alanine nitrogen base transferase reagent, be made up of the following components by the content of every liter,

[0114] Tris buffer 100mM pH7.15, 37°C,

[0115] L~alanine 500mM,

[0116] Lactate dehydrogenase 2.7ku / L,

[0117] α-ketoglutaric acid 15mM,

[0118] EDTA.Na 2 .2H 2 O 60mM

[0119] NAD 0.27mM

[0120] D~glucose 50mM.

[0121] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700U / L,

[0122] Dipotassium hydrogen phosphate 5mM

[0123] Sodium azide 1.5mM

[0124] According to the method of the above reagent solution, after the preparation of the above alanine aminotransferase reagent is completed, place it at 2-8°C for 1-3 days, and the reagent blank absorbance is ≥1.10, 340nm, 10mm optical path. When measuring the sample, use the rate method, the sample reagent ratio is 1:12, the temperature is 37°C, the delay time is 90 seconds, the measurement time is 180 seconds, and the number of readings is ≥ 6 points.

Embodiment 2

[0125] Embodiment 2: preparation aspartate aminotransferase reagent, is made up of the following components by the content of every liter,

[0126] Tris buffer 80mM, pH7.50, 37°C,

[0127] L~aspartic acid 240mM,

[0128] Malate dehydrogenase 600U / L

[0129] Lactate dehydrogenase 1000U / L

[0130] a~Ketoglutarate 12mM,

[0131] EDTA.Na 2 .2H 2 O 6mM

[0132] NAD 0.28mM

[0133] D~glucose 80mM.

[0134] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700U / L,

[0135] Dipotassium hydrogen phosphate 5mM

[0136] Sodium azide 1.5mM

[0137] According to the above method for preparing the reagent solution, after the preparation of the above aspartate aminotransferase reagent is completed, place it at 2-8°C for 1-5 days, and the reagent blank absorbance is ≥1.20, 340nm, 10mm optical path. When measuring the sample, use the rate method, the sample reagent ratio is 1:12, the temperature is 37°C, the delay time is 90 seconds, the measurement time is 180 seconds, and the numbe...

Embodiment 3

[0138] Embodiment 3: prepare urea reagent, be made up of following component by the content of every liter,

[0139] Tris buffer pH7.50, 37°C, 14.54g

[0140] Alpha-ketoglutarate 1.43g

[0141] Bovine Serum Acid 5.0g

[0142] Adenosine diphosphate potassium salt 0.65

[0143] Glutamate dehydrogenase (microbial source) 550u

[0144] Urease 12000u

[0145]NAD 0.192g

[0146] D~glucose 18.0g

[0147] Glucose-6-phosphate dehydrogenase (Leuconostoc) 3700u

[0148] Dipotassium hydrogen phosphate 0.87g

[0149] EDTA.Na 2 .2H 2 O 0.37g

[0150] Sodium azide 0.26g

[0151] According to the method for preparing the reagent solution above, after the preparation of the above urea reagent is completed, place it at 2-8°C for 1-7 days, and the reagent blank absorbance is ≥1.20, 340nm, 10mM optical path. When measuring the sample or calibrator, use the two-point method, the sample reagent ratio is 1:100, the temperature is 37°C, the delay time is 30 seconds, the measurement time i...

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