Double-reagent ammonia determination kit and determination method of ammonia content in sample

Abstract

The invention provides a double-reagent ammonia determination kit and a determination method of ammonia content in a sample. The double-reagent ammonia determination kit comprises a reagent R1 and a reagent R2, and each of the reagent R1 and the reagent R2 comprises reduced coenzyme, alpha-ketoglutaric acid and glutamate dehydrogenase; the concentration ratio of the reduced coenzyme in the R1 reagent to the R2 reagent is (2-5): 1, and the final concentration of the reduced coenzyme after the detection sample, the R1 reagent and the R2 reagent are mixed is 0.1-0.5 mmol / L; the active concentration ratio of the glutamate dehydrogenase in the R1 reagent to the R2 reagent is 1: (2-100), and the final concentration of the glutamate dehydrogenase after the detection sample, the R1 reagent and the R2 reagent are mixed is 10-15KU / L; the final concentration of the alpha-ketoglutaric acid is 1-90 mmol / L after the detection sample, the R1 reagent and the R2 reagent are mixed. The method can solve the problems of low airborne bottle opening stability and poor anti-interference performance of an ammonia determination method and a kit in the prior art, and is suitable for the field of ammonia detection.

Description

Technical Field

[0001] This invention relates to the field of ammonia detection, and more specifically, to a two-reagent ammonia assay kit and a method for determining the ammonia content in a sample. Background Technology

[0002] Ammonia is a neurotoxic substance produced during the breakdown of proteins and amino acids, and its concentration is of great significance for the diagnosis and treatment of diseases such as hepatic encephalopathy. There are various methods for detecting blood ammonia, including ion-selective electrode methods, gas chromatography, dry chemistry methods, and enzymatic methods. Among these, enzymatic methods are widely used for blood ammonia determination. The principle of blood ammonia determination utilizes a reaction catalyzed by glutamate dehydrogenase (GLDH). In aqueous solution, GLDH catalyzes the reductive amination of α-ketoglutarate with NH4+ and NADPH / NADH, thereby forming glutamate and NADP. + The details are as follows:

[0003]

[0004] Taking blood ammonia detection as an example, current blood ammonia detection reagents include single-reagent and dual-reagent detection systems. In single-reagent systems, because the single reagent detects the signal early in the reaction, there is insufficient time to eliminate interfering substances such as pyruvate and fats in the sample that affect signal detection. Dual-reagent systems, on the other hand, use an endpoint method for detection, which can effectively solve the interference problem. However, in practical applications, dual-reagent detection systems face onboard stability issues. The main reason for the poor onboard stability of blood ammonia reagents is the generation of impurity ammonia. The sources of impurity ammonia are mainly twofold: absorption of ammonia from the environment during use and ammonia generated from the degradation of various proteins and raw materials such as NADH.

[0005] Blood ammonia detection operates at the μmol level, and even trace amounts of ammonia (AMM) can significantly interfere with the results. CN113075139B discloses a stable, enzyme-kinetic-based two-reagent blood ammonia assay kit that eliminates interference from ammonia impurities. This method utilizes L-glutamine synthase and disodium glutarate to ensure reagent stability without affecting the main reaction. However, this enzyme has a large Michaelis constant and insufficient sensitivity to ammonia, resulting in poor effectiveness in eliminating ammonia impurities in practice. Furthermore, the L-glutamine synthase used in existing methods is expensive, which may limit its widespread use.

[0006] Therefore, developing a low-cost ammonia assay kit that can efficiently remove impurities and has good stability and anti-interference properties is the main challenge facing current technology. Summary of the Invention

[0007] The main objective of this invention is to provide a dual-reagent ammonia assay kit with good onboard opening stability and strong anti-interference performance, as well as a method for determining the ammonia content in a sample, in order to solve the problems of low onboard opening stability and poor anti-interference performance of existing methods and kits for ammonia determination.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a dual-reagent ammonia assay kit is provided, comprising reagent R1 and reagent R2, both of which contain reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase; wherein the concentration ratio of reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L; the activity concentration ratio of glutamate dehydrogenase in reagent R1 to reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L; and the final concentration of α-ketoglutarate after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L.

[0009] Further, in the dual-reagent ammonia assay kit, reagent R1 comprises: a buffer solution with a pH of 7.5-9.5, 0.15-0.5 mmol / L reduced coenzyme, 2-100 mmol / L α-ketoglutarate, and 1-10 KU / L glutamate dehydrogenase; reagent R2 comprises: a buffer solution with a pH of 7.5-9.5, 0.05-0.1 mmol / L reduced coenzyme, 2-100 mmol / L α-ketoglutarate, and 20-100 KU / L glutamate dehydrogenase; preferably, the reduced coenzyme comprises N... One or more of ADH, NADPH, Thio-NADH, Thio-NADPH, or APADH; preferably, reagent R1 further includes lactate dehydrogenase, the concentration of which in reagent R1 is 5-50 KU / L; preferably, reagent R1 further includes a first surfactant, the first surfactant including one or more of laurate, isodecyl alcohol polyoxyethylene ether, or stilbene phenol polyoxyethylene ether-18.8; preferably, the concentration of which isodecyl alcohol polyoxyethylene ether in reagent R1 is 1wt%-5wt%.

[0010] Furthermore, reagent R1 also includes a second surfactant, which includes one or more of polyoxyethylene fatty alcohol ether, Brij-35, isotridecyl alcohol ether or tridecyl alcohol polyether-12; preferably, the concentration of the second surfactant in reagent R1 is 0.1wt%-1wt%.

[0011] Furthermore, reagent R1 and / or reagent R2 also include an enzyme protectant, which includes one or more of sucrose, sorbitol, ADP or EDTA; preferably, the concentration of the enzyme protectant in reagent R1 and / or reagent R2 is 0.01wt%-0.5wt%.

[0012] Furthermore, reagent R1 and / or reagent R2 also include a preservative, which includes one or more of NaN3, PC300 or gentamicin sulfate; preferably, the concentration of the preservative in reagent R1 and / or reagent R2 is 0.01wt%-0.5wt%.

[0013] Furthermore, reagent R1 also includes a metal salt, which includes any one or more of the following: sodium chloride, potassium chloride, magnesium sulfate, sodium phosphate, or potassium phosphate; preferably, the concentration of the metal salt in reagent R1 is 1-10 g / L.

[0014] Furthermore, the buffer solution is selected from one or more of Tris, CHES, CAPSO, or Bistris propane.

[0015] To achieve the above objective, according to a second aspect of the present invention, a method for determining ammonia content in a sample analyzer is provided, the method comprising detecting ammonia content in a test sample in the sample analyzer using the above-described dual-reagent ammonia assay kit; the ammonia content includes the total concentration of free ammonia and ammonium ions in the test sample.

[0016] Further, the test sample includes a blood sample or a body fluid sample; preferably, the blood sample includes a whole blood sample, a serum sample, or a plasma sample; preferably, the test sample includes a urine sample; preferably, the method includes: mixing the test sample with reagent R1 and incubating at 37°C to obtain a first incubation system; mixing the first incubation system with reagent R2, and performing a first absorbance detection before performing a second incubation, recording the absorbance values ​​A1 corresponding to the main wavelength 340nm and the secondary wavelength 405nm; then performing a second incubation at 37°C to obtain a second incubation system, performing a second absorbance detection on the second incubation system, and recording the absorbance values ​​A2 corresponding to the main wavelength 340nm and the secondary wavelength 405nm; calculating the difference between A1 and A2 or the rate of change of A1 and A2 to obtain the ammonia content.

[0017] To achieve the above objectives, according to a third aspect of the present invention, a sample analyzer for detecting ammonia content is provided, the sample analyzer comprising: i) a sample storage mechanism including a sample tube for storing a sample containing a test sample; ii) a reagent storage mechanism for storing reagents, the reagents including reagent R1 and reagent R2, both reagent R1 and reagent R2 comprising reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase; wherein the concentration ratio of reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L; R The ratio of glutamate dehydrogenase activity concentration in reagent 1 to reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L; the final concentration of α-ketoglutarate after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L; iii) a reaction apparatus having at least one placement position for placing a reaction cup and incubating the incubation system in the reaction cup; iv) a pipette for transferring the test sample and reagents into the reaction cup; v) a measuring device for photometric measurement of the incubation system in the reaction cup to obtain the test result of the test sample.

[0018] By applying the technical solution of this invention and utilizing the aforementioned dual-reagent ammonia assay kit, a complete reaction system is constructed within the dual detection reagents, successfully overcoming the technical bottleneck in the field of blood ammonia detection: effectively eliminating interference from ammonia generated by reagent component degradation and from ambient ammonia absorbed during onboard opening. This solution significantly improves the reagent's opening stability, resulting in a breakthrough improvement in the accuracy of ammonia content detection results. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 A graph showing the results of the change in ammonia content of reagent R1 in an airborne stability test according to the present invention is presented.

[0021] Figure 2 A graph showing the results of the change in ammonia content of reagent R2 in an airborne stability test according to the present invention is presented.

[0022] Figure 3 A graph showing the results of the change in quality control level 1 in the airborne stability test according to the present invention is presented.

[0023] Figure 4 A graph showing the results of the change in quality control level 2 in the airborne stability test according to the present invention is presented. Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0025] As mentioned in the background section, existing reagent kits for detecting ammonia levels in blood samples (i.e., blood ammonia testing) exhibit low stability and interference resistance. Therefore, in this application, the inventors have attempted to develop a highly stable ammonia assay kit, and based on this, have proposed a series of protection schemes for this application.

[0026] In a first typical embodiment of this application, a dual-reagent ammonia assay kit is provided. The kit includes reagent R1 and reagent R2, both of which contain reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase. The concentration ratio of reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L. The activity concentration ratio of glutamate dehydrogenase in reagent R1 to reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L. The final concentration of α-ketoglutarate after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L.

[0027] In existing technologies, the stability of reduced coenzymes such as NADH (reduced coenzyme I) and NADPH (reduced coenzyme II) is pH-dependent, exhibiting good stability at pH values ​​above 9.5, but significant ammonia production still occurs during long-term storage. GLDH, if stored in alkaline conditions above pH 9.5, becomes increasingly unstable with increasing pH. GLDH only achieves sufficient stability, shelf life, and usability below pH 8. Therefore, to ensure the stability of NADH and GLDH, existing dual-reagent assay kits typically separate GLDH and NADH into different reagents. The difference between this application and existing technologies lies in the use of a complete reaction system for both reagents. The inventors discovered that the most significant factor affecting detection accuracy during the assay process is the absorption of ammonia from the air upon opening the bottle. Using a complete reaction system for both reagents effectively eliminates the absorption of ammonia from the air upon opening, improving stability during airborne assays. Furthermore, it eliminates impurities such as ammonia produced by enzyme and NADH degradation in the reagents. The dual-reagent ammonia assay kit of this application consists of two parts: reagent R1 and reagent R2. Both reagents contain reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase (GLDH). These key components work synergistically to ensure high efficiency and accuracy in the assay process.

[0028] In reagents R1 and R2, the concentration ratio of reduced coenzyme is carefully designed to be 2-5:1. This ratio is based on in-depth research into the balance between chemical reaction rate, detection sensitivity, and reagent stability. Specifically, when the final concentration of the reduced coenzyme introduced from reagents R1 and R2 in the mixed system reaches 0.1-0.5 mmol / L after mixing the test sample, reagent R1, and reagent R2, it ensures sufficient reaction substrate during the assay while avoiding excessive light absorption due to excessively high coenzyme concentration. This ensures the detection signal is within the instrument's optimal detection range, improving the accuracy and reliability of the results.

[0029] Regarding the glutamate dehydrogenase (GLDH) activity concentration ratio, in the above kit, the ratio of GLDH activity concentration in reagent R1 to reagent R2 is 1:2-100. This ensures that after mixing the test sample, reagent R1, and reagent R2, the final GLDH concentration in the mixed system remains stable between 10-15 KU / L. This concentration ratio is chosen to fully consider the relationship between detection sensitivity and reagent stability. Within this concentration range, the low concentration of GLDH in R1 can stably eliminate impurity ammonia over a long period, while avoiding excessively high enzyme activity that would lead to rapid consumption of the analyte during detection, affecting the analytical sensitivity of the reagent, or insufficient enzyme activity that would result in inadequate efficiency in eliminating impurity ammonia, thus failing to achieve the effect of improving the onboard stability of the reagent after opening.

[0030] The concentration of glutamate dehydrogenase used in this application is measured in kU / L. Its detection principle is the α-ketoglutarate substrate method, whereby, under the catalytic action of glutamate dehydrogenase in the sample, ammonia reacts with α-ketoglutarate and NADH to produce glutamate and NAD+. + The rate of decrease in absorbance of NADH at a specific wavelength is directly proportional to the activity of glutamate dehydrogenase in the sample. The specific reaction is: α-ketoglutarate + ammonia + NADH → glutamate + NAD. + +H2O. Detection procedure: Add 100μL of reagent containing α-ketoglutaric acid and ammonium acetate to 15μL of sample, incubate at 37℃ for 5 minutes, then add 25μL of reagent containing NADH, incubate for 70s, continuously monitor the absorbance change within 3min, and calculate the absorbance change rate (ΔA / min).

[0031] α-Ketoglutarate, an important substrate in the blood ammonia assay reaction, has its final concentration optimized to 1-90 mmol / L in the mixed system of the test sample, reagent R1, and reagent R2 in the two-reagent ammonia assay kit of this application. This concentration range is designed to balance the reaction rate and reagent stability. Too low a concentration of α-ketoglutarate may lead to insufficient reaction, affecting the sensitivity and accuracy of the detection; while too high a concentration may reduce the stability of GLDH in the reagent, decreasing the long-term stability of the reagent. Precise control of the α-ketoglutarate concentration ensures efficient reaction and improves the reliability of the detection.

[0032] The inventors discovered that by setting up a complete reaction system in both reagents R1 and R2, with the concentration ratio of reduced coenzyme in reagent R1 to that in reagent R2 being 2-5:1 and the ratio of GLDH activity concentration in reagent R1 to that in reagent R2 being 1:2-100, the excess reduced coenzyme and appropriate amount of GLDH in R1 and the appropriate amount of reduced coenzyme and excess GLDH in R2 can effectively eliminate the problem of increased ammonia content caused by ammonia absorption. At the same time, it overcomes the problem of impurity ammonia generated by the decay of reduced coenzyme and GLDH in traditional dual-reagent systems, ensuring the accuracy and stability of the measurement results without affecting the sensitivity of the detection.

[0033] Compared to the high-cost L-glutamine synthase in existing technologies, the GLDH enzyme used in this technical solution is less expensive. This technical solution breaks away from the design principle of dual-reagent blood ammonia assays requiring separate storage of the reduced coenzyme and GLDH, proposing that all dual-reagent ammonia assay kits incorporate a complete reaction system. This approach reduces costs and improves economic efficiency while ensuring detection accuracy, thus possessing greater potential for widespread application.

[0034] In a preferred embodiment, reagent R1 comprises: a buffer solution with a pH of 7.5-9.5, 0.15-0.5 mmol / L reduced coenzyme, 2-100 mmol / L α-ketoglutarate, and 1-10 KU / L glutamate dehydrogenase; reagent R2 comprises: a buffer solution with a pH of 7.5-9.5, 0.05-0.1 mmol / L reduced coenzyme, 2-100 mmol / L α-ketoglutarate, and 20-100 KU / L glutamate dehydrogenase. Experimental results show that setting the pH of both detection reagents to 7.5-9.5 maintains the stability of reduced coenzyme and GLDH.

[0035] In a preferred embodiment of the present invention, the components of the dual-reagent ammonia assay kit are carefully designed and optimized to maximize reagent stability while ensuring detection accuracy and interference resistance. The dual-reagent ammonia assay kit includes reagents R1 and R2, each containing specific concentrations of key active ingredients to create an efficient and stable detection environment.

[0036] Reagent R1 is formulated to contain the following key components:

[0037] A buffer solution with a pH of 7.5-9.5: This pH range is designed to ensure that glutamate dehydrogenase (GLDH) and its reduced coenzyme function under suitable conditions, preventing the enzyme activity from being affected by acidic or alkaline environments, while maintaining the stability of the coenzyme and reducing its degradation during storage. The buffer solution can be one or more of Tris, CHES, CAPSO, or Bistris propane. These buffer systems exhibit good buffering capacity over a wide pH range of 7.5-9.5, which is beneficial for long-term reagent storage and stability after opening.

[0038] 0.15-0.5 mmol / L reduced coenzyme:

[0039] The reduced coenzyme in R1 is mainly used to eliminate impurities such as ammonia, eliminate interference from pyruvate, and provide a substrate for the main reaction. When the concentration is below 0.15 mmol / L, it cannot provide sufficient substrate to eliminate impurities such as ammonia and pyruvate (pyruvate reacts with NADH to produce L-lactic acid under the action of lactate dehydrogenase (LDH)) within the shelf life. When the concentration is above 0.5 mmol / L, it will cause the absorbance to be too high after adding R2, which will exceed the optimal absorbance detection range of the biochemical analyzer and reduce the precision of the test.

[0040] 2-100 mmol / L α-Ketoglutarate: α-Ketoglutarate is another important substrate in the detection of blood ammonia, and its concentration directly affects the reaction rate and efficiency. In reagent R1, the concentration of α-ketoglutarate is optimized to 2-100 mmol / L to ensure sufficient reaction during onboard, storage, and detection processes.

[0041] 1-10 KU / L Glutamate Dehydrogenase: When the GLDH enzyme activity concentration in R1 is below 1 KU / L, the effect of eliminating impurity ammonia is not good. When the GLDH enzyme activity concentration is above 10 KU / L, it will cause the analyte to be consumed too much after being added to R1, resulting in low reagent reaction sensitivity and poor precision.

[0042] The R2 reagent was also designed to contain the following key components:

[0043] Buffer solution with pH value of 7.5-9.5: Similar to the buffer solution in reagent R1, the pH value of the buffer solution in reagent R2 is also controlled at 7.5-9.5 to ensure that the pH environment of the entire detection system is suitable after mixing, so as to promote enzyme activity and coenzyme stability.

[0044] 0.05-0.1 mmol / L Reduced Coenzyme: The reduced coenzyme in R2 mainly provides a reaction substrate for eliminating ammonia impurities in R2 and provides a partial reaction substrate for the main reaction. When the concentration is below 0.05 mmol / L, it cannot provide sufficient reaction substrate for eliminating ammonia impurities and the main reaction within the shelf life. When the concentration is above 0.1 mmol / L, it will cause the absorbance of the reaction system to be too high, exceeding the optimal absorbance limit of the instrument, and the test precision will be reduced.

[0045] 2-100 mmol / L α-ketoglutarate: The concentration of α-ketoglutarate in reagent R2 is the same as that in reagent R1, ensuring that the reaction proceeds fully during onboard or storage and during detection.

[0046] 20-100 KU / L Glutamate Dehydrogenase: A GLDH enzyme activity concentration in R2 below 20 KU / L or above 100 KU / L will lead to decreased analytical sensitivity and reduced precision. A GLDH enzyme activity concentration below 20 KU / L is due to insufficient enzyme activity and incomplete reaction, resulting in decreased analytical sensitivity. A GLDH enzyme activity concentration above 100 KU / L is due to excessive enzyme activity, which causes excessive consumption of the analyte upon addition to R2, leading to decreased analytical sensitivity during monitoring.

[0047] In this preferred embodiment, both reagents R1 and R2 contain glutamate dehydrogenase, reduced coenzyme, α-ketoglutarate, and a buffer solution, forming a "complete reaction system". This design breaks with the conventional practice of separating and storing enzymes and coenzymes in traditional dual-reagent systems or not setting up a complete reaction system for either reagent. By precisely controlling the concentration of each component in reagents R1 and R2, it is beneficial to improve the sensitivity and accuracy of detection.

[0048] In summary, this preferred embodiment has constructed a stable and efficient two-reagent ammonia assay kit by finely controlling the concentration and pH value of the reagent components. It has demonstrated excellent performance in automated testing in clinical laboratories and provided strong technical support for the accurate determination of blood ammonia levels.

[0049] In a preferred embodiment, the reduced coenzyme includes one or more of NADH, NADPH, Thio-NADH, Thio-NADPH, and APADH; preferably, reagent R1 further includes lactate dehydrogenase, and the concentration of lactate dehydrogenase in reagent R1 is 5-50 KU / L.

[0050] Reduced coenzymes, including but not limited to nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), thionicotinamide adenine dinucleotide (Thio-NADH), thionicotinamide adenine dinucleotide phosphate (Thio-NADPH), and 3-acetylpyridinyl adenine dinucleotide (APADH), can be used in the above kits because of their ability to undergo reductive amination catalyzed by glutamate dehydrogenase (GLDH).

[0051] To further enhance the kit's ability to eliminate interference, especially when dealing with potential pyruvate interference in samples, this invention adds lactate dehydrogenase (LDH) to reagent R1. The LDH concentration is set at 5-50 KU / L, a range chosen based on comprehensive experimental data. The presence of LDH, in conjunction with reduced coenzymes NADH or NADPH, effectively converts pyruvate to lactate, eliminating its interference with blood ammonia detection. At concentrations below 5 KU / L, LDH conversion efficiency is insufficient to completely eliminate pyruvate's influence, while concentrations above 50 KU / L may lead to excessive enzyme activity and unnecessary resource waste. Excessive enzyme dosage may even increase impurity ammonia production, resulting in excessive coenzyme consumption and affecting reagent stability.

[0052] In a preferred embodiment of the present invention, the R1 reagent not only contains the components mentioned above, but also contains a first surfactant. This innovative design aims to further enhance the anti-interference performance of the reagent kit.

[0053] The first surfactant, including but not limited to one or more of laurate, isodecyl alcohol polyoxyethylene ether, or stilbene phenol polyoxyethylene ether-18.8, can effectively reduce the surface tension between water and the organic phase, promote the dissolution of lipid-soluble substances, and thus reduce the influence of lipid-soluble interfering substances in the sample on the detection results. In the blood ammonia detection process, emulsified samples (samples rich in fat emulsions) are a common interfering factor. The addition of the first surfactant, by disrupting the surface tension of fat particles and accelerating their dissolution, ensures that the detection of glutamate dehydrogenase (GLDH), reduced coenzyme (NADH or NADPH), and blood ammonia, etc., is not affected by fat particles, thereby improving the accuracy and reliability of the detection.

[0054] In this preferred embodiment, the concentration of the first surfactant is optimized to 1 wt%-5 wt%. This concentration range is based on in-depth research into surfactant performance, enzyme activity, and overall reagent stability. At concentrations below 1 wt%, the surfactant's emulsifying and dissolving abilities are limited, potentially failing to effectively eliminate lipid-soluble interferences and thus affecting the accuracy of the detection results. Conversely, at concentrations exceeding 5 wt%, excessive surfactant may affect enzyme activity and conformation, leading to detection failure. Therefore, controlling the concentration of the first surfactant between 1 wt% and 5 wt% not only ensures its effective disruption of emulsified fat particles in the sample, improving the detection's anti-interference capability, but also maintains the long-term stability of the kit and enzyme activity, avoiding negative impacts on the detection process.

[0055] In a preferred embodiment of the present invention, in addition to the aforementioned components, reagent R1 also includes a second surfactant to further enhance the effect of the first surfactant. This innovative addition aims to further improve the performance of the reagent kit, particularly in reducing interference from complex lipid-soluble substances in blood samples. The second surfactant includes, but is not limited to, polyoxyethylene fatty alcohol ethers, Brij-35, isotridecyl alcohol ether, or tridecyl alcohol polyether-12. These compounds are selected as important adjuvants in this preferred embodiment due to their unique hydrophilic-lipophilic balance properties.

[0056] In a preferred embodiment, the concentration of the second surfactant in reagent R1 is strictly controlled between 0.1 wt% and 1 wt%. This concentration range ensures optimal emulsification and dispersion while avoiding negative impacts on enzyme activity. When the concentration of the second surfactant is below 0.1 wt%, the interfering-eliminating effect of surfactant 1 will not be achieved. Conversely, when the concentration exceeds 1 wt%, the interfering-eliminating effect of surfactant 1 will be inhibited.

[0057] In a preferred embodiment, reagent R1 and / or reagent R2 further include an enzyme protectant, which includes one or more of Triton X-100, sucrose, sorbitol, ADP, or EDTA; preferably, the concentration of the enzyme protectant in reagent R1 and / or reagent R2 is 0.01 wt% to 0.5 wt%. More preferably, the enzyme protectant is in reagent R2. The addition of the enzyme protectant is based on in-depth research on the maintenance of enzyme activity under complex storage and detection conditions, as well as comprehensive consideration of improving the overall performance of the kit.

[0058] In this preferred embodiment, the concentration of the enzyme protectant in reagents R1 and / or R2 is optimized to 0.01 wt%–0.5 wt%. This concentration range is designed to achieve the best balance between enzyme protection and overall reagent performance. Too low a concentration of enzyme protectant may not provide sufficient stability enhancement, leading to a significant decrease in enzyme activity after long-term storage or repeated opening and use; while too high a concentration may alter the chemical environment of the reagent, inhibiting enzyme activity and affecting the sensitivity and accuracy of the detection.

[0059] In the above technical solutions, enzyme protectants include, but are not limited to, one or more of sucrose, sorbitol, ADP, and EDTA. Triton X-100 primarily functions to prevent enzyme molecules from agglomerating and precipitating, thus reducing denaturation. Sucrose and sorbitol mainly increase solution viscosity to slow enzyme molecule movement and help maintain enzyme molecule structure. ADP is an activator of GLDH and can also maintain GLDH conformational stability. EDTA primarily chelates metal ions, preventing metal ions from causing changes in enzyme activity or denaturation.

[0060] In a preferred embodiment, reagent R1 and / or reagent R2 further include a preservative, which includes one or more of NaN3, PC300, or gentamicin sulfate; preferably, the concentration of the preservative in reagent R1 and / or reagent R2 is 0.01wt%-0.5wt%.

[0061] In a preferred embodiment of the present invention, to ensure the stability of the blood ammonia assay kit under long-term storage conditions and to effectively prevent the impact of microbial contamination on reagent performance, one or more preservatives are added to reagents R1 and / or R2. The addition of preservatives inhibits microbial growth, reduces reagent deterioration caused by microbial contamination, and extends the shelf life of the kit.

[0062] In a preferred embodiment, reagent R1 further includes a metal salt, which includes any one or more of the following: sodium chloride, potassium chloride, magnesium sulfate, sodium phosphate, or potassium phosphate; preferably, the concentration of the metal salt in reagent R1 is 1-10 g / L.

[0063] In the above kit, by adding a metal salt to reagent R1 and optimizing the concentration to 1-10 g / L, the ionic strength of the reagent can be increased, thereby improving the efficiency of enzyme catalytic reaction.

[0064] In a preferred embodiment, the buffer solution is selected from one or more of Tris, CHES, CAPSO, or Bis-trispropane.

[0065] Preferably, the buffer solutions contained in reagents R1 and R2 are the same buffer solution, which can ensure the stability of reduced coenzymes such as glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), and NADH. The pH of the buffer solution is set to 7.5-9.5, preferably 9.0. At this pH, GLDH and LDH have high catalytic efficiency and good stability. When the buffer solution pH is below 7.5, NADH will decay too quickly, resulting in poor long-term stability; when the buffer solution pH is above 9.5, GLDH stability will deteriorate, and enzyme activity will decrease excessively during long-term storage, leading to the failure of the ammonia elimination reaction and the main reaction.

[0066] In a preferred embodiment, the linear range of the above kit for detecting ammonia content in a sample is 10-1000 μmol / L.

[0067] In a preferred embodiment, the components of the above-mentioned kit include:

[0068] R1: Buffer solution, pH 7.5-9.5, concentration 200 mmol / L; NADH, concentration 0.15-0.25 mmol / L; α-ketoglutarate, concentration 2-100 mmol / L; glutamate dehydrogenase (GLDH), concentration 1-10 KU / L; enzyme protectant, concentration 0.01%-0.5%; lactate dehydrogenase (LDH), concentration 5-50 KU / L; primary surfactant, concentration 1%-5%; secondary surfactant, concentration 0.1%-1%; sodium chloride, concentration 1-10 g / L; preservative, concentration 0.01%-0.5%.

[0069] R2: Buffer, pH 7.5-9.5, concentration 200 mmol / L; NADH, concentration 0.5-1.5 mmol / L; α-ketoglutarate, concentration 2-100 mmol / L; GLDH, concentration 20-100 KU / L; enzyme protectant, concentration 0.01%-0.5%; preservative, concentration 0.01%-0.5%.

[0070] In a second typical embodiment of this application, a method for determining ammonia content in a sample analyzer is provided. The method includes: using the above-mentioned dual-reagent ammonia assay kit to detect the ammonia content of a sample in the sample analyzer; the ammonia content includes the total concentration of free ammonia and ammonium ions in the sample.

[0071] In a preferred embodiment, the above-mentioned determination method includes, but is not limited to:

[0072] 1) Control the pipette to draw R1 reagent and test sample from the sample storage mechanism and reagent storage mechanism in the sample analyzer into the reaction cup to form the first incubation system, wherein R1 reagent includes reduced coenzyme, α-ketoglutarate and glutamate dehydrogenase;

[0073] 2) After incubating in the first incubation system in step 1) for 3-5 minutes, use a pipette to draw reagent R2 from the reagent storage mechanism into the reaction cup to form the second incubation system; reagent R2 also includes reduced coenzyme, α-ketoglutarate and glutamate dehydrogenase;

[0074] 3) The ammonia content in the test sample is determined by measuring the change in absorbance in the second incubation system. The ammonia content includes the total concentration of free ammonia and ammonium ions in the test sample.

[0075] In the test sample, the concentration ratio of reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of reduced coenzyme after mixing with reagent R1 and reagent R2 is 0.1-0.5 mmol / L; the activity concentration ratio of glutamate dehydrogenase in reagent R1 to reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing with reagent R1 and reagent R2 is 10-15 KU / L; the final concentration of α-ketoglutarate after mixing with reagent R1 and reagent R2 is 1-90 mmol / L.

[0076] It's important to note that the term "reaction cup" mentioned in the above assay methods is actually a broad concept, encompassing all containers or devices that can be used for chemical reactions. This term is not intended to limit the shape of reaction containers or devices in sample analyzers, but rather to emphasize their functionality. This means that containers of any shape—cup-shaped, box-shaped, or otherwise—can be considered "reaction cups" as long as they provide a suitable environment for a chemical reaction. Therefore, the focus of this term is on the function and purpose of the container, not its appearance or shape. This definition provides greater flexibility in experimental design, allowing researchers to choose the most suitable container type based on the specific needs of the experiment, whether it's a traditional cup-shaped container or a more innovative box-shaped or other shaped device.

[0077] In a preferred embodiment, the test sample includes a blood sample or a body fluid sample; preferably, the blood sample includes a whole blood sample, a serum sample, or a plasma sample; preferably, the test sample includes a urine sample.

[0078] Preferably, step 3) determining the ammonia content in the sample by measuring the change in absorbance in the second incubation system includes: before the second incubation, controlling the reaction vessel to perform a first absorbance test and recording the absorbance values ​​A1 corresponding to the main wavelength of 340nm and the secondary wavelength of 405nm; then, the second incubation system is carried out at 37°C for 3-5 minutes, controlling the reaction vessel to perform a second absorbance test and recording the absorbance values ​​A2 corresponding to the main wavelength of 340nm and the secondary wavelength of 405nm; calculating the difference between A1 and A2 or the rate of change of A1 and A2 to obtain the ammonia content.

[0079] In the above calculation methods, the endpoint method is used to calculate the difference between A1 and A2, and the rate method is used to calculate the rate of change of A1 and A2.

[0080] In a third typical embodiment of this application, a sample analyzer for detecting ammonia content is provided, comprising:

[0081] i) A sample storage mechanism, including a sample tube for storing the sample to be tested;

[0082] ii) A reagent storage facility for storing reagents, including reagent R1 and reagent R2. Both reagent R1 and reagent R2 contain reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase. The concentration ratio of reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L. The activity concentration ratio of glutamate dehydrogenase in reagent R1 to reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L. The final concentration of α-ketoglutarate after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L.

[0083] iii) A reaction apparatus having at least one placement position for placing a reaction vessel and incubating the incubation system in the reaction vessel;

[0084] iv) A pipette, used to transfer test samples and reagents into a reaction vessel;

[0085] v) A measuring device for photometric determination of the incubation system in the reaction vessel to obtain the detection results of the test sample.

[0086] In a preferred embodiment, the sample analyzer further includes: vi) a control device electrically connected to the sample storage mechanism, reagent storage mechanism, reaction device, measuring device, and pipette, configured for:

[0087] Receive test instructions, the test instructions include detecting the ammonia content of the analyte in the sample; and

[0088] Response to test commands:

[0089] 1) Control the pipette to draw R1 reagent and test sample from the sample storage unit and reagent storage unit respectively into the reaction cup to form the first incubation system. R1 reagent includes reduced coenzyme, α-ketoglutarate and glutamate dehydrogenase.

[0090] 2) After incubating in the first incubation system in step 1 for 3-5 minutes, control the pipette to draw R2 reagent from the reagent storage mechanism into the reaction cup to form the second incubation system; R2 reagent also includes reduced coenzyme, α-ketoglutarate and glutamate dehydrogenase.

[0091] 3) The ammonia content in the test sample is determined based on the change in absorbance in the second incubation system measured by the measuring device. The ammonia content includes the total concentration of free ammonia and ammonium ions in the test sample.

[0092] As one implementation method, a sample storage mechanism is used to store sample tubes containing test samples.

[0093] Sample tubes are used to hold samples collected from patients, which in this protocol are test samples. Sample tubes can be stored in sample racks for storing bulk sample tubes or in sample containers for storing individual sample tubes.

[0094] It's important to clarify that the term "sample tube" is a broad concept, encompassing all containers or tubular structures that can be used for storing and processing samples. This term is not intended to limit the specific shape of sample containers or tubular structures in the laboratory, but rather to emphasize their functionality. This means that whether a container is test tube-shaped, bottle-shaped, or any other shape, as long as it provides a suitable environment for the storage, transportation, and analysis of samples, it can be considered a "sample tube." Therefore, the focus of this term is on the function and purpose of the container, not its appearance or shape.

[0095] The sample storage mechanism includes a sample storage component and a sample loading component. The sample storage component stores sample tubes; specifically, it stores multiple sample racks or sample containers. The sample loading component transports the sample tubes to the aspiration position for pipettes to aspirate samples. Specifically, the sample loading component removes the sample racks or sample containers containing the sample tubes from the sample storage component and transports them to the aspiration position. Alternatively, in specific applications, the sample storage mechanism may not include a sample loading component. For example, as an alternative implementation, the sample tubes containing the samples are placed in the aspiration position by an operator for pipette aspiration.

[0096] As one implementation, a reagent storage mechanism is provided for storing reagents, including reagent R1 and reagent R2.

[0097] In some embodiments, the reagent storage mechanism can be a reagent compartment with multiple reagent positions, each position holding the reagents required for the sample analyzer to detect the reagents. A pipette moves to the corresponding reagent position to draw the required reagent. In some embodiments, the reagent storage mechanism can also be a reagent tray, which has a disc-shaped structure and multiple reagent positions for holding the reagents required for the sample analyzer to detect the reagents. The reagent storage mechanism can rotate, causing the reagents it carries to rotate, to rotate the reagent positions to a specific location, such as the position where a pipette draws the reagent. The number of reagent storage mechanisms can be one or more.

[0098] In one embodiment, the reaction apparatus has at least one placement position for placing a reaction cup and incubating the reaction liquid within it. For example, the reaction apparatus can be a reaction disk, which has a disc-shaped structure and one or more placement positions for placing reaction cups. The reaction disk is rotatable, causing the reaction cups in its placement positions to rotate, thereby managing the reaction cups within the reaction disk and incubating the reaction liquid within them. The incubation temperature can be 25-37°C. In a single sample analysis, there can be one or more reaction apparatuses.

[0099] In one embodiment, a measuring device is used to perform photometric measurements on the incubated reaction solution to obtain the detection result of the sample to be tested. For example, based on the information obtained from the photometric measurements of the reaction solution, the concentration of the analyte in the sample is calculated using a calibration curve; in this application, the analyte is the ammonia content.

[0100] In one implementation, a pipette is used to transfer the whole blood sample or the reagent into the reaction vessel. The pipette is driven by a two-dimensional or three-dimensional mechanism to move in two-dimensional or three-dimensional space, allowing it to move to the sample or reagent aspiration position. It should be noted that one or more pipettes may be used in this analytical method.

[0101] In some embodiments of the present invention, the method for determining ammonia content includes, but is not limited to, the following steps:

[0102] 1. Sample Preparation: Users collect test samples, ensuring that the collection, processing, and storage conditions meet clinical standards and avoiding any external factors that may introduce additional ammonia or ammonium ions. Users load the collected samples into the sample storage mechanism of the sample analyzer, and load the blood ammonia R1 and R2 reagents into the reagent storage mechanism of the sample analyzer.

[0103] 2. Pre-incubation: Based on the test command, the sample analyzer controls the pipette to mix the sample with the R1 reagent in the reaction vessel and pre-incubate at a specific temperature (e.g., 37°C) for a certain period of time (usually 3-5 minutes). This step aims to ensure that the components in the R1 reagent are in full contact with the sample, eliminate most interfering substances, and provide a clean and stable substrate environment for the subsequent main reaction.

[0104] 3. R2 Main Reaction: After pre-incubation, the sample analyzer controls the pipette to add reagent R2 to the reaction vessel. The excess GLDH and appropriate amount of NADH in R2 ensure the smooth progress of the main reaction. That is, under the stronger enzyme catalysis, the remaining ammonia or ammonium ions in the sample are completely converted into glutamate, while NADH is converted into NAD. + The oxidation process. This conversion can be quantified by monitoring changes in absorbance at specific wavelengths.

[0105] 4. Data Recording and Analysis: The measuring device of the sample analyzer records the absorbance values ​​at the main wavelength (e.g., 340 nm) and the secondary wavelength (e.g., 405 nm) at specified time intervals. By calculating the difference between the absorbance value A1 when R2 is added and the absorbance value A2 at the end of the reaction, or the rate of change of A1 and A2, the total concentration of free ammonia and ammonium ions in the sample is deduced by comparing it with the standard curve.

[0106] The ammonia determination method provided in this embodiment, through the ingenious use of a dual-reagent system, not only significantly improves the onboard stability of the test reagents after opening, but also effectively overcomes the limitations of traditional single-reagent systems in terms of anti-interference, precision, accuracy, and long-term stability. Specifically, the components in reagent R1 pre-emptively remove interfering substances from the sample, while the excess enzyme activity in reagent R2 ensures the complete reaction of ammonia or ammonium ions, making the final measured ammonia content more accurately reflect the original state of the sample.

[0107] In a preferred embodiment, the above determination method includes, but is not limited to: adding 24 μL of sample and 160 μL of reagent R1, mixing well and incubating at 37°C for 5 min, adding 32 μL of reagent R2, mixing well, reading absorbance A1, incubating at 37°C for 5 min, reading absorbance A2, and calculating the change in absorbance.

[0108] In a preferred embodiment, the linear range of the above determination method for detecting ammonia content in a sample is 10-1000 μmol / L. If the original ammonia content in the sample is not within the above linear range, the sample needs to be diluted or concentrated before detection to ensure the accuracy of the detection results.

[0109] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0110] Detection example

[0111] The detection method for the dual-reagent blood ammonia test kit is as follows: First, add the sample and reagent R1, mix thoroughly, and incubate at 37°C for 3-5 minutes. Then, add reagent R2 and record the absorbance values ​​A1 at the main wavelength of 340nm and the secondary wavelength of 405nm. After mixing, incubate at 37°C for 5 minutes and record the absorbance values ​​A2 at the main wavelength of 340nm and the secondary wavelength of 405nm. Calculate the change in absorbance.

[0112] The configurations of the example and comparative sample are as follows:

[0113] The common components of the examples and comparative examples are: 10 g / L NaCl and 0.01% NaN3. The remaining components are prepared according to the formula shown in Table 1.

[0114] Table 1

[0115]

[0116]

[0117]

[0118] Note: In Table 1, "%" refers to the mass fraction "wt%".

[0119] Introduction to experimental methods for verifying technical effects:

[0120] 1. Airborne stability evaluation:

[0121] ① The reagents of Examples 1 to 14 and Comparative Examples 1 to 3 at 10℃ were opened and placed in a fully automated biochemical analyzer. During the onboard process, the instrument was always running. The changes in ammonia content in reagents R1 and R2 were tracked on days 0, 2, 4, 6, 8, 10, 12 and 14. If the ammonia content was less than 7 μmol / L, it was considered that there was no dissolved ammonia in the reagent.

[0122] ② The reagents of Examples 1 to 14 and Comparative Examples 1 to 3 at 10℃ were opened and placed in a fully automated biochemical analyzer. During the onboard process, the instrument was always running. The changes of two quality control levels (60 μmol / L and 185 μmol / L) were continuously tracked on days 0, 2, 4, 6, 8, 10, 12 and 14. When the deviation of the quality control level was less than 15%, the onboard stability was considered to be good.

[0123] 2. Accelerated stability evaluation at 37℃:

[0124] ① The reagents of Examples 1 to 14 and Comparative Examples 1 to 3 were sealed and placed in a 37°C constant temperature biochemical incubator. On the 4th, 7th and 11th days, all reagents were taken out and tested with ammonia-free water (the reagents were calibrated with calibrators before testing). The changes in blank absorbance and blank reactivity (absorbance at the endpoint of the reaction minus absorbance after adding R2) of the reagents were tracked. If the change in blank absorbance was less than 15% and the change in blank reactivity was less than 15% and there was no continuous upward trend, the long-term storage stability of the reagents was considered good. If the change in analytical sensitivity was less than 10% and there was no continuous downward trend, the long-term stability of the reagents was considered good.

[0125] ② Examples 6, 7, and Comparative Examples 1 to 3 were placed in a 37°C constant temperature biochemical incubator and removed on the 7th and 11th days. The samples with added sodium pyruvate in plasma were tested using a fully automated biochemical analyzer (Shenzhen New Industries Biomedical Engineering Co., Ltd., Biossays C8). The anti-pyruvate interference performance of the reagent was evaluated by calculating the deviation from the test results of the sample without interfering substances. A test deviation of less than 10% was considered to have strong anti-interference ability.

[0126] ③Analyze changes in sensitivity

[0127] The reagents from Examples 5, 12, and 13 were sealed and placed in a 37°C constant temperature biochemical incubator. On days 4, 7, and 11, the reagents were taken out to test 100 μmol / L samples and ammonia-free water (the reagents were calibrated with calibrators before testing). The reactivity of the 100 μmol / L sample minus the reactivity of the ammonia-free water was the analytical sensitivity of the reagent. The changes in the analytical sensitivity of the reagent were tracked. If the change in analytical sensitivity was less than 10%, and there was no continuous upward trend, the reagent was considered to have good long-term storage stability. If the change in analytical sensitivity was less than 10% and there was no continuous downward trend, the reagent was considered to have good long-term stability.

[0128] 3. Evaluation of resistance to pyruvate and lipid interference:

[0129] ① Anti-pyruvate interference: The reagents of Example 6, Example 7, and Comparative Examples 1 to 3 were tested with a fully automated biochemical analyzer (Shenzhen New Industries Biomedical Engineering Co., Ltd., Biossays C8) on samples with added sodium pyruvate in plasma. The anti-pyruvate interference performance of the reagents was evaluated by calculating the deviation from the test results of the sample without interfering substances. A test deviation of less than 10% was considered to have strong anti-interference ability.

[0130] ② Performance against lipid turbidity: The reagents of Examples 6, 8 to 11, 14, and Comparative Examples 1 to 3 were tested using a fully automated biochemical analyzer on samples with added lipid emulsion in plasma. The performance of the reagents against lipid turbidity was evaluated by calculating the deviation from the test results of the samples without interfering substances. A test deviation of less than 10% was considered to indicate strong anti-interference ability.

[0131] The performance comparison results of the embodiments and comparative examples are as follows:

[0132] 1) Airborne stability evaluation

[0133] ① Changes in ammonia content of airborne reagents

[0134] Data related to the changes in ammonia content of airborne reagents in Examples 1 to 14 and Comparative Examples 1 to 3, such as... Figure 1 and Figure 2 As shown.

[0135] The results showed that the R1 reagent in Examples 1 to 14 had the complete reaction system required for the ammonia reaction. It can be seen that the ammonia content in the R1 reagent did not exceed 7 μmol / L until day 14 of the airborne test (this level is the blank limit of the kit; the kit's detection result is greater than 7 μmol / L, indicating the presence of dissolved ammonia in the reagent). However, Comparative Examples 1 and 2 did not include the complete reaction system, and the detection result on day 2 of the airborne test was already greater than 7 μmol / L. Dissolved ammonia was already present in the R1 reagent on day 2, and the ammonia content even exceeded 90 μmol / L until day 14 of the airborne test.

[0136] When R2 reagent was tested, the results were similar to those of R1 reagent. The R2 reagents of Examples 1 to 14 and Comparative Example 1 had the complete reaction system required for the ammonia reaction. It can be seen that the ammonia content in the R1 reagent did not exceed 7 μmol / L until the 14th day on the air. However, Comparative Example 2 did not include the complete reaction system, and the test result on the 2nd day on the air was already greater than 7 μmol / L. Until the 14th day on the air, the ammonia content had also exceeded 90 μmol / L. The dissolved ammonia in both R1 and R2 reagents can seriously affect the accuracy of the test results.

[0137] ②Airborne quality control test deviation

[0138] Data related to changes in airborne quality control in Examples 1 to 14 and Comparative Examples 1 to 3, such as Figure 3 and Figure 4 As shown.

[0139] Among them, the R1 reagent in Examples 1 to 14 all have the complete reaction system required for the ammonia reaction, but based on Figure 1 and Figure 2The results showed that the deviation of different levels of quality control samples was less than 15% during the airborne 0-14 days. In other words, although the sample is incubated with reagent R1 containing the complete reaction system in the scheme provided in this application, the incubation in advance will not affect the accuracy of the final reagent test results.

[0140] The results showed that Comparative Examples 1 and 2 were both dual-reagent systems without a complete reaction system. It can be seen that when detecting 60 μmol / L of quality control 1 (this level represents the upper limit of the reference range for blood ammonia concentration in normal population samples; in clinical practice, a test sample exceeding this upper limit indicates a positive sample), the deviation between Comparative Examples 1 and 2 exceeded 15% by day 4 of onboard testing. This fluctuation during use could lead to the test sample being falsely identified as a positive sample, and the deviation even reached 90% by day 14. In contrast, Examples 1 to 14, by setting up a dual-reagent system containing a complete reaction system, excess R1 reduced coenzyme, and sufficient R2 glutamate dehydrogenase, maintained a relative deviation of less than 15% until day 14. Therefore, the scheme of this application can effectively avoid false positives in the test results of samples at the reference range threshold.

[0141] When testing quality control 2 at 185 μm0 L / L (a reference level requiring further treatment by physicians), Comparative Examples 1 and 2 showed a deviation exceeding 15% starting on day 10 of onboard testing. This fluctuation during use could lead physicians to misjudge samples that had not yet met diagnostic criteria as having reached a high level, resulting in premature treatment. In contrast, Examples 1 through 14 maintained an onboard test until day 14, with relative deviations less than 15%, improving testing accuracy and preventing overtreatment.

[0142] 2) High-temperature accelerated stability evaluation

[0143] ① The absorbance of the reagent blank after acceleration at 37℃ is shown in Table 2.

[0144] Table 2

[0145]

[0146]

[0147] As shown in Table 2 above, the blank absorbance changes of less than 15% in Examples 1 to 14 by day 11 indicate that the NADH, GLDH, LDH, and other substances in the reagents provided in this application are relatively stable under the reagent combination relationship. The NADH and GLDH in Comparative Examples 1 to 3 are also relatively stable under their respective buffer conditions, with blank absorbance changes of less than 15% by day 11. Furthermore, the fact that the onboard quality control of Comparative Examples 1 and 2 deviated by more than 15% starting from day 10 further illustrates that, under the reagent combination relationship of Comparative Examples 1 and 2, the ammonia absorbed from the air has a far greater impact on the accuracy of the detection results than the impurity ammonia generated by the degradation of the raw materials, addressing the issue of inaccurate reagent detection.

[0148] ②The reagent blank reactivity test was performed at 37℃. The test data are shown in Table 3.

[0149] Table 3

[0150]

[0151] In Comparative Example 1 (R1) and Comparative Example 2 (R1 and R2), the absence of a complete reaction system failed to eliminate the ammonia impurity. As shown in Table 3, after 4 days of accelerated reaction at 37°C with the reagents sealed, the change in reagent blank reactivity exceeded 40%, and after 7 or 11 days of accelerated reaction, the change even exceeded 100%. This indicates that although the raw materials appear relatively stable based on the blank absorbance values, slight degradation can still lead to inaccurate detection. Such fluctuations significantly affect the detection results for samples at the μmol / L level.

[0152] Due to its low analytical sensitivity, the blank reactivity of the single-reagent comparative example 3 was greater than 5% after 7 days of acceleration, and even greater than 15% after 11 days of acceleration, indicating a large fluctuation in the blank reactivity test.

[0153] In Examples 1 to 14, the change in blank reactivity was less than 5% within an 11-day acceleration period. This indicates that by setting up a dual-reagent system for the complete reaction, this application can further eliminate impurities such as ammonia generated by the unstable degradation of reagent components, which is beneficial to improving the detection stability of the reagent.

[0154] Based on the experimental results of Examples 1 to 14 and Comparative Example 1, it can be seen that R1 is only the Comparative Example 1 with GLDH. The change in blank reactivity after the accelerated experiment is mainly caused by the degradation of GLDH, which proves that the stability of GLDH should not be ignored.

[0155] ③The change in reagent analysis sensitivity after acceleration at 37℃ is shown in Table 4.

[0156] Table 4

[0157]

[0158] As shown in Table 4 above, the analytical sensitivity of Examples 5 and 12 decreased by about 10% after accelerating to 11 days, while the analytical sensitivity of Example 13 remained within ±3% after accelerating to 11 days. This indicates that adding an enzyme protectant in R2 has a good effect on accelerating the stability of the reagents, which is more conducive to ensuring the analytical sensitivity of the detection reagents.

[0159] 3) Evaluation of resistance to pyruvate and lipid interference

[0160] ① Test results on resistance to pyruvate interference are shown in Table 5.

[0161] Table 5

[0162]

[0163] As shown in Table 5 above, Comparative Examples 1 to 3 exhibited test deviations of <10% when detecting samples containing 400 μmol / L pyruvate interference. However, with increasing interference concentration, the test deviations could reach 50%, demonstrating their inability to eliminate high-concentration pyruvate interference. Even with the addition of LDH, Comparative Example 3 lacked sufficient reaction time to completely eliminate high-concentration pyruvate interference. In contrast, under the condition of adding LDH in R1 with a concentration not less than 5 KU / L, even with high concentrations of sodium pyruvate in the sample, the test deviation was far less than 10%, proving that adding LDH in R1 effectively eliminated pyruvate interference. Examples 6 and 7 were not affected by airborne ammonia, raw material degradation ammonia, or pyruvate in the samples.

[0164] ② Resistance to lipophilic interference test, the test data are shown in Table 6.

[0165] Table 6

[0166]

[0167] As shown in Table 6 above, adding laurate alone to R1 in the reagent can eliminate low-concentration fat emulsion interference, and the elimination effect is better as the amount of laurate added increases (Examples 8 and 9); adding isotretinoin ether in addition to laurate significantly improves the elimination effect (Examples 6, 10, and 11); however, if the reagent system contains only isotretinoin ether, it cannot eliminate fat emulsion interference (Example 14), proving that it is laurate that achieves the fat-reducing effect, and isotretinoin ether alone cannot reduce fat. However, adding isotretinoin ether in addition to laurate greatly improves the fat-reducing effect, indicating that laurate and isotretinoin ether have a synergistic effect.

[0168] ③ The test results on the resistance to pyruvate interference after acceleration at 37℃ are shown in Table 7.

[0169] Table 7

[0170]

[0171]

[0172] As shown in Table 7 above, under the condition of adding LDH in R1 and the LDH concentration not less than 5 KU / L, even if the sample contains a high concentration of sodium pyruvate, the test deviation is much less than 10%, and LDH is very stable in the system. The test deviation is less than 10% after 11 days of acceleration at 37℃. However, commercially available blood ammonia reagents (Comparative Examples 1 to 3) cannot eliminate samples containing more than 400 μmol / L of interfering substances. In Comparative Examples 1 and 2, the deviations after 7 and 11 days of acceleration are basically unchanged because GLDH and NADH in the system are relatively stable. In Comparative Example 3, due to the poor stability of the reagent, the reagent analysis sensitivity decreases significantly with the increase of acceleration time, and the deviation of testing for pyruvate interference also increases.

[0173] As can be seen from the above description, the embodiments of the present invention have achieved the following technical effects: This application has developed a dual-reagent ammonia assay kit and detection method. By constructing a complete reaction system within the dual detection reagents, the concentrations of GLDH and NADH in reagents R1 and R2 are cleverly controlled, and the pH value of the reagents is maintained within a specific range. This successfully overcomes the long-standing problem faced by blood ammonia detection reagents, namely, effectively suppressing the serious positive interference caused by ammonia generation due to reagent component degradation and ammonia absorption from the air. It significantly improves the stability of the reagents after opening, and greatly enhances the accuracy and reliability of the detection results, bringing new breakthroughs and optimization directions for ammonia detection reagents.

[0174] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A two-reagent ammonia assay kit, characterized in that, The dual-reagent ammonia assay kit includes reagent R1 and reagent R2, both of which contain reduced coenzyme, α-ketoglutarate, and glutamate dehydrogenase. The concentration ratio of the reduced coenzyme in reagent R1 to reagent R2 is 2-5:1, and the final concentration of the reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L. The ratio of the activity concentration of glutamate dehydrogenase in reagent R1 to that in reagent R2 is 1:2-100, and the final concentration of glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L. The final concentration of α-ketoglutarate after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L.

2. The dual-reagent ammonia assay kit according to claim 1, characterized in that, In the dual-reagent ammonia assay kit The R1 reagent comprises: a buffer solution with a pH of 7.5-9.5, 0.15-0.5 mmol / L of the reduced coenzyme, 2-100 mmol / L of the α-ketoglutarate and 1-10 KU / L of the glutamate dehydrogenase; The R2 reagent comprises: the buffer solution with a pH of 7.5-9.5, 0.05-0.1 mmol / L of the reduced coenzyme, 2-100 mmol / L of α-ketoglutarate and 20-100 KU / L of the glutamate dehydrogenase; Preferably, the reduced coenzyme includes one or more of NADH, NADPH, Thio-NADH, Thio-NADPH, or APADH; Preferably, the R1 reagent further comprises lactate dehydrogenase, wherein the concentration of lactate dehydrogenase in the R1 reagent is 5-50 KU / L; Preferably, the R1 reagent further includes a first surfactant, which includes one or more of laurate, isodecyl alcohol polyoxyethylene ether, or stilbene phenol polyoxyethylene ether-18.8; Preferably, the concentration of the first surfactant in the R1 reagent is 1wt%-5wt%.

3. The dual-reagent ammonia assay kit according to claim 2, characterized in that, The R1 reagent also includes a second surfactant, which includes one or more of polyoxyethylene fatty alcohol ether, Brij-35, isotridecyl alcohol ether or tridecyl alcohol polyether-12. Preferably, the concentration of the second surfactant in the R1 reagent is 0.1 wt% to 1 wt%.

4. The dual-reagent ammonia assay kit according to any one of claims 1-3, characterized in that, The R1 reagent and / or the R2 reagent further include an enzyme protectant, which includes one or more of sucrose, sorbitol, ADP or EDTA; Preferably, the concentration of the enzyme protectant in reagent R1 and / or reagent R2 is 0.01wt%-0.5wt%.

5. The dual-reagent ammonia assay kit according to any one of claims 1-2, characterized in that, The R1 reagent and / or the R2 reagent further include a preservative, which includes one or more of NaN3, PC300 or gentamicin sulfate; Preferably, the concentration of the preservative in reagent R1 and / or reagent R2 is 0.01wt%-0.5wt%.

6. The dual-reagent ammonia assay kit according to any one of claims 1-2, characterized in that, The R1 reagent also includes a metal salt, which includes any one or more of the following: sodium chloride, potassium chloride, magnesium sulfate, sodium phosphate, or potassium phosphate; Preferably, the concentration of the metal salt in the R1 reagent is 1-10 g / L.

7. The dual-reagent ammonia assay kit according to claim 2, characterized in that, The buffer solution is selected from one or more of Tris, CHES, CAPSO or Bis-trispropane.

8. A method for determining ammonia content in a sample analyzer, characterized in that, The determination method includes using the dual-reagent ammonia assay kit according to any one of claims 1-7 to detect the ammonia content in the test sample in the sample analyzer; The ammonia content includes the total concentration of free ammonia and ammonium ions in the test sample.

9. The determination method according to claim 8, characterized in that, The test samples include blood samples or body fluid samples; Preferably, the blood sample includes a whole blood sample, a serum sample, or a plasma sample; Preferably, the test sample includes a urine sample; Preferably, the method includes: The test sample was mixed with the R1 reagent and incubated at 37°C to obtain the first incubation system; After mixing the first incubation system with the R2 reagent, a first absorbance test is performed before the second incubation, and the absorbance values ​​A1 corresponding to the main wavelength of 340 nm and the secondary wavelength of 405 nm are recorded. Then, the system is incubated at 37°C to obtain a second incubation system. A second absorbance test is performed on the second incubation system, and the absorbance values ​​A2 corresponding to the main wavelength of 340 nm and the secondary wavelength of 405 nm are recorded. The ammonia content is obtained by calculating the difference between A1 and A2 or the rate of change of A1 and A2.

10. A sample analyzer for detecting ammonia content, characterized in that, The sample analyzer includes: i) A sample storage mechanism, including a sample tube for storing a sample containing a test sample; ii) A reagent storage mechanism for storing reagents, the reagents including reagent R1 and reagent R2, wherein both reagent R1 and reagent R2 contain reduced coenzyme, α-ketoglutarate and glutamate dehydrogenase; The concentration ratio of the reduced coenzyme in reagent R1 to that in reagent R2 is 2-5:

1. The final concentration of the reduced coenzyme after mixing the test sample, reagent R1, and reagent R2 is 0.1-0.5 mmol / L; The ratio of glutamate dehydrogenase activity concentration in reagent R1 to that in reagent R2 is 1:2-100. The final concentration of the glutamate dehydrogenase after mixing the test sample, reagent R1, and reagent R2 is 10-15 KU / L; The final concentration of α-ketoglutaric acid after mixing the test sample, reagent R1, and reagent R2 is 1-90 mmol / L; iii) A reaction apparatus having at least one placement position for placing a reaction vessel and incubating the incubation system in the reaction vessel; iv) A pipette for transferring the test sample and the reagent into the reaction vessel; v) A measuring device for performing photometric measurements on the incubation system in the reaction vessel to obtain the detection results of the test sample.

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