Triglyceride determination kit

Abstract

The invention provides a triglyceride determination kit, the kit comprises a reagent 1 and a reagent 2, the reagent 1 comprises the following main components: piperazine-1, 4-diethyl sulfonic acid (PIPES) buffer solution (pH 6.8), glycerophosphate oxidase (GPO), peroxidase (POD), triadenosine phosphate (ATP), chromogen, a surfactant, a stabilizer and a preservative; and the reagent 2 comprises the following main components: a piperazine-1, 4-diethyl sulfonic acid (PIPES) buffer solution (pH is 6.5), lipoprotein esterase (LPL), glycerol kinase (GK), 4-aminoantipyrine (4-AA), a surfactant, a stabilizer and a preservative. By optimizing a chromogen substrate in the reagent and adding potassium ferrocyanide, the linear range of the prepared kit is wider, and the bottle opening test is more stable.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection. The triglyceride determination kit provided by the invention improves the test linear range while significantly improving the opening stability, and specifically relates to a triglyceride determination kit and a preparation method thereof. Background Art

[0002] Triglyceride (abbreviated as TRIG) is a fat molecule formed by three molecules of long-chain fatty acids and glycerol. Triglyceride is the most abundant lipid in the human body. Most tissues can use the decomposition products of triglyceride to supply energy. At the same time, tissues such as the liver and fat can also synthesize triglycerides and store them in adipose tissue.

[0003] Triglycerides are used for auxiliary diagnosis of diseases such as atherosclerosis, diabetes, hypothyroidism, and nephrotic syndrome. Elevated TG can be seen in: familial hypertriglyceridemia, familial mixed hyperlipidemia; secondary diseases: diabetes, glycogen storage disease, hypothyroidism, nephrotic syndrome, pregnancy, etc.; in high-risk states of acute insulitis, TG is significantly elevated, and hypertension, cerebrovascular disease, coronary heart disease, diabetes, obesity, and hyperlipoproteinemia often have familial clustering. Simple hypertriglyceridemia is not an independent risk factor for coronary heart disease, and it only has pathological significance when accompanied by high TC, high LDL-C, and low HDL-C. Decreased TG is seen in: hyperthyroidism, adrenocortical hypofunction, severe liver dysfunction, and is very likely to cause fatty liver and other symptoms.

[0004] At present, common laboratory diagnostic methods include enzymatic method, chemical method, chromatography, etc. Chemical method and chromatography are not suitable for widespread application in routine clinical testing due to the numerous operating steps and high technical requirements. At present, the most widely used method in clinical practice is the enzymatic method for detecting serum TG levels. The principle of the enzymatic reaction is as follows: lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides into glycerol and fatty acids, glycerol kinase (GK) catalyzes the phosphorylation of glycerol to produce glycerol-3-phosphate, which is oxidized to dihydroxyacetone phosphate and H2O2 under the catalysis of glycerol phosphate oxidase (GPO). H2O2 reacts with 4-aminoantipyrine (4-AAP) and chromogen substrate under the action of peroxidase (POD) to form quinone imine compounds, the color depth of which is proportional to the concentration of triglycerides in the sample.

[0005] The triglyceride detection kits currently on the market have the characteristics of good stability, strong anti-interference ability, and high sensitivity. However, their test linearity is narrow. The reagent opening is prone to oxidation reaction, resulting in poor opening stability and other performance indicators that have not been improved. Therefore, it is of great significance to provide a triglyceride detection kit with wide test linearity, good opening stability, and strong anti-interference ability. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a triglyceride determination kit, which has a wider linear range by selecting a composite ratio of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS). Potassium ferrocyanide is added to the reagent to reduce the impact caused by oxidation reaction caused by the reagent being opened for a long time.

[0007] A triglyceride determination kit, characterized in that the kit comprises reagent 1 and reagent 2, wherein the main components of reagent 1 are: piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8), glycerol phosphate oxidase (GPO), peroxidase (POD), triadenosine phosphate (ATP), complex chromogen, surfactant, stabilizer, and preservative; the main components of reagent 2 are: piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.5), lipoprotein lipase (LPL), glycerol kinase (GK), 4-aminoantipyrine (4-AAP), potassium ferrocyanide, surfactant, stabilizer, and preservative.

[0008] Furthermore, in the reagent 1, the concentration of piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8) is 50 mmol / L; the concentration of glycerol phosphate oxidase (GPO) is 0.8 KU / L; the concentration of peroxidase (POD) is 5 KU / L; the concentration of adenosine triphosphate (ATP) is 0.5 mmol / L; the concentration of complex chromogen is 0.6 g / L; the concentration of surfactant is 1-10 g / L; the concentration of stabilizer is 1-10 g / L; and the concentration of preservative is 0.1-0.5%.

[0009] Furthermore, the composite chromogen in the reagent 1 is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS), and the optimal weight ratio is 1:1.

[0010] Furthermore, the surfactant in the reagent 1 is Tween 20 with a concentration of 5 g / L.

[0011] Furthermore, the reagent 1 stabilizer selects bovine serum albumin (BSA) with a concentration of 2 g / L.

[0012] Furthermore, the preservative of reagent 1 is proclin 300 with a concentration of 0.4 ml / L.

[0013] Furthermore, the concentration of the reagent 2 buffer piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8) is 35 mmol / L; the concentration of lipoprotein lipase (LPL) is 2 KU / L; the concentration of glycerol kinase (GK) is 5 KU / L; the concentration of 4-aminoantipyrine (4-AAP) is 0.5 mmol / L; the concentration of the surfactant is 1-10 g / L; the concentration of the stabilizer is 1-10 g / L; and the concentration of the preservative is 0.1-0.5%.

[0014] Furthermore, the surfactant of reagent 2 is selected as Triton X-100 with a concentration of 5 g / L.

[0015] Furthermore, the reagent 2 stabilizer selects a sucrose concentration of 2 g / L.

[0016] Furthermore, the preservative of reagent 2 is proclin 300 with a concentration of 0.4 ml / L.

[0017] The present invention has the following beneficial effects: 1. The present invention optimizes the chromogen substrate, thereby making the linear range of the triglyceride detection kit wider.

[0018] 2. The present invention obtains a triglyceride detection kit for opening stability test by adding potassium ferrocyanide reducing agent into the kit. DETAILED DESCRIPTION

[0019] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0020] Example 1 The present invention provides a triglyceride detection kit, the main components of which are shown in Table 1: Table 1. Reagent components of Example 1 The selection grouping of the chromogen substrate is set as shown in Table 2: Table 2. Chromogen substrate composition and concentration Group Chromogen Substrate concentration 1 DAOS 0.6g / L 2 MAOS 0.6g / L 3 DAOS:MAOS=1:1 0.6g / L Performance evaluation: Samples with a theoretical concentration range of 0-15mmol / L were selected to calculate the correlation coefficient r and the deviation between the measured mean and the theoretical concentration. The results are shown in Table 3: Table 3. Test results of samples with different chromogen substrate groups According to the results in the above table, when DAOS and MAOS are prepared separately, the highest detection value of the triglyceride detection reagent cannot reach 15mmol / L, and the correlation coefficient is less than 0.99, and the deviation value is greater than ±10%, that is, the linear range is narrow and the linearity is poor, which cannot meet the needs of clinical use. When the concentration ratio of the chromogen substrate is 1:1, the linear range of the kit is wide, the highest detection value can reach 15mmol / L, and the correlation coefficient is greater than 0.99, and the deviation value is within ±10%, that is, the linear correlation is good and the measurement is accurate, which meets the needs of clinical use.

[0021] Embodiment 2: The present invention provides a triglyceride detection kit, the main components of which are shown in Table 4: Table 4. Reagent components of Example 2 The difference between the comparative example and the embodiment is that potassium ferrocyanide is not added to reagent 2. At the same time, the embodiment and the comparative example are calibrated. After calibration, performance tests are performed on the 0th, 10th, 20th, 25th, 30th, 32nd, 34th, 36th, 38th, 40th, 42nd and 44th days after opening the bottle, without recalibration during the period. The results are compared. The results are shown in Table 5: Table 5. Open bottle stability test results As shown in Table 5, the measured value deviation of the comparative reagent is less than 10% after 32 days of opening, and the performance can meet the detection requirements. The measured value deviation is greater than 10% after 34 days, which no longer meets the detection requirements, and the measured value is constantly rising; the measured value deviation of the example reagent is less than 10% after 42 days of opening, and the performance still meets the detection requirements. The time when the measured value deviation is less than 10% after the reagent 2 is added with potassium ferrocyanide is opened and the opening stability of the reagent is effectively improved.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A triglyceride determination kit, characterized in that The kit comprises reagent 1 and reagent 2. The main components of reagent 1 are: piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8), glycerol phosphate oxidase (GPO), peroxidase (POD), triadenosine phosphate (ATP), complex chromogen, surfactant, stabilizer, and preservative; the main components of reagent 2 are: piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.5), lipoprotein lipase (LPL), glycerol kinase (GK), 4-aminoantipyrine (4-AAP), potassium ferrocyanide, surfactant, stabilizer, and preservative.

2. A triglyceride determination kit according to claim 1, characterized in that: The concentration of piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8) in the reagent 1 is 50 mmol / L; the concentration of glycerol phosphate oxidase (GPO) is 0.8 KU / L; the concentration of peroxidase (POD) is 5 KU / L; the concentration of adenosine triphosphate (ATP) is 0.5 mmol / L; the concentration of complex chromogen is 0.6 g / L; the concentration of surfactant is 1-10 g / L; the concentration of stabilizer is 1-10 g / L; and the concentration of preservative is 0.1-0.5%.

3. A triglyceride determination kit according to claim 1 or 2, characterized in that: The composite chromogen in the reagent 1 is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS), and the optimal concentration ratio is 1:

1.

4. A triglyceride determination kit as claimed in claim 1 or 2, characterized in that: The surfactant in the reagent 1 is Tween 20 with a concentration of 5 g / L.

5. A triglyceride determination kit as claimed in claim 1 or 2, characterized in that: The stabilizer of reagent 1 is bovine serum albumin (BSA) with a concentration of 2 g / L.

6. A triglyceride determination kit as claimed in claim 1 or 2, characterized in that: The preservative of reagent 1 is proclin-300 with a concentration of 0.4 ml / L.

7. A triglyceride determination kit as claimed in claim 1, characterized in that: The reagent 2 buffer piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH 6.8) has a concentration of 35 mmol / L; a lipoprotein lipase (LPL) concentration of 2 KU / L; a glycerol kinase (GK) concentration of 5 KU / L; a 4-aminoantipyrine (4-AAP) concentration of 0.5 mmol / L; a surfactant concentration of 1-10 g / L; a stabilizer concentration of 1-10 g / L; and a preservative concentration of 0.1-0.5%.

8. A triglyceride determination kit according to claim 1 or 7, characterized in that: The surfactant selected in reagent 2 is Triton X-100 with a concentration of 5 g / L.

9. A triglyceride determination kit as claimed in claim 1 or 7, characterized in that: The reagent 2 stabilizer selected sucrose concentration is 2 g / L.

10. A triglyceride determination kit according to claim 1 or 7, characterized in that: The preservative of reagent 2 is proclin-300 with a concentration of 0.4 ml / L.

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