阅读说明：本技术 一种白介素-6检测试剂盒及其制备方法 (Interleukin-6 detection kit and preparation method thereof ) 是由 袁嘉扬 张勇 于 2019-09-12 设计创作，主要内容包括：本发明涉及生物技术领域,涉及一种白介素-6检测试剂盒,尤其涉及一种白介素-6检测试剂盒的制备方法,公开了包括试剂R1和试剂R2的组分和浓度,所述试剂R2由第二缓冲液、保存液以及胶乳微球抗体偶联物组成,其保存液由N,N-二羟乙基甘氨酸、第二稳定剂、第二防腐剂构成,胶乳微球抗体偶联物由胶乳微球、白介素-6抗体、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐构成。本发明的优点是：该方法使用的样本量较少,与其他检测方法相比具有简单、准确、快速的特点,并可在全自动生化分析仪上批量分析实现高通量检测；该试剂盒具有稳定性好、灵敏度高、线性范围宽、特异性高、精密度好、检测时间短等特点,且价格低廉。(The invention relates to the technical field of biology, relates to an interleukin-6 detection kit, and particularly relates to a preparation method of the interleukin-6 detection kit, and discloses components and concentrations including a reagent R1 and a reagent R2, wherein the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, the preservation solution consists of N, N-dihydroxyethyl glycine, a second stabilizer and a second preservative, and the latex microsphere antibody conjugate consists of a latex microsphere, an interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride. The invention has the advantages that: the method uses a small amount of samples, has the characteristics of simplicity, accuracy and rapidness compared with other detection methods, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer; the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision, short detection time and the like, and is low in price.)

1. An interleukin-6 detection kit, which 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 is composed of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution is composed of N, N-dihydroxyethyl glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate is composed of latex microspheres, interleukin-6 antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of each component is as follows:

2. the interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

3. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.

4. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the electrolyte in the reagent R1 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.

5. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the surfactant in the reagent R1 is one or a mixture of more than one of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

6. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.

7. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the second buffer solution in the reagent R2 is one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.

8. The interleukin-6 detection kit according to claim 1, wherein the kit comprises: the latex microsphere in the reagent R2 has a selected particle size of 80-250nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde group and amino group.

9. The method for preparing the interleukin-6 detection kit according to any one of claims 1 to 8, wherein the method comprises the following steps: the method comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, an electrolyte, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, 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 8.30-10.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;

B) reagent R2 preparation:

preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;

preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethylglycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;

b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting an interleukin-6 antibody to the concentration of 0.5-3.0g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, shaking and dripping the mixture into the mixed solution while reacting for 120 minutes at room temperature, adding a second stabilizer for sealing, shaking and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the supernatant after the last time, adding the preservation solution, ultrasonically suspending, filling the prepared R2 into a finished product tank, and marking.

Technical Field

The invention relates to the technical field of biology, relates to an interleukin-6 detection kit, and particularly relates to a preparation method of the interleukin-6 detection kit.

Background

Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of functions. The interleukin-6 has physiological activity on B cells, T cells, hematopoietic stem cells, liver cells and brain cells, can induce the differentiation of the B cells to generate immunoglobulin, promote the proliferation and growth of the T cells, promote the proliferation of bone marrow hematopoietic stem cells, and enhance the differentiation of blood cells and the anti-tumor effect thereof. The biological function of interleukin-6 plays an important role in resisting infection, tumor, immune diseases, immunoregulation and the like.

Studies have shown that interleukin-6 is rapidly produced during acute inflammatory reactions in the presence of trauma, surgery, stress, infection, brain death, tumor production and other conditions. The continuous monitoring of the interleukin-6 content in the serum or plasma of an intensive care patient can effectively evaluate the severity of Systemic Inflammatory Response Syndrome (SIRS), and the prognosis of sepsis and septic shock. Interleukin-6 can be used as early warning index of sepsis, and simultaneously, interleukin-6 also plays an important role in chronic inflammatory response.

At present, in China, the detection of interleukin-6 mainly takes foreign imported reagents and enzyme-linked immunosorbent assay (ELISA) as main clinical application, the imported reagents are expensive, the economic burden is large for patients, and the primary popularization is not facilitated; the ELISA method has low detection sensitivity, narrow linear range and long detection process, is usually only suitable for manual operation and is not beneficial to detection of a full-automatic detection instrument, thereby limiting the popularization and application of the method. At present, the domestic interleukin-6 immunochemiluminescence kit with independent intellectual property rights is less, so that the interleukin-6 domestic kit with high analysis sensitivity and wide linear range has important practical significance in clinical application.

Disclosure of Invention

The purpose of the invention is: aiming at the defects, the interleukin-6 detection kit and the preparation method thereof are provided, wherein the interleukin-6 detection kit has the advantages of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time.

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

the interleukin-6 detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight:

the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of N, N-dihydroxyethyl glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate consists of a latex microsphere, an interleukin-6 antibody and 1- (3-dimethylamino propyl) -3-ethyl carbodiimide hydrochloride, and the concentration of each component is as follows:

0.01-0.3g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

The first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

The first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.

The electrolyte in the reagent R1 is one or a mixture of more than two of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate.

The surfactant in the reagent R1 is one or more of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

The first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.

The second buffer solution in the reagent R2 is one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.

The latex microsphere in the reagent R2 has a selected particle size of 80-250nm, and the surface modification group is one of carboxyl, hydroxyl, aldehyde group and amino group.

A preparation method of an interleukin-6 detection kit comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, an electrolyte, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, 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 8.30-10.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;

B) reagent R2 preparation:

preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;

preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding N, N-dihydroxyethylglycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-7.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;

b3 preparation of latex microsphere antibody conjugate: diluting the latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting the interleukin-6 antibody to the concentration of 0.5-3.0g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, dripping the mixture into the mixed solution while shaking the mixture, reacting the mixture at room temperature for 120 minutes, adding a second stabilizer for sealing, shaking the mixture for uniformly mixing, reacting the mixture at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending the mixture, repeatedly centrifuging and cleaning the mixture for 3 times, centrifuging the supernatant for the last time, adding a resuspension solution, ultrasonically suspending the suspension, filling the prepared R2 into a finished product tank, and marking the finished product tank.

Compared with the prior art, the invention achieves the technical effects that: the invention realizes the detection of the interleukin-6 content on a biochemical analyzer for the first time, and the detection range can reach 1.5-3000 pg/mL; the method uses a small amount of samples, has the characteristics of simplicity, accuracy and rapidness compared with other detection methods, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer; the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision, short detection time and the like, is low in price, and can be widely applied to large, medium and small hospitals.

Drawings

FIG. 1 is a calibration curve diagram of indirect coupling of latex microspheres with interleukin-6 antibody in example 4 of the present invention. Wherein the X-axis represents standard concentration and the Y-axis represents absorbance.

FIG. 2 is a graph showing the measurement of the linear range in example 4 of the present invention.

Detailed Description

The invention is further described with reference to the following figures and examples: