阅读说明：本技术 一种脂肪酶试剂盒及其制备方法 (Lipase kit and preparation method thereof ) 是由 黄益峰 于 2019-11-07 设计创作，主要内容包括：本发明涉及一种脂肪酶试剂盒及其制备方法,包括试剂R1、试剂R2和试剂R3,所述试剂R1包括下述原料：80～120mmol/l酒石酸缓冲溶液(pH为7.0)；3.0～5.0mmol/l牛磺脱氧胆酸；0.05-0.08mmol/l苯丁酸；450～550KU/l共脂肪酶；10-15mmol/l氯化钙；2.0-3.0mmol/l脱氧胆酸；所述试剂R2包括下述原料：80～120mmol/l酒石酸缓冲溶液(pH为7.0)；0.5～1.0mmol/l表面活性剂；0.2～0.8mmol/蛋白变性剂；2.0-3.0mmol/l乙醚；所述试剂R3包括下述原料：80～120mmol/l酒石酸缓冲液(pH为7.0)；0.15～0.35mmol/l6-甲基试卤灵酯；3.0～5.0mmol/l牛磺脱氧胆酸。提供一种持续性灵敏度高,检测精度高,反应一段时间后,能够持续维持脂肪酶的活性,提高了后阶段的反应准确度。(The invention relates to a lipase kit and a preparation method thereof, wherein the lipase kit comprises a reagent R1, a reagent R2 and a reagent R3, and the reagent R1 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 3.0-5.0 mmol/l taurodeoxycholic acid; 0.05-0.08mmol/l phenylbutyric acid; 450-550 KU/l colipase; 10-15mmol/l calcium chloride; 2.0-3.0mmol/l deoxycholic acid; the reagent R2 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 0.5 to 1.0mmol/l surfactant; 0.2-0.8 mmol/protein denaturant; 2.0-3.0mmol/l diethyl ether; the reagent R3 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 0.15-0.35 mmol/l 6-methyl resorufin ester; 3.0-5.0 mmol/l taurodeoxycholic acid. The method has high continuous sensitivity and high detection precision, can continuously maintain the activity of the lipase after reacting for a period of time, and improves the reaction accuracy of the later stage.)

1. A lipase kit comprises a reagent R1, a reagent R2 and a reagent R3, and is characterized in that the reagent R1 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

3.0-5.0 mmol/l taurodeoxycholic acid;

0.05-0.08mmol/l phenylbutyric acid;

450-550 KU/l colipase;

10-15mmol/l calcium chloride;

2.0-3.0mmol/l deoxycholic acid;

the reagent R2 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

0.5 to 1.0mmol/l surfactant;

0.2-0.8 mmol/protein denaturant;

2.0-3.0mmol/l diethyl ether;

the reagent R3 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

0.15-0.35 mmol/l 6-methyl resorufin ester;

3.0-5.0 mmol/l taurodeoxycholic acid.

2. The lipase kit as claimed in claim 1, wherein the reagent R1 comprises the following raw materials:

100mmol/l tartaric acid buffer solution (pH 7.0);

3.4mmol/l taurodeoxycholic acid;

0.06mmol/l phenylbutyric acid;

500KU/l colipase;

12mmol/l calcium chloride;

2.6mmol/l deoxycholic acid;

the reagent R2 comprises the following raw materials:

100mmol/l tartaric acid buffer solution (pH 7.0);

0.8mmol/l surfactant;

0.6 mmol/protein denaturant;

2.5mmol/l diethyl ether;

the reagent R3 comprises the following raw materials:

100mmol/l tartaric acid buffer (pH 7.0);

0.27mmol/l 6-methyl resorufin ester;

3.4mmol/l taurodeoxycholic acid.

3. The lipase kit as claimed in claim 1, wherein the surfactant is PEG2000, PEG6000 or PEG 8000.

4. The lipase kit as claimed in claim 1, wherein the volume ratio of the reagent R1, the reagent R2 and the reagent R3 is 1:1: 1.

5. The lipase kit as claimed in claim 1, wherein the protein denaturant is guanidine hydrochloride.

6. A method for producing a lipase kit according to any of claims 1 to 5, characterized in that it comprises the following steps: step S1, weighing each component of the reagent R1, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S2, weighing each component of the reagent R2, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, weighing each component of the reagent R3, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, checking the semi-finished product; step S4, cleaning a packaging appliance, drying, and filling reagents R1, R2 and R3 respectively; and step S5, packaging, inspecting finished products, and warehousing for storage.

7. The method of claim 6, wherein in step S2, the surfactant, the protein denaturant and the ether are mixed and stirred, and then the tartaric acid buffer is added into the system.

8. The method for preparing the lipase kit according to claim 7, wherein the surfactant, the protein denaturant and the ether are heated for 10min while being mixed and stirred, the heating temperature is 40-50 ℃, and the mixture is allowed to stand to room temperature after the heating is finished.

9. The method of claim 8, wherein the tartaric acid buffer is added to the system in multiple times.

The technical field is as follows:

the invention relates to the technical field of medical detection reagents, in particular to a lipase kit and a preparation method thereof.

Background art:

lipase (Lip), also known as triacylglycerol hydrolase, is a generic name for enzymes that hydrolyze triacylglycerol of long-chain fatty acids. The main synthesis site of human lipase is in the pancreatic acinus, and this kind of lipase is also called pancreatic lipase, and is the main source of serum lipase. In addition, some digestive organs such as stomach, duodenum, esophagus, and white blood cells, adipose tissue, lung, vascular endothelium may also secrete small amounts of lipase and enter the blood. Serum lipase activity assays are useful for the diagnosis of pancreatic disease, particularly in acute pancreatitis. The level difference of human serum lipase of patients with acute pancreatitis, patients with non-acute pancreatitis and patients with normal pancreatitis is larger, and lipase has better sensitivity and specificity in diagnosis of acute pancreatitis compared with amylase.

The detection of the existing lipase usually uses a lipase kit, for example, the invention patent with the application number of CN201811635803.9 discloses a lipase detection kit and a production process, the lipase detection kit comprises a reagent R1 and a reagent R2 which are independent from each other, and the reagent R1 comprises the following components: the reagent R2 comprises the following components: the lipase detection kit is ready to use. The principle of lipase is: the 1, 2-o-dilauryl-rac-glycerol-3-glutaric acid (6 '-methyl resorufin) ester is hydrolyzed under the action of lipase to produce 1, 2-o-dilauryl-rac-glycerol and glutaric acid (6' -methyl resorufin) ester. Wherein glutaric acid (6' -methyl resorufin) ester is hydrolyzed by further action of lipase to produce glutaric acid and methyl resorufin, which appears red in solution. The rate of production of red methylprednisolone detected at a wavelength of 570nm, which is proportional to the lipase activity in the sample, is calculated to give the lipase activity in the sample.

However, in the above technical scheme, because the lipase activity is detected for a period of time, after the substrate is subjected to enzymolysis for a period of time, the enzymolysis reaction speed is slowed down due to the production of reactants, so the color deepening degree is slowed down, the reaction color change cannot be observed at the later stage, the absorbance change is not obvious, and the reference significance of the later measured data is small.

The invention content is as follows:

the invention aims to solve the technical problem of providing a method which has high continuous sensitivity and high detection precision, can continuously maintain the activity of lipase after reacting for a period of time and improves the reaction accuracy of the later stage.

In order to solve the technical problems, the invention adopts a lipase kit which comprises a reagent R1, a reagent R2 and a reagent R3, and is characterized in that the reagent R1 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

3.0-5.0 mmol/l taurodeoxycholic acid;

0.05-0.08mmol/l phenylbutyric acid;

450-550 KU/l colipase;

10-15mmol/l calcium chloride;

2.0-3.0mmol/l deoxycholic acid;

the reagent R2 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

0.5 to 1.0mmol/l surfactant;

0.2-0.8 mmol/protein denaturant;

2.0-3.0mmol/l diethyl ether;

the reagent R3 comprises the following raw materials:

80-120 mmol/l tartaric acid buffer solution (pH 7.0);

0.15-0.35 mmol/l 6-methyl resorufin ester;

3.0-5.0 mmol/l taurodeoxycholic acid.

Adopt above-mentioned technical scheme: the phenylbutyric acid can keep the activity of lipase cells, and after reacting for a period of time, the activity of the lipase can be continuously maintained, so that the reaction accuracy of the later stage is improved; in addition, the phenylbutyric acid can be removed to reduce protein, the protein can affect a substrate, the stability of the reagent can be enhanced after the phenylbutyric acid is removed, and the accuracy of a test result can be improved; glutaric acid can be dissolved in ether in a large amount, and the ether component is arranged, so that on one hand, a large amount of glutaric acid is absorbed, the reaction product is reduced, the reaction is promoted to be rapidly continued, the absorbance difference in unit time is obviously improved, on the other hand, a competitive inhibitor is arranged between the generated glutaric acid and the lipase, and the situation that the glutaric acid and the lipase compete for a binding site on the lipase is avoided by reducing the contact between the glutaric acid and the lipase, so that the binding reaction between the lipase and the substrate is realized; therefore, the activity of the lipase can be combined with the continuous sites of the substrate, the enzymolysis reaction is continued, and the reaction accuracy of the later stage is improved.

Preferably, the reagent R1 comprises the following raw materials:

100mmol/l tartaric acid buffer solution (pH 7.0);

3.4mmol/l taurodeoxycholic acid;

0.06mmol/l phenylbutyric acid;

500KU/l colipase;

12mmol/l calcium chloride;

2.6mmol/l deoxycholic acid;

the reagent R2 comprises the following raw materials:

100mmol/l tartaric acid buffer solution (pH 7.0);

0.8mmol/l surfactant;

0.6 mmol/protein denaturant;

2.5mmol/l diethyl ether;

the reagent R3 comprises the following raw materials:

100mmol/l tartaric acid buffer (pH 7.0);

0.27mmol/l 6-methyl resorufin ester;

3.4mmol/l taurodeoxycholic acid.

Preferably, the protein denaturant is guanidine hydrochloride.

Preferably, the surfactant is PEG2000, PEG6000 or PEG 8000.

Preferably, the volume ratio of the reagent R1 to the reagent R2 to the reagent R3 is 1:1: 1.

In order to solve the technical problems, the invention discloses a preparation method of a lipase kit, which comprises the following steps: it comprises the following steps: step S1, weighing each component of the reagent R1, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S2, weighing each component of the reagent R2, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, weighing each component of the reagent R3, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, checking the semi-finished product; step S4, cleaning a packaging appliance, drying, and filling reagents R1, R2 and R3 respectively; and step S5, packaging, inspecting finished products, and warehousing for storage.

Preferably, in step S2, the surfactant, the protein denaturant, and the ether are mixed and stirred, and then the tartaric acid buffer is added to the system.

Preferably, the surfactant, the protein denaturant and the ether are heated for 10min in the process of mixing and stirring, the heating temperature is 40-50 ℃, and the mixture is kept stand to room temperature after the heating is finished.

Preferably, the tartaric acid buffer is added to the system in multiple portions.

Compared with the prior art, the invention has the following advantages: calcium chloride is an enzyme reaction accelerator, can activate the activity of enzyme and provide the ionic environment required by the reaction, and can increase the salt ion effect in the reagent. PEG2000, PEG6000 or PEG 8000 belong to surfactants, have stronger emulsification, improve the melting property and stability of various raw materials, and the stability of the reagent in opening the bottle and the long-term storage stability are better. The sodium taurodeoxycholate plays a role of a stabilizer and an accelerator in the reaction, accelerates the lipase reaction and ensures the high-efficiency reaction. Colipases are catalytic enzymes of reactions required in the reaction. Guanidine hydrochloride is a protein denaturant, removes the influence of hybrid proteins on a substrate, enhances the stability of the reagent, and is beneficial to improving the accuracy of a test result. 1, 2-o-dilauryl-rac-glycerol-3-glutaric acid (6 '-methyl resorufin) ester is a substrate for 6' -methyl resorufin reaction, is mainly used for generating methyl resorufin, and is a chromogen in colorimetry.

The specific implementation mode is as follows:

the present invention will be further described with reference to the following specific examples.