阅读说明：本技术 一种对氧磷酶1的检测试剂盒及制备方法 (Detection kit for paraoxonase 1 and preparation method thereof ) 是由 袁嘉扬 单以朗 于 2019-11-07 设计创作，主要内容包括：本发明涉及一种对氧磷酶1的检测试剂盒,其特征在于：包括试剂R1和试剂R2,试剂R1的各组分及浓度包括：20-100mmol/L的第一缓冲液、0.5-2g/L的稳定剂、5-20mmol/L的激活剂、1-5g/L的抗干扰剂A、20-60ml/L的表面活性剂、5-20g/L的抗干扰剂B以及0.8-2.0ml/L的防腐剂；试剂R2的各组分及浓度包括：20-100mmol/L的第二缓冲液,2-10mmol/L的对硝基苯乙酸乙酯,5-20g/L的电解质,1-5ml/L的助溶剂,5-10g/L的促进剂以及0.8-2.0ml/L的防腐剂。本发明的优点是：稳定性好、线性范围宽、准确度高、精密度好。(The invention relates to a detection kit for paraoxonase 1, which is characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: 20-100mmol/L of first buffer solution, 0.5-2g/L of stabilizer, 5-20mmol/L of activator, 1-5g/L of anti-interference agent A, 20-60ml/L of surfactant, 5-20g/L of anti-interference agent B and 0.8-2.0ml/L of preservative; the components and concentrations of the reagent R2 include: 20-100mmol/L of second buffer solution, 2-10mmol/L of ethyl p-nitrophenylacetate, 5-20g/L of electrolyte, 1-5ml/L of cosolvent, 5-10g/L of accelerant and 0.8-2.0ml/L of preservative. The invention has the advantages that: good stability, wide linear range, high accuracy and good precision.)

1. A detection kit for paraoxonase 1 is characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:

the reagent R2 comprises the following components in percentage by weight:

2. the detection kit for paraoxonase 1 according to claim 1, wherein: the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of PBS buffer solution, HEPES buffer solution, GOOD' S buffer solution, MES buffer solution and Tris buffer solution.

3. The detection kit for paraoxonase 1 according to claim 1 or 2, wherein: the preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.

4. The detection kit for paraoxonase 1 according to claim 3, wherein: the stabilizer in the reagent R1 is one or a mixture of more than two of casein, mannitol and bovine serum albumin.

5. The detection kit for paraoxonase 1 according to claim 4, wherein: the surfactant in the reagent R1 is one or a mixture of more than two of 2-pyrrolidone, Tween 20, triton, polyvinylpyrrolidone, octyl phenyl polyoxyethylene ether and NP-40.

6. The detection kit for paraoxonase 1 according to claim 4 or 5, wherein: the activator in the reagent R1 is calcium chloride, the anti-interference agent A is potassium ferrocyanide, and the anti-interference agent B is urea.

7. The detection kit for paraoxonase 1 according to claim 6, wherein: the electrolyte in the reagent R2 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.

8. The detection kit for paraoxonase 1 according to claim 7, wherein: the cosolvent in the reagent R2 is one or a mixture of more than two of glycerol, sodium benzoate, sodium salicylate, p-aminobenzoic acid, urea, nicotinamide and acetamide.

9. The detection kit for paraoxonase 1 according to claim 7 or 8, wherein: the accelerant in the reagent R2 is one or a mixture of more than two of polyethylene glycol 8000, polyethylene glycol 6000, polyethylene glycol 4000 and polyethylene glycol 2000.

10. The method for producing the paraoxonase 1 detection kit according to any one of claims 1 to 9, characterized in that: comprises the following steps of (a) carrying out,

(1) preparation of a reagent R1;

adding a proper amount of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a stabilizer, an activator, an anti-interference agent A, a surfactant, an anti-interference agent B and an antiseptic while stirring according to the concentration requirement in claim 1, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.30-9.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;

(2) preparation of a reagent R2;

adding a proper amount of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution, ethyl p-nitrophenylacetate, an electrolyte, a cosolvent, an accelerant and a preservative while stirring according to the concentration requirement in claim 1, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.30-9.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking.

Technical Field

The invention relates to the technical field of biology, in particular to a detection reagent for paraoxonase 1 and a preparation method thereof.

Background

The PON1 gene product, also called serum paraoxonase, plays an important role in the detoxification of organophosphorus nerve agents and has close relation with the diseases such as atherosclerosis, coronary heart disease, type 2 diabetes and the like.

Animal and in vivo experiments on serum paraoxonase show that the protein may have important pathophysiological and pharmacological effects. With the development of molecular biology technology, the in vitro expression and gene therapy of the PON1 gene product become possible, so that the PON1 gene product plays an important role in the treatment of organophosphorus pesticide poisoning, cardiovascular diseases, diabetic vascular complications and the like.

The existing reagent and the existing detection method for detecting paraoxonase 1 have the defects of poor sensitivity and repeatability, long detection time and low accuracy, and limit the application of the reagent and the detection method. Therefore, a new technical solution should be provided to solve the above problems.

Disclosure of Invention

The purpose of the invention is: provides a paraoxonase 1 detection kit with good stability and high accuracy and a preparation method thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

the detection kit of paraoxonase 1 comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:

the reagent R2 comprises the following components in percentage by weight:

the further technical scheme is as follows:

the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of PBS buffer solution, HEPES buffer solution, GOOD' S buffer solution, MES buffer solution and Tris buffer solution.

The preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.

The stabilizer in the reagent R1 is one or a mixture of more than two of casein, mannitol and bovine serum albumin.

The surfactant in the reagent R1 is one or a mixture of more than two of 2-pyrrolidone, Tween 20, triton, polyvinylpyrrolidone, octyl phenyl polyoxyethylene ether and NP-40.

Specifically, the method comprises the following steps:

the activator in the reagent R1 is calcium chloride, the anti-interference agent A is potassium ferrocyanide, and the anti-interference agent B is urea.

The electrolyte in the reagent R2 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.

The cosolvent in the reagent R2 is one or a mixture of more than two of glycerol, sodium benzoate, sodium salicylate, p-aminobenzoic acid, urea, nicotinamide and acetamide.

The accelerant in the reagent R2 is one or a mixture of more than two of polyethylene glycol 8000, polyethylene glycol 6000, polyethylene glycol 4000 and polyethylene glycol 2000.

The preparation method of the detection kit of the paraoxonase 1 comprises the following steps,

(1) preparation of a reagent R1;

adding a proper amount of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a stabilizer, an activator, an anti-interference agent A, a surfactant, an anti-interference agent B and a preservative while stirring according to the concentration requirements, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.30-9.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;

(2) preparation of a reagent R2;

adding a proper amount of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution, p-nitrophenylacetic acid ethyl ester, an electrolyte, a cosolvent, an accelerant and a preservative while stirring according to the concentration requirements, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.30-9.50, finally fixing the volume to the final volume, storing in a finished product tank, and identifying.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

1. the method for detecting the activity of the paraoxonase 1 on the full-automatic biochemical analyzer is firstly realized, the used sample amount is less, compared with other detection methods, the method has the characteristics of simplicity, accuracy and rapidness, and the high-throughput detection can be realized on the full-automatic biochemical analyzer through batch analysis.

2. Aiming at the defects of more interference substances and poorer sensitivity in the detection process of the existing detection reagent, the 2-pyrrolidone, the potassium ferrocyanide and the urea are simultaneously added into the R1 reagent, so that the interference near the wavelength of 405nm can be effectively removed, the linear range can reach 2-300U/L, the sensitivity is improved, the accuracy of the detection result is ensured, and the detection reagent can be widely applied to large, medium and small hospitals.

Drawings

FIG. 1 is a calibration curve of a calibration standard

Wherein: the X-axis represents calibrator concentration and the Y-axis represents absorbance.

FIG. 2 is a linear plot of accuracy

Wherein: the X-axis represents the theoretical concentration and the Y-axis represents the mean of the assay results.

Detailed Description

The invention is further described below with reference to specific examples: