阅读说明：本技术 一种微量白蛋白免疫比浊法检测试剂盒及其制备方法 (Microalbumin immunoturbidimetry detection kit and preparation method thereof ) 是由 袁嘉扬 单以朗 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,涉及一种微量白蛋白免疫比浊法检测试剂盒,尤其涉及一种微量白蛋白免疫比浊法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包括反应液、保存液以及微量白蛋白单克隆抗体-生物素-链霉亲和素的抗体偶联物。本发明的优点是：经多次研发实验后,我司采用微量白蛋白单克隆抗体1:2连接生物素,与链霉亲和素二者1:1混合反应,可明显改善重复性和灵敏度,线性范围可做到5.0-800mg/L。(The invention relates to the technical field of biology, relates to a microalbumin immunoturbidimetry detection kit, and particularly relates to a preparation method of the microalbumin immunoturbidimetry detection kit, and discloses components and concentrations including a reagent R1 and a reagent R2, wherein the reagent R2 comprises a reaction liquid, a preservation liquid and a microalbumin monoclonal antibody-biotin-streptavidin antibody conjugate. The invention has the advantages that: after multiple research and development experiments, the trace albumin monoclonal antibody 1:2 is adopted to be connected with biotin, and the biotin and streptavidin are subjected to 1:1 mixed reaction, so that the repeatability and the sensitivity can be obviously improved, and the linear range can be 5.0-800 mg/L.)

1. a microalbumin immunoturbidimetry detection kit 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 a reaction liquid, a preservation liquid and a microalbumin monoclonal antibody-biotin-streptavidin antibody conjugate, wherein the reaction liquid is composed of a second buffer solution, the preservation liquid is composed of N, N-dihydroxyethylglycine, a first stabilizing agent and a second preservative, the microalbumin monoclonal antibody-biotin-streptavidin antibody conjugate is composed of biotin, streptavidin, a microalbumin monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component comprises:

2. The microalbumin immunoturbidimetry assay kit of claim 1, wherein: the first buffer solution A and the first buffer solution B in the reagent R1 comprise one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution, Tris buffer solution and glycine buffer system.

3. the microalbumin immunoturbidimetry assay kit of claim 1, wherein: the first preservative in the reagent R1 comprises one of sodium azide, Proclin-950 and Proclin-300.

4. the microalbumin immunoturbidimetry assay kit of claim 1, wherein: the second buffer solution in the reagent R2 comprises one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution, MOPSO buffer solution and Tris buffer solution.

5. The microalbumin immunoturbidimetry assay kit of claim 1, wherein: the stabilizer in the reagent R2 comprises one or more of bovine serum albumin, casein and mannitol.

6. the microalbumin immunoturbidimetry assay kit of claim 1, wherein: the second preservative of the reagent R2 comprises one of sodium azide, Proclin-950 and Proclin-300.

7. the microalbumin immunoturbidimetry assay kit of claim 1, wherein: the pH value of the reagent R1 is between 6.80 and 8.00, and the pH value of the reagent R2 is between 6.30 and 8.50.

8. The microalbumin immunoturbidimetry assay kit of claim 1, wherein: the ratio of the biotin-microalbumin monoclonal antibody to the streptavidin-microalbumin monoclonal antibody is 1:1-3:1, and the ratio of the streptavidin-biotin-microalbumin monoclonal antibody to the biotin-microalbumin monoclonal antibody is 0.5:1-4: 1.

9. The method for preparing the microalbumin immunoturbidimetry assay kit of any one of claims 1 to 8, comprising: the method comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank, adding a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm, keeping stirring in a constant speed state, adding a first buffer solution A and a first buffer solution B while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG6000 and a first preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B) Reagent R2 preparation:

Preparation of B1 reaction solution: adding part of purified water into the liquid preparation tank, and adjusting the rotating speed of the stirrer to 200-300rpm to keep the stirring in a constant speed state. Adding MES while stirring, stirring until the materials are completely dissolved, and the mixture is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 4.50-6.50, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

b2 preparation of preserving fluid: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding N, N-dihydroxyethyl glycine while stirring until the material is completely dissolved, adding a stabilizer, adding a second preservative after the material is completely dissolved, keeping the solution clear and transparent, adjusting the pH value to 6.30-8.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;

preparation of B3 antibody conjugate: diluting the microalbumin monoclonal antibody to the concentration of 0.05-3.0g/L by using reaction liquid, dialyzing with biotin at a ratio of 1:1-3 overnight, mixing with streptavidin at a ratio of 1:0.5-4, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the reaction solution to a concentration of 0.005-0.3g/L, shaking up and dropping into the mixed solution, reacting at room temperature for 120 min, adding bovine serum albumin, sealing, shaking, mixing, reacting at room temperature for 60 min, centrifuging the mixture at 14000rpm for 30 min, removing supernatant, adding storage solution, repeatedly centrifuging and cleaning for 3 times, centrifuging for the last time to remove supernatant, adding the preservation solution, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.

Technical Field

the invention relates to the technical field of biology, relates to a microalbumin immunoturbidimetry detection kit, and particularly relates to a preparation method of the microalbumin immunoturbidimetry detection kit.

background

Microalbumin refers to the microalbumin found in human urine, albumin being a normal protein in blood, but very small amounts of albumin are found in urine under physiological conditions. Under normal conditions, the concentration of the microalbumin in the urine is not more than 20mg/L, and when the concentration of the microalbumin in the urine reaches 20-200mg/L, the urine is microalbuminuria, which reflects abnormal leakage protein of the kidney of a human body. The detection of microalbumin is the most sensitive and reliable diagnostic index for early detection of kidney diseases. The disease condition can be accurately diagnosed by the detection result of the urine microalbumin and the statement of the disease condition, symptoms and medical history. Regular detection of urine microalbumin can play a positive role in preventing and treating nephropathy at an early stage.

disclosure of Invention

The purpose of the invention is: aiming at the defects, the microalbumin immunoturbidimetry detection kit for improving repeatability and sensitivity and the preparation method thereof are provided.

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

The microalbumin immunoturbidimetry detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration:

the reagent R2 comprises a reaction liquid, a preservation liquid and a microalbumin monoclonal antibody-biotin-streptavidin antibody conjugate, wherein the reaction liquid is composed of a second buffer solution, the preservation liquid is composed of N, N-dihydroxyethylglycine, a first stabilizing agent and a second preservative, the microalbumin monoclonal antibody-biotin-streptavidin antibody conjugate is composed of biotin, streptavidin, a microalbumin monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component comprises:

The first buffer solution A and the first buffer solution B in the reagent R1 comprise one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution, Tris buffer solution and glycine buffer system.

the first preservative in the reagent R1 comprises one of sodium azide, Proclin-950 and Proclin-300.

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

the stabilizer in the reagent R2 comprises one or more of bovine serum albumin, casein and mannitol.

The second preservative of the reagent R2 comprises one of sodium azide, Proclin-950 and Proclin-300.

the pH value of the reagent R1 is between 6.80 and 8.00, and the pH value of the reagent R2 is between 6.30 and 8.50.

the ratio of the biotin-microalbumin monoclonal antibody to the streptavidin-microalbumin monoclonal antibody is 1:1-3:1, and the ratio of the streptavidin-biotin-microalbumin monoclonal antibody to the biotin-microalbumin monoclonal antibody is 0.5:1-4: 1.

A preparation method of a microalbumin immunoturbidimetry detection kit comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank, adding a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm, keeping stirring in a constant speed state, adding a first buffer solution A and a first buffer solution B while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG6000 and a first preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B) reagent R2 preparation:

preparation of B1 reaction solution: adding part of purified water into the liquid preparation tank, and adjusting the rotating speed of the stirrer to 200-300rpm to keep the stirring in a constant speed state. Adding MES while stirring, stirring until the materials are completely dissolved, and the mixture is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 4.50-6.50, and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B2 preparation of preserving fluid: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding N, N-dihydroxyethyl glycine while stirring until the material is completely dissolved, adding a stabilizer, adding a second preservative after the material is completely dissolved, keeping the solution clear and transparent, adjusting the pH value to 6.30-8.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;

Preparation of B3 antibody conjugate: diluting the microalbumin monoclonal antibody to the concentration of 0.05-3.0g/L by using reaction liquid, dialyzing with biotin at a ratio of 1:1-3 overnight, mixing with streptavidin at a ratio of 1:0.5-4, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the reaction solution to a concentration of 0.005-0.3g/L, shaking up and dropping into the mixed solution, reacting at room temperature for 120 min, adding bovine serum albumin, sealing, shaking, mixing, reacting at room temperature for 60 min, centrifuging the mixture at 14000rpm for 30 min, removing supernatant, adding storage solution, repeatedly centrifuging and cleaning for 3 times, centrifuging for the last time to remove supernatant, adding the preservation solution, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.

compared with the prior art, the invention achieves the technical effects that: after multiple research and development experiments, the trace albumin monoclonal antibody 1:2 is adopted to be connected with biotin, and the biotin and streptavidin are subjected to 1:1 mixed reaction, so that the repeatability and the sensitivity can be obviously improved, and the linear range can be 5.0-800 mg/L.

Drawings

FIG. 1 is a graph showing the direct calibration of a microalbumin monoclonal antibody in example 4 of the present invention. Where the X-axis represents standard concentration and the Y-axis represents dOD.

FIG. 2 is a graph showing the calibration curve of the antibody conjugate using microalbumin monoclonal antibody-biotin-streptavidin in example 5 of the present invention. Where the X-axis represents standard concentration and the Y-axis represents dOD.

Detailed Description

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