阅读说明：本技术 一种甘胆酸检测试剂盒及其制备方法 (Glycocholic acid 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,涉及一种甘胆酸检测试剂盒,尤其涉及一种甘胆酸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,其中R2中加入了生物素、甘胆酸抗体以及链霉亲和素,其中生物素结合甘胆酸抗体比例为1:1-4:1之间,链霉亲和素结合生物素-甘胆酸抗体比例为0.5:1-4:1之间。本发明的优点是：采用级联放大反应,即抗甘胆酸单克隆抗体连接生物素1:2,再与链霉亲和素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0.5-80mg/L。(the invention relates to the technical field of biology, relates to a glycocholic acid detection kit, and particularly relates to a preparation method of the glycocholic acid detection kit, and discloses components and concentrations including a reagent R1 and a reagent R2, wherein biotin, a glycocholic acid antibody and streptavidin are added into R2, the ratio of the biotin to the glycocholic acid antibody is 1:1-4:1, and the ratio of the streptavidin to the biotin-glycocholic acid antibody is 0.5:1-4: 1. The invention has the advantages that: by adopting cascade amplification reaction, namely, the monoclonal antibody of the anti-glycocholic acid is connected with the biotin 1:2 and then is mixed with the streptavidin 1:1 for reaction, the repeatability and the sensitivity can be obviously improved, and the linear range can be 0.5-80 mg/L.)

1. A glycocholic acid 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 comprises the following components in percentage by weight:

2. the glycocholic acid detection kit according to 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 glycocholic acid detection kit according to claim 1, wherein: the first preservative in the reagent R1 comprises one of sodium azide, Proclin-950 and Proclin-300.

4. The glycocholic acid detection kit according to claim 1, wherein: the second buffer solution A and the second buffer solution B in the reagent R2 comprise 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 glycocholic acid detection kit according to claim 1, wherein: the stabilizing agent in the reagent R2 comprises one or more of bovine serum albumin, casein, gelatin and mannitol.

6. The glycocholic acid detection kit according to claim 1, wherein: the suspending agent of the reagent R2 comprises one or the combination of more than two of glucose, D-trehalose, mannitol, sucrose, maltose, lactose and glycerol.

7. The glycocholic acid detection kit according to claim 1, wherein: the second preservative of the reagent R2 comprises one of sodium azide, Proclin-950, Proclin-300 and the like.

8. the glycocholic acid detection kit according to claim 1, wherein: the pH value of the reagent R1 is between 6.00 and 8.50, and the pH value of the reagent R2 is between 7.0 and 8.0.

9. The glycocholic acid detection kit according to claim 1, wherein: the ratio of the biotin-glycocholic acid antibody to the biotin-glycocholic acid antibody is 1:1-4:1, and the ratio of the streptavidin-biotin-glycocholic acid antibody to the streptavidin-glycocholic acid antibody is 0.5:1-4: 1.

10. The method for preparing a glycocholic acid assay kit according to any one of claims 1 to 9, which comprises: 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 while stirring according to a standard of 0.8-3.2g/L, adding a first buffer solution B according to a standard of 8.75-24.31g/L, stirring for 5-30 minutes until the materials are completely dissolved and no precipitate is left at the bottom of a clear and transparent solution preparation tank, adjusting the pH value to 6.00-8.50, adding sodium chloride according to a standard of 5.8-20.0g/L, sequentially adding PEG 6000 and a first preservative according to the feeding mode after the materials are completely dissolved, wherein the standards of the PEG 6000 and the first preservative are respectively 20-70g/L and 0.8-2.0ml/L, continuously stirring for 5-30 minutes until all the materials are completely dissolved and the solution is clear and transparent after the feeding is finished, no precipitate is left at the bottom of the liquid preparation tank, and the volume is fixed 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:

Adding part of purified water into a liquid preparation tank on a magnetic stirrer, turning on a power switch of the magnetic stirrer, adjusting a stirrer to a middle-gear rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution A while stirring according to a standard of 0.5-2.8g/L, adding a second buffer solution B according to a standard of 11.5-23.4g/L, taking care not to splash water, stirring for 5-30 minutes until the material is completely dissolved, adjusting the pH value to 7.00-8.00 after no precipitate is left at the bottom of a clear and transparent liquid preparation tank of the solution, adding sodium chloride according to a standard of 5.8-20.0g/L, and sequentially adding a stabilizer, a suspending agent and a second preservative according to the feeding mode after the material is completely dissolved, wherein the feeding standards of the stabilizer, the suspending agent and the second preservative are respectively as follows: 1-10g/L, 5-30g/L and 0.8-2.0ml/L, after the feeding is finished, continuously stirring for 5-30 minutes until all materials are completely dissolved, the solution is clear and transparent, and no precipitate is left at the bottom of the liquid preparation tank;

Adding biotin into a beaker according to the standard of 5-20g/L on a magnetic stirrer, turning on a power switch of the magnetic stirrer, adjusting a stirrer to a medium-speed rotation speed, keeping the solution in a medium-speed rotation state, adding an anti-glycocholic acid monoclonal antibody according to the standard of 25-75mg/L while stirring, stirring for 1-4h for reaction, dialyzing to remove unbound free antibody after the reaction is finished, adding streptavidin according to the standard of 5-20g/L for reaction for 1-4h, and dialyzing to remove unbound biotin-antibody after the reaction is finished;

Finally, adding the streptavidin-biotin-anti-glycocholic acid monoclonal antibody into a solution preparation tank, and continuously stirring for 5-30 minutes until the solution is clear and transparent and no precipitate is left at the bottom of the solution preparation tank;

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

Technical Field

The invention relates to the technical field of biology, relates to a glycocholic acid detection kit, and particularly relates to a preparation method of the glycocholic acid detection kit.

background

glycocholic acid (CG) is one of the binding forms of cholic acid that is formed by binding cholic acid and glycine. The normal metabolic pathway of CG is the entero-hepatic circulation, which is synthesized by liver cells, discharged into the gall bladder through the bile capillary and bile duct, enters the duodenum along with bile, and helps digestion of food. 95% of the bile acid is reabsorbed at the terminal of ileum, returned to the liver via portal vein, and taken up by hepatocytes for reuse. In serum, it is predominantly present in protein-bound form, with less than 1% of the total amount spilled into the systemic circulation. Under normal conditions, peripheral blood cholesterol levels are very low and serum CG concentrations stabilize at lower levels.

when the liver cells are damaged, the CG uptake capacity of the liver cells is reduced, so that the content of CG in blood is increased; when bile stagnates, bile acid excretion by the liver becomes difficult, and the content of CG in the blood circulation is increased, which also increases the content of CG in the blood. Therefore, the determination of serum glycocholic acid (SCG) by RIA (radioimmunoassay) method is one of the sensitive indicators for evaluating the function of hepatocytes and the circulating function of hepatobiliary substances.

the content of glycocholic acid in serum of normal people is 1.3 +/-0.8 mg/L, the range can fluctuate within 0.4-2.98 mg/L, and the hepatitis diagnosis lower limit value is less than 3.18 mg/L. The blood CG of patients with various hepatitis, primary liver cancer and liver cirrhosis is obviously higher than that of normal people, and the presentation is increased; the biliary duct and gallbladder excretion dysfunction of patients with cholelithiasis and jaundice can cause the significant increase of serum CG; the serum CG level of the liver cirrhosis, the obstructive liver disease and the intestinal-hepatic circulatory disturbance is higher than that of normal people.

glycocholic acid is the most predominant bile acid component in serum during the late gestation. The CG level of the pregnant woman serum is gradually increased along with the gestational period during normal pregnancy until the CG value of the full-term pregnancy is increased by 30-60 percent compared with that during non-pregnancy. Some pregnant women may have physiologically elevated serum CG values and clinically no obvious ICP symptoms (intrahepatic cholestasis during pregnancy). As the CG value is increased to different degrees, the injury to the mother and the baby is caused to different degrees, and the damage is larger as the CG value is higher.

in current clinical application, methods for measuring glycocholic acid mainly include RIA (radioimmunoassay) method and enzyme-linked immunosorbent assay. The common characteristics of the methods are that special measuring instruments are needed, the operation is complex, the test time is long, the methods cannot be applied to the clinical large-flow automatic analysis, and the cost is high. Due to the limitation of the defects, glycocholic acid is not a routine item for clinical detection at present.

disclosure of Invention

The purpose of the invention is: aiming at the defects, the glycocholic acid detection kit and the preparation method thereof are provided, and the repeatability and the sensitivity are improved.

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

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

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

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

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

the suspending agent of the reagent R2 comprises one or the combination of more than two of glucose, D-trehalose, mannitol, sucrose, maltose, lactose and glycerol.

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

the pH value of the reagent R1 is between 6.00 and 8.50, and the pH value of the reagent R2 is between 7.0 and 8.0.

The ratio of the biotin-glycocholic acid antibody to the biotin-glycocholic acid antibody is 1:1-4:1, and the ratio of the streptavidin-biotin-glycocholic acid antibody to the streptavidin-glycocholic acid antibody is 0.5:1-4: 1.

A preparation method of a glycocholic acid 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 while stirring according to a standard of 0.8-3.2g/L, adding a first buffer solution B according to a standard of 8.75-24.31g/L, stirring for 5-30 minutes until the materials are completely dissolved and no precipitate is left at the bottom of a clear and transparent solution preparation tank, adjusting the pH value to 6.00-8.50, adding sodium chloride according to a standard of 5.8-20.0g/L, sequentially adding PEG 6000 and a first preservative according to the feeding mode after the materials are completely dissolved, wherein the standards of the PEG 6000 and the first preservative are respectively 20-70g/L and 0.8-2.0ml/L, continuously stirring for 5-30 minutes until all the materials are completely dissolved and the solution is clear and transparent after the feeding is finished, no precipitate is left at the bottom of the liquid preparation tank, and the volume is fixed 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:

Adding part of purified water into a liquid preparation tank on a magnetic stirrer, turning on a power switch of the magnetic stirrer, adjusting a stirrer to a middle-gear rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution A while stirring according to a standard of 0.5-2.8g/L, adding a second buffer solution B according to a standard of 11.5-23.4g/L, taking care not to splash water, stirring for 5-30 minutes until the material is completely dissolved, adjusting the pH value to 7.00-8.00 after no precipitate is left at the bottom of a clear and transparent liquid preparation tank of the solution, adding sodium chloride according to a standard of 5.8-20.0g/L, and sequentially adding a stabilizer, a suspending agent and a second preservative according to the feeding mode after the material is completely dissolved, wherein the feeding standards of the stabilizer, the suspending agent and the second preservative are respectively as follows: 1-10g/L, 5-30g/L and 0.8-2.0ml/L, after the feeding is finished, continuously stirring for 5-30 minutes until all materials are completely dissolved, the solution is clear and transparent, and no precipitate is left at the bottom of the liquid preparation tank;

Adding biotin into a beaker according to the standard of 5-20g/L on a magnetic stirrer, turning on a power switch of the magnetic stirrer, adjusting a stirrer to a medium-speed rotation speed, keeping the solution in a medium-speed rotation state, adding an anti-glycocholic acid monoclonal antibody according to the standard of 25-75mg/L while stirring, stirring for 1-4h for reaction, dialyzing to remove unbound free antibody after the reaction is finished, adding streptavidin according to the standard of 5-20g/L for reaction for 1-4h, and dialyzing to remove unbound biotin-antibody after the reaction is finished;

Finally, adding the streptavidin-biotin-anti-glycocholic acid monoclonal antibody into a solution preparation tank, and continuously stirring for 5-30 minutes until the solution is clear and transparent and no precipitate is left at the bottom of the solution preparation tank;

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

Compared with the prior art, the invention achieves the technical effects that: by adopting cascade amplification reaction, namely, the monoclonal antibody of the anti-glycocholic acid is connected with the biotin 1:2 and then is mixed with the streptavidin 1:1 for reaction, the repeatability and the sensitivity can be obviously improved, and the linear range can be 0.5-80 mg/L.

Drawings

FIG. 1 is a plot of a calibration curve for reagent R2 without the use of a cascade amplification reaction 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 of the calibration curve of reagent R2 using a cascade amplification reaction in example 4 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: