Triglyceride determination single reagent, kit and determination method thereof

Abstract

The invention belongs to the technical field of medical examination and determination, and relates to a triglyceride determination single reagent, a kit and a determination method thereof, the single reagent comprises the following components: a buffer solution, magnesium ions, a complexing agent, a stabilizer, a color developing agent, an adenosine triphosphate compound, a phenolic compound, a catalytic reaction enzyme, an antioxidant, an anti-bilirubin interference agent and an anti-hemoglobin interference agent; wherein the catalytic reaction enzyme comprises lipoprotein esterase, glycerol kinase, glycerophosphate oxidase and peroxidase. The stability of the reagent can be improved, the enzyme activity is ensured, the accuracy of the reagent is improved, the stability of the kit is improved, and the application range, accuracy and utilization rate of the kit are improved.

Description

Technical Field

[0001] The present application relates to the field of medical testing and determination technology, and in particular to a single reagent and a kit for determining triglyceride and a determination method thereof. Background Art

[0002] Triglyceride (TG) is composed of three fatty acids and one glycerol molecule, and is mainly derived from the decomposition of fat in food. If triglyceride is excessive, it will accumulate under the skin and cause obesity, accumulate in the blood vessel wall and cause arteriosclerosis, accumulate in the heart and cause cardiac hypertrophy, and accumulate in the liver and cause fatty liver.

[0003] Elevated serum triglycerides are a common phenomenon in the middle-aged and elderly population, and elevated serum triglycerides are an important risk factor for coronary heart disease, so controlling triglycerides is an important measure to reduce ischemic heart disease attacks. Currently, triglyceride levels are divided into four levels: normal level <1.69mmol / L; critical high level 1.69-2.25mmol / L; high level 2.26-5.63mmol / L; extremely high level ≥5.64mmol / L. Patients with critical high and high levels of triglycerides often have lipid disorders that increase the risk of coronary heart disease, such as familial combined hyperlipidemia and diabetic dyslipidemia. Patients with triglyceride levels higher than 11.3mmol / L have a greatly increased risk of acute pancreatitis.

[0004] Therefore, increased triglyceride levels in human serum are specific indicators of primary or secondary hypertriglyceridemia, and are of great value in the diagnosis of atherosclerosis, diabetes, nephrotic syndrome, fatty liver and other liver diseases, systemic lupus erythematosus, glycogen storage disease, congenital lipoprotein deficiency, and pancreatitis crisis.

[0005] Serum TG determination methods can generally be divided into three categories: chemical method, enzymatic method and chromatography.

[0006] The chemical method uses an organic solvent to extract TG from the specimen, removes interferences such as phospholipids in the extract, then uses alkali to hydrolyze (saponify) TG, oxidizes glycerol with periodic acid to generate formaldehyde, and then uses a color reaction to measure formaldehyde. The more accurate method is the dichloromethane-silicic acid-chromotropic acid method (Van Handel-Caslson method), which can completely extract, remove interferences from phospholipids and glycerol, and has high color stability of chromotropic acid. However, it is not suitable for routine work applications due to the numerous operating steps and high technical requirements.

[0007] Radionuclide dilution / gas chromatography / mass spectrometry (ID / GC / MS) is mainly used to establish deterministic methods in reference systems and to prepare and determine the value of reference materials. This method is expensive, sample processing is complex, and it is difficult to promote and apply.

[0008] At present, almost all clinical laboratories use the enzymatic method to detect serum TG levels. Although this method has the advantages of being simple, rapid, trace, and highly precise, during the development of the triglyceride determination kit (oxidase method), it was found that the reagents in the kit were unstable and had high requirements for storage conditions and storage containers. When unopened, the shelf life is 12 months when stored at 2-8°C; after opening the bottle, its shelf life is only 14 days, and in the accelerated test at 37°C, after 7 days, the reagent changes from a colorless clear liquid to a red clear liquid, and the enzyme activity decreases, which in turn affects the accuracy of the reagent. Summary of the invention

[0009] The purpose of the embodiments of the present application is to provide a single reagent, a kit and a method for determining triglycerides, so as to solve the technical problem in the prior art that the reagents are unstable and affect the accuracy of the reagents.

[0010] In order to solve the above technical problems, the present application provides a single reagent for triglyceride determination, which adopts the following technical solution:

[0011] The components of the single reagent include buffer, magnesium ions, complexing agent, stabilizer, color developer, adenosine triphosphate compound, phenolic compound, catalytic enzyme, antioxidant, anti-bilirubin interfering agent and anti-hemoglobin interfering agent;

[0012] Wherein, the catalytic reaction enzymes include lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase and peroxidase.

[0013] Further, the concentration of the buffer is 50-100 mmol / L;

[0014] The concentration of the magnesium ion is 2.0-3.0 g / L;

[0015] The concentration of the complexing agent is 2.0-3.0 mmol / L;

[0016] The concentration of the stabilizer is 1.0g-1.5 / L;

[0017] The concentration of the developer is 0.3-0.5 mmol / L;

[0018] The concentration of the adenosine triphosphate compound is 2.0-3.0 mmol / L;

[0019] The concentration of the phenolic compound is 4.0-4.5 mmol / L;

[0020] The concentration of the lipoprotein esterase is 100-120 KU / L;

[0021] The concentration of the glycerol kinase is 1.5-1.8 KU / L;

[0022] The concentration of the glycerol phosphate oxidase is 8-10 KU / L;

[0023] The concentration of the peroxidase is 5-7 KU / L;

[0024] The concentration of the antioxidant is 0.03-0.05 g / L;

[0025] The anti-bilirubin interfering agent includes potassium ferrocyanide and lauramide propyl betaine, the concentration of the potassium ferrocyanide is 0.15-0.20 mmol / L, and the concentration of the lauramide propyl betaine is 3.0-5.0 g / L;

[0026] The anti-hemoglobin interfering agent is selected from sodium oxamate, and the concentration of the sodium oxamate is 2.0-3.0 mmol / L.

[0027] Furthermore, the buffer is selected from one of tris(hydroxymethylaminomethane) buffer, imidazole buffer, 2-(N-morpholine)ethanesulfonic acid buffer and 1,4-piperazine diethanesulfonic acid buffer.

[0028] Further, the magnesium ion is selected from at least one of magnesium acetate, magnesium aspartate, magnesium chloride and magnesium sulfate;

[0029] The developer is selected from one of 4-aminoantipyrine and TOOS.

[0030] Furthermore, the antioxidants include vitamin C and coenzyme Q10.

[0031] Furthermore, the components of the single reagent also include an activator, and the activator is selected from one of mercaptoglycerol, N-acetyl-L-cysteine, L-cysteine ​​and Triton X-100.

[0032] Furthermore, the components of the single reagent also include a preservative, and the preservative is selected from one of sodium azide, methylisothiazolinone and Proclin series preservatives.

[0033] Furthermore, the components of the single reagent include 1,4-piperazine diethanesulfonic acid buffer, magnesium chloride, ethylenediaminetetraacetic acid disodium salt, bovine serum albumin, 4-aminoantipyrine, adenosine triphosphate-disodium salt, 4-chloro-phenol, lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase, peroxidase, vitamin C, coenzyme Q10, potassium ferrocyanide, lauramidopropyl betaine and sodium oxamate.

[0034] In order to solve the above technical problems, the present application also provides a triglyceride determination kit, which adopts the following technical solution:

[0035] Includes a triglyceride assay single reagent as described above.

[0036] In order to solve the above technical problems, the present application also provides a method for determining triglycerides in a kit for determining triglycerides, which adopts the following technical solution:

[0037] Collecting a serum sample, adding a single reagent to the serum sample, mixing, incubating for 5 to 10 minutes, and measuring the absorbance at a main wavelength of 500 nm and a secondary wavelength of 700 nm;

[0038] The triglyceride content was calculated based on the absorbance.

[0039] Compared with the prior art, this application has the following beneficial effects:

[0040] The single reagent provided in the present application has buffer, magnesium ions, chelating agents, stabilizers, color developers, adenosine triphosphate compounds, phenolic compounds, catalytic reaction enzymes, antioxidants, anti-bilirubin interfering agents and anti-hemoglobin interfering agents added to its components. By improving the reagent formula, the stability of the reagent can be improved, the enzyme activity can be ensured, and the accuracy of the reagent can be improved, thereby improving the stability of the kit, and improving the scope of use, accuracy and utilization of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 It is a data diagram of the clinical correlation experimental results of the triglyceride determination kit and the sample of the embodiment of the present application;

[0043] Figure 2 It is a reaction curve diagram of the comparative example of the present application;

[0044] Figure 3 is a reaction curve diagram of an embodiment of the present application;

[0045] Figure 4 This is a linear graph after 7 days acceleration at 37°C in the example of the present application. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0047] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0048] In order to make the technical personnel in this technical field better understand the scheme of this application, the technical scheme in the embodiment of this application will be clearly and completely described below in conjunction with the accompanying drawings. The experimental methods used in the embodiments of this application are all common methods unless otherwise specified; the reagents used in the embodiments of this application can be obtained from commercial channels unless otherwise specified.

[0049] The present application provides a single reagent for triglyceride determination, wherein the components of the single reagent R include a buffer, magnesium ions, a complexing agent, a stabilizer, a color developer, an adenosine triphosphate compound, a phenolic compound, a catalytic reaction enzyme, an antioxidant, an anti-bilirubin interfering agent, and an anti-hemoglobin interfering agent;

[0050] The enzymes catalyzing the reaction include lipoprotein lipase, glycerol kinase, glycerol phosphate oxidase and peroxidase.

[0051] Among them, the buffer provides a suitable pH buffer environment for the reagent reaction; magnesium ions are used to activate lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase and peroxidase in the reagent, especially glycerol kinase and peroxidase, to promote triglyceride reaction; the complexing agent is an important chelating agent, which can chelate metal ions in the solution to form a soluble chelate without any precipitation, thereby improving the antioxidant property of the reagent; the stabilizer protects the enzyme and prevents its decomposition and non-specific adsorption; the color developer reacts with phenolic compounds in the presence of an oxidant to generate a red dye; lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase and peroxidase play a catalytic role in the reaction of triglycerides; the antioxidant is used to prevent the components in the reagent from being oxidized; the anti-bilirubin interfering agent and the anti-hemoglobin interfering agent can eliminate the interference of bilirubin and hemoglobin on the reagent, significantly improving the stability and anti-interference ability of the reagent.

[0052] Glycerol in serum reacts with adenosine triphosphate (ATP) in the adenosine triphosphate compound in single reagent R to generate glycerol-3-phosphate and adenosine diphosphate (ADP) under the action of glycerol kinase (GK). Glycerol-3-phosphate reacts with O2 to generate dihydroxyacetone phosphate (DAP) and H2O2 under the action of glycerol phosphate oxidase (GPO). The color developer reacts with H2O2 to generate red quinone imine pigment under the action of peroxidase (POD). The absorbance of the red quinone imine pigment generated by the reaction is proportional to the concentration of triglycerides in the sample. The absorbance can be measured at a wavelength of 500nm using a fully automatic biochemical analyzer to calculate the content of triglycerides. The principle reaction is as follows:

[0053]

[0054]

[0055]

[0056]

[0057] In some embodiments, the concentration of the buffer is 50-100 mmol / L; the concentration of the magnesium ion is 2.0-3.0 g / L; the concentration of the complexing agent is 2.0-3.0 mmol / L; the concentration of the stabilizer is 1.0 g-1.5 / L; the concentration of the developer is 0.3-0.5 mmol / L; the concentration of the adenosine triphosphate compound is 2.0-3.0 mmol / L; the concentration of the phenolic compound is 4.0-4.5 mmol / L; the concentration of the lipoprotein lipase is 100-120 KU / L; the concentration of the glycerol kinase is 100-120 KU / L; the concentration of the glycerol kinase is 100-120 KU / L; the concentration of the phosphodiester ... The concentration of glycerol phosphate oxidase is 8-10 KU / L; the concentration of peroxidase is 5-7 KU / L; the concentration of antioxidant is 0.03-0.05 g / L; the anti-bilirubin interfering agent includes potassium ferrocyanide and lauramide propyl betaine, the concentration of potassium ferrocyanide is 0.15-0.20 mmol / L, and the concentration of lauramide propyl betaine is 3.0-5.0 g / L; the anti-hemoglobin interfering agent is selected from sodium oxamate, and the concentration of sodium oxamate is 2.0-3.0 mmol / L.

[0058] The interference of bilirubin in blood samples is eliminated by adding potassium ferrocyanide. Potassium ferrocyanide has strong oxidizing properties and can convert bilirubin into biliverdin, avoiding inaccurate results of jaundice sample measurements. However, although potassium ferrocyanide alone can eliminate the interference of bilirubin, it affects the accuracy, analytical sensitivity and linear range of the reagent. By adding Emulgen series surfactants such as lauramide propyl betaine, the decline in linearity and accuracy can be effectively avoided. Potassium ferrocyanide can effectively eliminate the interference of bilirubin, but it will reduce the reaction rate of the substrate. The Emulgen series surfactants have a protective effect on the substrate reaction. The two interact with each other, solving the problem of anti-bilirubin interference of the reagent without affecting the accuracy and linear performance indicators.

[0059] By adding sodium oxamate to the single reagent, the stability of hemoglobin can be inhibited, thus avoiding the negative interference of hemoglobin on the test results.

[0060] In a specific example, the concentration of the buffer is 50mmol / L; the concentration of magnesium ions is 2.0g / L; the concentration of the chelating agent is 2mmol / L; the concentration of the stabilizer is 1.0g / L; the concentration of the color developer is 0.3mmol / L; the concentration of the adenosine triphosphate compound is 2mmol / L; the concentration of the phenolic compound is 4mmol / L; the concentration of lipoprotein lipase is 100KU / L; the concentration of glycerol kinase is 1.5KU / L; the concentration of glycerol phosphate oxidase is 8KU / L; the concentration of peroxidase is 5KU / L; the concentration of the antioxidant is 0.04g / L; the concentration of potassium ferrocyanide is 0.15mmol / L, the concentration of lauroylamidopropyl betaine is 4.0g / L; and the concentration of sodium oxamate is 2.5mmol / L.

[0061] In some embodiments, the buffer is selected from one of tris (hydroxymethyl) aminomethane buffer (Tris buffer), imidazole buffer, 2-(N-morpholinoline) ethanesulfonic acid buffer (MES buffer), and 1,4-piperazine diethanesulfonic acid buffer (PIPES buffer). Among them, the buffer is preferably PIPES buffer.

[0062] The above buffer type can provide a suitable pH buffer environment for the reagent reaction, with a pH value of 7.0±0.15.

[0063] In some embodiments, the magnesium ion is selected from at least one of magnesium acetate, magnesium aspartate, magnesium chloride and magnesium sulfate, wherein the magnesium ion is preferably derived from magnesium chloride.

[0064] In some embodiments, the color developer is selected from one of 4-aminoantipyrine and TOOS, wherein TOOS is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

[0065] In some embodiments, antioxidants include vitamin C and coenzyme Q10.

[0066] Lipoprotein lipase, glycerol kinase, glycerol phosphate oxidase, peroxidase and coenzyme Q10 are all enzymes, and commercial enzymes can be used.

[0067] In some embodiments, the components of the single reagent R further include an activator, and the activator is selected from one of mercaptoglycerol, N-acetyl-L-cysteine, L-cysteine ​​and Triton X-100.

[0068] In some embodiments, the concentration of the activator is 15-20 mmol / L.

[0069] Among them, the activator can preferably be N-acetyl-L-cysteine, which has the advantages of increasing solubility, activating enzymes, reducing interference from serum lipid substances, reducing the hysteresis effect of the detection platform, facilitating the use of reagents, and reducing nonspecific adsorption and antibacterial effects on solid surfaces in immunoassays.

[0070] In some embodiments, the components of the single reagent R further include a preservative, and the preservative is selected from one of sodium azide, methylisothiazolinone and Proclin series preservatives.

[0071] In some embodiments, the concentration of the preservative is 0.5-0.8 g / L.

[0072] Among them, the preservative is preferably sodium azide, which has the advantages of strong solubility, being tasteless and odorless, and having excellent preservative and bactericidal properties without affecting the performance and appearance of the reagent, and can be prepared into an aqueous solution for use as needed.

[0073] In some embodiments, the components of the single reagent R include 1,4-piperazine diethanesulfonic acid buffer, magnesium chloride, ethylenediaminetetraacetic acid disodium salt, bovine serum albumin, 4-aminoantipyrine, adenosine triphosphate-disodium salt, 4-chloro-phenol, lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase, peroxidase, vitamin C and coenzyme Q10.

[0074] Among them, the concentration of 1,4-piperazine diethanesulfonic acid buffer is 50mmol / L; the concentration of magnesium chloride (magnesium ion) is 2.0g / L; the concentration of ethylenediaminetetraacetic acid disodium salt (chelating agent) is 2mmol / L; the concentration of bovine serum albumin (stabilizer) is 1.0g / L; the concentration of 4-aminoantipyrine is 0.3mmol / L; the concentration of adenosine triphosphate-disodium salt (adenosine triphosphate compound) is 2mmol / L; the concentration of 4-chloro-phenol (phenolic compound) is 4mmol / L; the concentration of lipoprotein lipase is 100KU / L; the concentration of glycerol kinase is 1.5KU / L; the concentration of glycerol phosphate oxidase is 8KU / L; the concentration of peroxidase is 5KU / L; the concentration of vitamin C (antioxidant) is 0.035g / L; the concentration of coenzyme Q10 (antioxidant) is 0.05g / L.

[0075] In some embodiments, the components of the single reagent R include 1,4-piperazine diethanesulfonic acid buffer, magnesium chloride, ethylenediaminetetraacetic acid disodium salt (complexing agent), bovine serum albumin (stabilizer), 4-aminoantipyrine, adenosine triphosphate-disodium salt, 4-chloro-phenol, lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase, peroxidase, vitamin C, coenzyme Q10, potassium ferrocyanide, lauramidopropyl betaine and sodium oxamate.

[0076] Among them, the concentration of 1,4-piperazine diethanesulfonic acid buffer is 50mmol / L; the concentration of magnesium chloride is 2.0g / L; the concentration of ethylenediaminetetraacetic acid disodium salt is 2mmol / L; the concentration of bovine serum albumin is 1.0g / L; the concentration of 4-aminoantipyrine is 0.3mmol / L; the concentration of adenosine triphosphate-disodium salt is 2mmol / L; the concentration of 4-chloro-phenol is 4mmol / L; the concentration of lipoprotein lipase is 100KU / L; the concentration of glycerol kinase is 1.5KU / L; the concentration of glycerol phosphate oxidase is 8KU / L; the concentration of peroxidase is 5KU / L; the concentration of vitamin C (antioxidant) is 0.035g / L; the concentration of coenzyme Q10 is 0.05g / L; the concentration of potassium ferrocyanide is 0.15mmol / L, the concentration of lauramidopropyl betaine is 4.0g / L; the concentration of sodium oxamate is 2.5mmol / L.

[0077] The preparation method of the single reagent R of the present application is a conventional method, that is, according to the above-mentioned formula ratio, each component except the enzyme in the single reagent is added to pure water, mixed and stirred to fully dissolve, and the pH is adjusted to 7.0±0.15, and then glycerol kinase, glycerol-3-phosphate oxidase, lipoprotein lipase, and peroxidase are added, mixed, and the single reagent R is prepared.

[0078] Buffer, magnesium ions, chelating agents, stabilizers and antioxidants are added to the components of the single reagent of the present application. By improving the reagent formula, the stability of the reagent can be improved, the enzyme activity can be guaranteed, and the accuracy of the reagent determination can be improved, thereby improving the stability of the kit, and improving the scope of use, accuracy and utilization of the kit.

[0079] Based on the above-mentioned single reagent for triglyceride determination, the present application also provides a triglyceride determination kit, which includes the above-mentioned single reagent for triglyceride determination.

[0080] The stability of the test kit of the present application is improved, thereby improving the detection accuracy of the test kit.

[0081] The present application also provides a triglyceride determination method of the kit, comprising the following steps:

[0082] Collect serum samples, add single reagents to the serum samples, mix well, incubate for 5 to 10 minutes, and then measure the absorbance at the main wavelength of 500 nm and the secondary wavelength of 700 nm;

[0083] The triglyceride content was calculated based on the absorbance.

[0084] Specifically, the measurement conditions are as follows:

[0085] Temperature: 37°C

[0086] The light path of the cuvette is 1.0 cm;

[0087] The main detection wavelength is 500nm and the secondary detection wavelength is 700nm.

[0088] The method for measuring the triglyceride (Tg) activity in a sample using the triglyceride assay kit of the present application is as follows:

[0089] Take 3 μL of serum sample (calibration tube uses calibrator as sample, blank tube uses purified water as sample) and add 200 μL of single reagent R and mix well. After incubation at 37°C for 5 min, adjust to zero with blank tube and detect the absorbance A of other tubes at main wavelength / sub-wavelength of 500 / 700 nm.

[0090] Table 1 Test samples

[0091]

[0092] The triglyceride (Tg) activity in the sample determined by the kit of this application is calculated according to the following formula:

[0093]

[0094] It should be noted that the test kit of this application is suitable for fully automatic biochemical analyzers such as Daan Stream SuperB-800 / 2000, Beckman AU680 / 5800, Hitachi HITACHI7180 / 7600, Hitachi LABOSPECT 008AS, Toshiba TBA120FR, Siemens ADVIA1200 / 2400, Sysmex BX-4000, Mindray BS800 / 830, Dirui CS-600B / CS-1200, and Yilanbei ES-480.

[0095] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0096] Example 1

[0097] This embodiment provides a triglyceride determination kit, which is based on a fully automatic biochemical analyzer, so that the detection system can directly and quickly perform detection.

[0098] The components and concentrations of the kit of this example are shown in Table 2 below.

[0099] Table 2

[0100]

[0101] The preparation method of the single reagent R in this embodiment is a conventional method, that is, according to the above-mentioned formula ratio, each component except the enzyme in the single reagent is added to pure water, mixed and stirred to fully dissolve, and the pH is adjusted to 7.15±0.15, and then glycerol kinase, glycerol-3-phosphate oxidase, lipoprotein esterase, and horseradish peroxidase are added and mixed to prepare the single reagent R.

[0102] Comparative Example 1

[0103] This comparative example provides a kit, the components and concentrations of the kit are shown in Table 3.

[0104] Table 3 Comparative Example Reagent Components and Concentrations

[0105]

[0106] The preparation method of the reagent in the comparative example is a conventional method, that is, each component of the reagent is added with distilled water and mixed and stirred, and the pH is adjusted to 7.15±0.15.

[0107] It is stipulated that the machine parameters, sample volume and reagent volume of Example 1 and Comparative Example 1 are the same, the same samples / quality control are tested, and the tests are carried out simultaneously on a Hitachi 7180 fully automatic biochemical analyzer.

[0108] After Example 1 and Comparative Example 1 were prepared, Randox CAL3 (target value: 2.83 mmol / L, batch number: 1214UE) was used for two-point linear calibration after the reagent system was stable, and then Randox CON-2 (target value: 1.00 mmol / L, acceptable range: 0.84-1.16 mmol / L, batch number: 1552UN) and CON-3 (target value: 2.97 mmol / L, acceptable range: 2.49-3.45 mmol / L, batch number: 1072UE) were used for quality control verification, and the calibration curve, reaction absorbance, and quality control measurement value were recorded.

[0109] The reagents of Example 1 and Comparative Example 1 were divided into two parts, one part was stored at 2-8°C, and the other part was accelerated in a constant temperature oven at 37°C for 7 days. Full performance testing of the 2-8°C and 37°C reagents was performed for 7 days, including calibration, quality control, precision, linearity and clinical comparison.

[0110] The repetitive samples used Randox quality control products of different concentrations, the clinical comparison samples were 200 random sera stored at -80°C, and the clinical comparison reagent was the triglyceride determination kit (GPO-PAP method) produced by Beijing Jiuqiang Biotechnology Co., Ltd. [Beijing Medical Device Registration No. 20162400564].

[0111] (1) According to the above measurement settings, the measurement was performed on the Hitachi 7180 fully automatic biochemical analyzer, and the measurement values ​​of 200 clinical samples were recorded. The results are shown in Table 4 and Figure 1 shown.

[0112] Table 4: Measured values ​​of 240 clinical samples from the embodiment and Beijing Jiuqiang (unit: mmol / L)

[0113]

[0114]

[0115]

[0116]

[0117] Figure 1 The following are the results of clinical correlation experiments between the embodiments and 240 samples from Beijing Jiuqiang Hospital.

[0118] The above results show that the embodiment is clinically compared with the triglyceride determination kit (GPO-PAP method) produced by Beijing Jiuqiang Biotechnology Co., Ltd., which has been mature on the market. 2 It reached 0.9988, with good linear fit and high negative and positive coincidence rates, which met the requirements of clinical trials.

[0119] (2) According to the above measurement settings, the measurement was carried out on a Hitachi 7180 fully automatic biochemical analyzer, and the reaction curves, quality control, repeatability, and linearity results of the embodiment and the control example after 7 days of acceleration at 37°C were shown in the following table. Figures 2 to 4 As shown in Tables 5 to 7.

[0120] Table 5 Landau CAL3 calibration data

[0121]

[0122] Table 6 Precision data after 7 days acceleration at 37°C in Example

[0123]

[0124]

[0125] Table 7 Linear data after 7 days acceleration at 37°C in Example

[0126]

[0127] The above results show that, compared with the comparative example, the embodiment after acceleration at 37°C for 7 days has no effect on the enzyme activity in the reagent and still has a good reaction, judging from the calibration results and the reaction curve of the calibrator; the detection quality control products CON-2\CON-3 are both under control, and the precision coefficient of variation CV% of the two concentrations does not exceed ±5.0%; the linearity is in the range of [0,16.0]mmol / L, the consistency rate is between 95.4%-100.8%, the relative deviation does not exceed ±10.0%, and the linear correlation coefficient r>0.990; the reagent blank absorbance is measured at A500nm (primary) / A600nm (secondary) <0.1000; after acceleration at 37°C for 7 days, the reagent does not obviously turn red and is still a light yellow clear liquid.

[0128] In summary, the triglyceride determination kit provided by the present application has high stability and good detection accuracy.

[0129] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A single reagent for determining triglycerides, characterized in that: The components of the single reagent include buffer, magnesium ions, complexing agent, stabilizer, color developer, adenosine triphosphate compound, phenolic compound, catalytic enzyme, antioxidant, anti-bilirubin interfering agent and anti-hemoglobin interfering agent; Wherein, the catalytic reaction enzymes include lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase and peroxidase.

2. The single reagent for determining triglycerides according to claim 1, characterized in that: The concentration of the buffer is 50-100 mmol / L; The concentration of the magnesium ion is 2.0-3.0 g / L; The concentration of the complexing agent is 2.0-3.0 mmol / L; The concentration of the stabilizer is 1.0g-1.5 / L; The concentration of the developer is 0.3-0.5 mmol / L; The concentration of the adenosine triphosphate compound is 2.0-3.0 mmol / L; The concentration of the phenolic compound is 4.0-4.5 mmol / L; The concentration of the lipoprotein esterase is 100-120 KU / L; The concentration of the glycerol kinase is 1.5-1.8 KU / L; The concentration of the glycerol phosphate oxidase is 8-10 KU / L; The concentration of the peroxidase is 5-7 KU / L; The concentration of the antioxidant is 0.03-0.05 g / L; The anti-bilirubin interfering agent includes potassium ferrocyanide and lauramide propyl betaine, the concentration of the potassium ferrocyanide is 0.15-0.20 mmol / L, and the concentration of the lauramide propyl betaine is 3.0-5.0 g / L; The anti-hemoglobin interfering agent is selected from sodium oxamate, and the concentration of the sodium oxamate is 2.0-3.0 mmol / L.

3. The single reagent for determining triglycerides according to claim 1, characterized in that: The buffer is selected from one of tris(hydroxymethylaminomethane) buffer, imidazole buffer, 2-(N-morpholine)ethanesulfonic acid buffer and 1,4-piperazine diethanesulfonic acid buffer.

4. The single reagent for determining triglycerides according to claim 1, characterized in that: The magnesium ion is selected from at least one of magnesium acetate, magnesium aspartate, magnesium chloride and magnesium sulfate; The developer is selected from one of 4-aminoantipyrine and TOOS.

5. The single reagent for determining triglycerides according to claim 1, characterized in that: The antioxidants include vitamin C and coenzyme Q10.

6. The single reagent for determining triglycerides according to claim 1, characterized in that: The components of the single reagent also include an activator, which is selected from one of mercaptoglycerol, N-acetyl-L-cysteine, L-cysteine ​​and Triton X-100.

7. The single reagent for determining triglycerides according to claim 1, characterized in that: The components of the single reagent also include a preservative, and the preservative is selected from one of sodium azide, methylisothiazolinone and Proclin series preservatives.

8. The single reagent for determining triglycerides according to any one of claims 1 to 7, characterized in that: The components of the single reagent include 1,4-piperazine diethanesulfonic acid buffer, magnesium chloride, ethylenediaminetetraacetic acid disodium salt, bovine serum albumin, 4-aminoantipyrine, adenosine triphosphate-disodium salt, 4-chloro-phenol, lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase, peroxidase, vitamin C, coenzyme Q10, potassium ferrocyanide, lauramidopropyl betaine and sodium oxamate.

9. A triglyceride determination kit, characterized in that: Comprising the single reagent for determining triglyceride as described in any one of claims 1 to 8.

10. A triglyceride determination method of a kit, characterized in that: The following steps are involved: Collecting a serum sample, adding a single reagent to the serum sample, mixing, incubating for 5 to 10 minutes, and measuring the absorbance at a main wavelength of 500 nm and a secondary wavelength of 700 nm; The triglyceride content was calculated based on the absorbance.

Images