Blood ammonia determination kit

Abstract

The invention discloses a blood ammonia determination kit. The kit comprises a reagent 1 (R1) and a reagent 2 (R2), the kit is characterized in that the reagent 1 consists of the following components: a buffer solution, inorganic salt, glutamic acid, ATP, TOOS, peroxidase, glutamine synthetase, pyruvate oxidase, pyruvic acid and a preservative; the reagent 2 is prepared from the following components: a buffer solution, a surfactant, MBTH, a deoxidant, BSA and a preservative. The kit disclosed by the invention takes Trinder reaction as a basic principle, develops color in an MBTH + chromogen mode, successfully solves the problem of poor airborne stability by adding a deoxidant and stabilizing MBTH by specific surface activity, and is relatively good in overall performance evaluation result of the reagent.

Description

Technical Field

[0001] This invention relates to the field of biochemical analysis, specifically to a blood ammonia (AMM) assay kit (Trinder method). Background Technology

[0002] Ammonia in the human body is produced during protein metabolism through amino acid deamination, the breakdown of glutamine in the kidneys, and the action of intestinal bacteria. Most ammonia is synthesized into urea in the liver via the ornithine cycle. A portion is used for the amination of keto acids to synthesize glutamine, which forms ammonium salts in the kidneys and is excreted in the urine. Clinically, elevated blood ammonia levels are seen in hepatic coma, severe hepatitis, liver tumors, shock, uremia, organophosphate poisoning, congenital hyperammonemia, and transient hyperammonemia in infants. Decreased blood ammonia levels are seen in low-protein diets and anemia. Blood ammonia plays an important role in the diagnosis and treatment of hepatic coma and hepatic encephalopathy. Methods for measuring blood ammonia include ion exchange resin methods, direct methods, electrode methods, and enzymatic methods, among which enzymatic methods are widely used due to their simplicity and high specificity.

[0003] Currently, most commercially available reagents for blood ammonia-enzyme assays utilize the following reaction involving glutamate dehydrogenase (GLDH), α-ketoglutarate (α-KG), and reduced nicotinamide adenine dinucleotide (NADH):

[0004] This method has high specificity and low cost, and is currently the mainstream detection method in the market. However, this method is susceptible to contamination from the detection environment and instrument status, resulting in poor repeatability. In addition, there is also the problem of increased blank reaction during airborne testing.

[0005] A search of Chinese patents revealed a patent (publication number CN1749755A) for a blood ammonia assay kit (Trinder method). This kit uses glutamine synthase as a catalyst and calculates blood ammonia levels by measuring the amount of hydrogen peroxide generated during the reaction. The principle is as follows:

[0006] This method has high sensitivity and good accuracy, but it has the following drawbacks: 1. When using the 4AA (4-aminoantipyrine) + chromogen method for colorimetric reaction, the sensitivity is lower than that of the MBTH (3-methyl-2-phenylthiazolinone hydrazone) + chromogen method, resulting in poor repeatability of low-value samples.

[0007] 2 When using MBTH (3-methyl-2-phenylthiazolinone hydrazone) + chromogen for colorimetric reaction, MBTH is easily oxidized, causing it to decompose due to air oxidation during the onboard process after the reagent is opened, resulting in a rapid decrease in sensitivity.

[0008] Both of the above methodologies have shortcomings, and there is currently no blood ammonia assay kit that is both highly accurate and stable. Summary of the Invention

[0009] To address the technical problems in existing methods, this invention provides a blood ammonia assay kit with high accuracy, strong anti-interference ability, and good stability.

[0010] This invention utilizes the basic principle of the Trinder method for determining blood ammonia, employing a colorimetric method using MBTH + chromogen. MBTH, as a highly sensitive colorimetric reagent, is standardized for industrial testing, including the determination of indicators such as formaldehyde and total acrylonitrile aldehydes. However, due to its susceptibility to oxidation and poor stability in aqueous solutions, it is generally used in freshly prepared laboratory tests and is difficult to apply to biochemical reagents with long shelf lives. To address the problem of poor onboard stability of MBTH caused by its susceptibility to oxidation, this invention improves the stability of MBTH in reagents by adding an oxygen scavenger and a specific surfactant, effectively solving its onboard stability problem.

[0011] The technical solution of the present invention is as follows: A blood ammonia (AMM) assay kit (Trinder method) includes reagent 1 and reagent 2. Reagent 1 contains glutamate, ATP, TOOS, peroxidase, glutamine synthase, pyruvate oxidase, and pyruvate; reagent 2 contains 3-methyl-2-benzothiazolinone hydrazone (MBTH), an oxygen scavenger, a surfactant, and BSA.

[0012] The reagent 1 consists of the following components at the following mass concentrations: Buffer solution 20~200mmol / L Inorganic salts 5g / L~40g / L Glutamic acid 0.1g / L~0.5g / L ATP 5g / L~10g / L TOOS 1g / L~5g / L Peroxidase 1KU / L~10KU / L Glutamine synthase 1KU / L~10KU / L Pyruvate oxidase 10KU / L~50KU / L Pyruvate 1g / L~5g / L Preservatives: 0.05g / L~2g / L The components and concentrations of reagent 2 are as follows: Buffer solution 20~100mmol / L Surfactant 5g / L~40g / L MBTH 1g / L~5g / L Oxygen scavenger 1 g / L~5 g / L BSA 1g / L~5g / L Preservatives: 0.05g / L~2g / L.

[0013] The buffer solutions in reagents 1 and 2 are each independently selected from one or more of the following: Tris buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, and glycine buffer. The inorganic salt in reagent 1 is selected from one or more of sodium chloride, potassium chloride, and calcium chloride. The surfactant in reagent 2 is selected from one or more of the following: alkoxyethylene hydroxyethanol series: NP7, NP9, NP10, NP40S; The oxygen scavenger in reagent 2 is selected from one or more of the following: acetone oxime, hydrazine, sodium sulfite, and sodium thiosulfate.

[0014] The preservatives in reagents 1 and 2 are selected from one or more of sodium azide, PC300, and sodium benzoate.

[0015] Furthermore, the kit, while meeting accuracy, linearity, repeatability, and clinical requirements, exhibits airborne stability of at least 14 days.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses the Trinder reaction as the basic measurement principle and MBTH + chromogen for color development. By adding an oxygen scavenger and a specific surfactant to stabilize MBTH, it successfully solves the problem of poor onboard stability of the blood ammonia reagent (Trinder method). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below with reference to four comparative examples and two embodiments.

[0018] Key differences between the different options:

[0019] Different preparation methods: Comparative Example 1: Preparation of a serum ammonia (AMM) assay kit (glutamate dehydrogenase method) Reagent 1 (R1) is: Tris buffer 50 mmol / L (pH 7.40) Sodium chloride 10g / L Disodium ethylenediaminetetraacetate 5g / L NADH 0.5g / L Ketoglutarate 1.5 g / L lactate dehydrogenase 10 kU / L Ascorbic acid oxidase 2KU / L Preservative: Sodium azide 0.5g / L Reagent 2 (R2) is: Tris buffer 25 mmol / L (pH 7.40) Triton 100 5g / L BSA 5g / L Glutamate dehydrogenase 15 KU / L Preservative: Sodium azide 0.5g / L Comparative Example 2: Preparation of a serum ammonia (AMM) assay kit (Trinder method) Reagent 1 (R1) is: Glycine buffer 50 mmol / L Sodium chloride 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthase 2KU / L Pyruvate oxidase 10 KU / L Pyruvate 1g / L~5g / L Sodium azide 0.5 g / L Reagent 2 (R2) is: Glycine buffer 50 mmol / L MBTH 1g / L BSA 5g / L Sodium azide 1g / L Control Example 3: Preparation of a Blood Ammonia (AMM) Assay Kit (Trinder Method) Reagent 1 (R1) is: Glycine buffer 50 mmol / L Sodium chloride 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthase 2KU / L Pyruvate oxidase 10 KU / L Pyruvate 1g / L~5g / L Sodium azide 0.5 g / L Reagent 2 (R2) is: Glycine buffer 50 mmol / L Surfactant 1: Brij-35 5g / L MBTH 1g / L Oxygen scavenger 1: Ascorbic acid 2g / L BSA 5g / L Sodium azide 1g / L Comparative Example 4: Preparation of a Blood Ammonia (AMM) Assay Kit (Trinder Method) Reagent 1 (R1) is: Glycine buffer 50 mmol / L Sodium chloride 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthase 2KU / L Pyruvate oxidase 10 KU / L Pyruvate 1g / L~5g / L Sodium azide 0.5 g / L Reagent 2 (R2) is: Glycine buffer 50 mmol / L Surfactant 2: TWEEN20 5g / L MBTH 1g / L Oxygen scavenger 2: Glutathione 2g / L BSA 5g / L Sodium azide 1g / L Example 1: Preparation of a blood ammonia (AMM) assay kit (Trinder method) Reagent 1 (R1) is: Glycine buffer 50 mmol / L Sodium chloride 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthase 2KU / L Pyruvate oxidase 10 KU / L Pyruvate 1g / L~5g / L Sodium azide 0.5 g / L Reagent 2 (R2) is: Glycine buffer 50 mmol / L NP7 10g / L MBTH 1g / L Acetone oxime 5g / L BSA 5g / L Sodium azide 1g / L Example 2: Preparation of a blood ammonia (AMM) assay kit (Trinder method) Reagent 1 (R1) is: Glycine buffer 50 mmol / L Sodium chloride 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthase 2KU / L Pyruvate oxidase 10 KU / L Pyruvate 1g / L~5g / L Sodium azide 0.5 g / L Reagent 2 (R2) is: Glycine buffer 50 mmol / L NP10 8g / L MBTH 1g / L Hydrazine 3g / L BSA 5g / L Sodium azide 1g / L 1. Performance evaluation was conducted on Comparative Examples 1-4 and Examples 1-2. The evaluation indicators and methods are as follows: 1.1 Calibration and quality control: the deviation between the quality control mean and the target value should not exceed ±10%. 1.2 Repeatability: Two plasma samples with high and low concentrations were tested, with each sample tested 10 times. The coefficient of variation was calculated. 1.3 Linearity: Within the range of [8, 300] μmol / L, select at least 5 uniformly distributed concentration levels, repeat the test 3 times for each level, and compare the linear correlation coefficient r ≥ 0.995. The relative deviation within the range of (100, 300] μmol / L should not exceed ±10%, and the absolute deviation within the range of [8, 300] μmol / L should not exceed ±10 μmol / L.

[0020] 1.4 Accuracy: Recovery test, recovery rate within the range of 90% to 110%. 1.5 Clinical comparison: Plasma samples from 20 random patients were measured, and the correlation and measurement deviations among the groups were compared. 1.6 Airborne stability test: Under airborne conditions, water quality is monitored regularly for 30 days, and no recalibration is performed at each time point during the monitoring process.

[0021] 1.7 Accelerated stability test: Under 37℃, the reagent values ​​at different time points were detected using the synchronous isochronous method. 2. Evaluation Results: 2.1 Calibration and Quality Control

[0022] 2.2 Repeatability

[0023] 2.3 Linear

[0024] 2.4 Accuracy

[0025] 2.5 Clinical Comparison

[0026] 2.6 Airborne Stability

[0027] 2.7 Accelerated stability (37℃ for 14 days)

[0028] 3. Summary of conclusions: 3.1 Calibration: Due to the use of the Trinder method, the parameters and calibration results of Comparative Examples 2 / 3 / 4 and Example 1 / 2 are significantly different from those of Comparative Example 1, but this difference does not affect practical use; 3.2 Quality control, linearity, accuracy, and clinical comparison were all satisfactory for all groups, with no significant differences among the groups; repeatability: control case 1 (glutamate dehydrogenase method) was worse than the other 5 groups (Trinder method).

[0029] 3.3 Key Improvements: 3.3.1 Airborne stability: In Control Example 1, the blank response increased significantly with prolonged airborne time, showing a significant increase starting from day 4. Due to the large variation in measured values, monitoring was only conducted for 14 days. In Control Examples 2 / 3 / 4, due to MBTH stability failure, the measured values ​​decreased significantly with prolonged time (more than 7 days). In Control Examples 3 / 4, the addition of some common surfactants and scavengers did not show significant improvement. In Examples 1 / 2, after the addition of specific surfactants and scavengers, the measured values ​​did not change significantly within 14 days of airborne operation, showing a significant improvement in airborne stability compared to Control Examples 1 / 2 / 3 / 4.

[0030] 3.3.3 Accelerated stability: Control Example 1 (glutamate dehydrogenase method) was stable for about 11 days after acceleration; Control Examples 2 / 3 / 4 showed a significant decrease in measured values ​​due to the failure of MBTH as the acceleration time at 37℃ increased. Examples 1 / 2 showed relatively stable measured values ​​after 14 days of acceleration with the addition of specific surfactants and scavengers.

[0031] Based on the above evaluation results, the present invention has significant improvements in the following aspects: 1. Compared with mainstream blood ammonia (AMM) assay kits (glutamate dehydrogenase method), it has significant improvements in repeatability and onboard stability; 2. Compared with known ammonia (AMM) assay kits (Trinder method), the addition of specific surfactants and oxygen scavengers significantly improved airborne stability.

[0032] Under the same conditions, if the applicant changes the type of surfactant and oxygen scavenger, such as sodium sulfite, sodium thiosulfate or a combination of two of acetone oxime, hydrazine, sodium sulfite, and sodium thiosulfate, or a combination of at least two of the alkoxyethylene hydroxyethanol series: NP7, NP9, NP10, and NP40S, its airborne stability can reach at least 14 days.

[0033] The specific examples described above are merely illustrative of the invention and are intended to aid in understanding the invention; they are not intended to limit the invention. Those skilled in the art to which this invention pertains can make various modifications and improvements based on the concept of this invention, and these modifications and improvements all fall within the scope of protection of this invention.

[0034] Any aspects not covered in this invention are applicable to existing technologies.

Claims

1. A blood ammonia measuring kit, characterized by comprising: Reagent 1 and reagent 2, the reagent 1 comprising glutamic acid, ATP, TOOS, peroxidase, glutamine synthetase, pyruvate oxidase, pyruvic acid; the reagent 2 comprising 3-methyl-2-benzothiazolone hydrazone MBTH, oxygen scavenger, surfactant, BSA.

2. The blood ammonia measuring kit according to claim 1, characterized by The surfactant in the reagent 2 is selected from one or more of the following: alkoxylated polyethylene hydroxy ethanol series: NP7, NP9, NP10, NP40S; the oxygen scavenger in the reagent 2 is selected from one or more of the following: acetone oxime, hydrazine, sodium sulfite, sodium thiosulfate.

3. A blood ammonia measuring kit characterized by comprising: The reagent 1 and the reagent 2, The components and concentrations of the reagent 1 are as follows: Buffer 20~200mmol / L Inorganic salt 5g / L~40g / L Glutamic acid 0.1g / L~0.5g / L ATP 5g / L~10g / L TOOS 1g / L~5g / L Peroxidase 1KU / L~10KU / L Glutamine synthetase 1KU / L~10KU / L Pyruvate oxidase 10KU / L~50KU / L Pyruvic acid 1g / L~5g / L Preservative 0.05g / L~2g / L The components and concentrations of the reagent 2 are as follows: Buffer 20~100mmol / L Surfactant 5g / L~40g / L MBTH 1g / L~5g / L Oxygen scavenger 1 g / L~5g / L BSA 1g / L~5g / L Preservative 0.05g / L~2g / L The surfactant in the reagent 2 is selected from one or more of the following: alkoxylated polyethylene hydroxy ethanol series: NP7, NP9, NP10, NP40S; The oxygen scavenger in the reagent 2 is selected from one or more of the following: acetone oxime, hydrazine, sodium sulfite, sodium thiosulfate.

4. The blood ammonia measuring kit according to claim 1, wherein The buffer in the reagent 1 and the reagent 2 is independently selected from one or more of the following: Tris buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 3-[N-N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, glycine buffer; The inorganic salt in the reagent 1 is selected from one or more of the following: sodium chloride, potassium chloride, calcium chloride; The preservative in the reagent 1 and the reagent 2 is selected from one or more of the following: sodium azide, PC300, sodium benzoate.

5. The blood ammonia measuring kit according to claim 3, wherein The reagent 1 and the reagent 2, The components and concentrations of the reagent 1 (R1) are as follows: Buffer 50mmol / L Inorganic salt 5g / L Glutamic acid 0.5g / L ATP 5g / L TOOS 1g / L Peroxidase 10KU / L Glutamine synthetase 2KU / L Pyruvate oxidase 10KU / L Pyruvic acid 1g / L~5g / L Preservative 0.5g / L The components and concentrations of the reagent 2 (R2) are as follows: Buffer 50mmol / L Surfactant 8g / L MBTH 1g / L Oxygen scavenger 3g / L BSA 5g / L Preservative 1g / L.

6. The blood ammonia measuring kit according to any one of claims 1 to 5, characterized by, The kit is catalyzed by enzyme The blood ammonia generates hydrogen peroxide, and then the Trinder reaction is carried out to develop color, and the degree of color development is proportional to the concentration of blood ammonia, which is specifically as follows:

7. The blood ammonia measuring kit according to any one of claims 1 to 5, characterized by, The kit has on-board stability of at least 14 days under the premise of meeting accuracy, linearity, repeatability, and clinical requirements. The kit has on-board stability of at least 14 days under the premise of meeting accuracy, linearity, repeatability, and clinical requirements.

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