CN112051354A

CN112051354A - Lipase determination kit and preparation method thereof

Info

Publication number: CN112051354A
Application number: CN202010775855.7A
Authority: CN
Inventors: 凡速朋; 赵畅; 龚婷; 梁艳; 吴年芬; 舒芹; 张雪娇; 赵愿安
Original assignee: Wuhan Life Origin Biotech Joint Stock Co ltd
Current assignee: Wuhan Life Origin Biotech Joint Stock Co ltd
Priority date: 2020-08-05
Filing date: 2020-08-05
Publication date: 2020-12-08
Anticipated expiration: 2040-08-05
Also published as: CN112051354B

Abstract

The invention discloses a lipase determination kit and a preparation method thereof, wherein an R1 reagent comprises a buffer solution, sodium deoxycholate, colipase, a metal salt, a protective agent, a surfactant and a preservative; the R2 reagent comprises L-tartaric acid, sodium taurodeoxycholate, 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester, a cosolvent, a stabilizer and a preservative, wherein the cosolvent comprises ethyl acetate, dilauryl glycerol sulfate and a Triton surfactant. According to the invention, ethyl acetate, dilauryl glycerol sulfate and Triton surfactant are preferably used as cosolvents together to exert the synergistic interaction of the three, so that the solubility of the substrate is greatly improved, and furthermore, mercaptoethanol and mannitol are used as composite stabilizers, so that the lipase determination kit with good stability, high accuracy and precision and remarkably improved linear range is obtained.

Description

Lipase determination kit and preparation method thereof

Technical Field

The invention belongs to the technical field of clinical biochemical detection, and particularly relates to a lipase determination kit and a preparation method thereof.

Background

Serum Lipase (LPS) was derived mainly from pancreas and a small amount from stomach and small intestine, and lipase activity was measured to reflect the dysregulation of pancreas. The increase of lipase in human body is commonly seen in acute pancreatitis, pancreatic cancer, calculous pancreatic duct obstruction, liver cirrhosis, intestinal obstruction, acute and chronic kidney diseases and the like. LPS is a group of enzymes that catalyse the hydrolysis of long chain fatty acid glycerides, which hydrolyse only the fatty acids in the alpha position of the fatty acid glycerides. LPS has a certain specificity to long-chain fatty glyceride, and the participation of cholate and colipase is required for the maximum activity and specificity of LPS to be exerted or expressed. The detection method of LPS mainly adopts a chromogen substrate method and an enzyme coupling color development method, wherein the basic principle of the chromogen substrate method is as follows: a colorimetric method using 1, 2-di-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin ester) as a substrate mainly decomposes the 1, 2-di-dilauryl-racemic-glycerol and glutaric acid (6-methyl resorufin) ester under the action of cholate and colipase, the glutaric acid (6-methyl resorufin) ester spontaneously hydrolyzes to produce glutaric acid and methyl resorufin which shows red color, and LPS activity can be measured by detecting the generation rate of the methyl resorufin.

For example, patent CN109490296A discloses a lipase detection kit and a production process, comprising reagent R1 and reagent R2 which are independent of each other, wherein reagent R1 comprises buffer solution, enzyme reaction accelerator, surfactant, sodium deoxycholate, colipase and sodium azide; the reagent R2 includes: buffer solution, enzyme reaction accelerator, sodium taurodeoxycholate, protein denaturant, 1, 2-di-lauroyl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester and sodium azide. However, in the preparation process of the R2 reagent, the chromogen substrate 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) is not sufficiently dissolved, so that the batch difference and the stability of the reagent are influenced, and the substrate is separated out from the R2 reagent along with the increase of the standing time, so that the measured value is reduced along with the increase of the standing time; meanwhile, the linear range of the lipase kit in the prior art is narrow, so that the detection is carried out after a sample needs to be diluted when a high value is detected, and the workload of clinical detection is increased.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a lipase determination kit, which is mainly used for optimizing an R2 reagent, and the solubility and the stability of a substrate are obviously improved by matching ethyl acetate, dilauryl glycerol sulfate and a Triton surfactant for use, and the accuracy, the precision and the linear range of the reagent are enhanced.

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

a lipase determination kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer solution, sodium deoxycholate, colipase, a metal salt, a protective agent, a surfactant and a preservative; the R2 reagent comprises L-tartaric acid, sodium taurodeoxycholate, 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester, a cosolvent, a stabilizer and a preservative, wherein the cosolvent comprises ethyl acetate, dilauryl glycerol sulfate and a Triton surfactant.

Due to the lipase substrate: 1, 2-O-dilauryl-racemization-glycerol-3-pentanoic acid- (6-methyl resorufin) ester is an ester substance and is not easy to dissolve in water, and lipase substrate precipitation is an important factor causing instability of a lipase determination kit and large batch difference, so that a proper cosolvent is preferably selected to increase the stability of the lipase substrate, and the cosolvent consists of ethyl acetate, dilauryl glycerol sulfate and a Triton surfactant. The lipase substrate as an ester substance has certain solubility in organic solvent ethyl acetate, but the ethyl acetate solution dissolved with the substrate can generate precipitation after being dissolved in water, and the substrate can be more fully dissolved by matching the ethyl acetate with the dilauryl glycerol sulfate. However, the dilauryl glycerol sulfate is insoluble in water and is not easy to disperse and dissolve when added into a solution, so a nonionic surfactant Triton with fat solubility and hydrophilicity is also needed to be added, and the ethyl acetate and the dilauryl glycerol sulfate in which substrates are dissolved are fully dissolved in water. The lipase determination kit has the advantages that the ethyl acetate, the dilauryl glycerol sulfate and the Triton surfactant are jointly used as cosolvents, so that the solubility and the stability of the substrate can be greatly improved, and the dissolution time of the substrate is reduced, so that the lipase determination kit with good stability, high accuracy and precision and wide linear range is obtained.

Preferably, the concentration of ethyl acetate in the cosolvent is 3-10 ml/L, the concentration of dilauryl glycerol sulfate is 0.5-2 g/L, and the mass percent of the Triton surfactant is 0.1-1%.

Preferably, the stabilizer in the R2 reagent is mercaptoethanol and mannitol in a mass ratio of 1: 1-3. Mercaptoethanol and mannitol are used as composite stabilizers, wherein mercaptoethanol is easily oxidized and hydrolyzed, and free radicals can be effectively removed by compounding mercaptoethanol and mannitol, so that the stability of the reagent is remarkably enhanced.

Preferably, the stabilizing agent in the R2 reagent is mercaptoethanol and mannitol in a mass ratio of 1: 2.

Preferably, the pH of the R1 reagent is 8.0, including in particular:

the pH value of the R2 reagent is 4.0, and the reagent specifically comprises

Wherein, the content percentages of the components in the R1 reagent and the R2 reagent are mass percentages.

Preferably, the buffer in the R1 reagent is one or more of BICINE buffer, Tris buffer, PIPES buffer and HEPES buffer.

Preferably, the metal salt in the R1 reagent is one or more of sodium chloride, potassium chloride, manganese dichloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, zinc sulfate, sodium tetraprocate, potassium carbonate, disodium ethylene diamine tetraacetate and sodium tartrate.

Preferably, the protective agent in the R1 reagent is an amino acid protective agent, and comprises one or more of glycine, L-serine, oxalic acid, aspartic acid, bovine serum albumin and sodium glutamate.

Preferably, the surfactants in the R1 reagent are: tween-20, tween-80, polyoxyethylene lauryl ether (brij-35), tetraethylene glycol monolauryl ether (Thesit), glycerol, castor oil, PVP K40 (polyvinylpyrrolidone K40), GENAPOLX-080(α -isotridecyl- ω -hydroxy-poly (oxy-1, 2-ethylene)), NP (nonylphenol polyoxyethylene ether) series, polyethylene glycol series, Emulgen series.

The invention also provides a preparation method of the lipase determination kit, which comprises the following steps: (1) preparation of R1 reagent: taking a proper amount of pure water, adding a buffer solution, adjusting the pH value to 8.0, then adding other components in proportion, and obtaining an R1 reagent after constant volume;

(2) preparation of R2 reagent: weighing a substrate 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester, adding the substrate into ethyl acetate, uniformly stirring, sequentially adding dilauryl glycerol sulfate and a Triton surfactant, and uniformly stirring to fully dissolve the substrate; weighing other components in the R2 reagent, dissolving the other components in pure water, adjusting the pH value to 4.0, then slowly adding the dissolved substrate obtained in the step I at a constant speed, continuously stirring in the liquid adding process, and finally fixing the volume to obtain the R2 reagent.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, ethyl acetate, dilauryl glycerol sulfate and Triton surfactant are preferably used as cosolvents together to exert the synergistic interaction of the three, so that the solubility and the dissolution efficiency of the lipase substrate are greatly improved; furthermore, mercaptoethanol and mannitol are used as composite stabilizers to enhance the stability of the reagent, so that the lipase determination kit with good stability, high accuracy and precision and remarkably improved linear range is obtained.

Drawings

FIG. 1 shows properties of R2 reagents prepared in example 1 and comparative example 1 of the present invention, wherein 1-1 is R2 reagent obtained in comparative example 1, and 1-2 is R2 reagent obtained in example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a lipase determination kit, which comprises an R1 reagent and an R2 reagent, wherein the pH of the R1 reagent is 8.0, and the lipase determination kit specifically comprises:

the pH value of the R2 reagent is 4.0, and the reagent specifically comprises

Wherein, the content percentages of the components in the R1 reagent and the R2 reagent are mass percentages, and the stabilizing agent is mercaptoethanol and mannitol with the mass ratio of 1: 2.

The preparation method of the kit comprises the following steps: (1) preparation of R1 reagent: measuring a proper amount of pure water, adding a BICINE buffer solution, adjusting the pH to 8.0, adding other component substances in the R1 reagent according to the proportion, stirring and dissolving, and fixing the volume to obtain the R1 reagent; (2) preparation of R2 reagent: weighing a substrate 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester, putting the substrate into a beaker filled with ethyl acetate, uniformly stirring, then sequentially adding dilauryl glycerol sulfate and Tritonx-114, stirring at a high speed to fully dissolve the substrate, and fully dispersing the solution in a particle state to prepare a stable emulsifier; weighing other components in the R2 reagent, dissolving the other components in pure water, adjusting the pH value to 4.0, slowly and uniformly adding the dissolved substrate obtained in the step I into the solution in a mode of an injector or an external capillary hose, continuously stirring in the liquid adding process to ensure that the substrate raw material is quickly and uniformly dissolved in the solution, and finally fixing the volume to obtain the R2 reagent.

Example 2

The present embodiment is different from embodiment 1 in that: the formula of the R1 reagent is as follows:

the pH value of the R2 reagent is 4.0, and the reagent specifically comprises

Example 3

The present embodiment is different from embodiment 1 in that: the stabilizer in the R2 reagent is mercaptoethanol and mannitol with the mass ratio of 1: 1.

Example 4

The present embodiment is different from embodiment 1 in that: the stabilizer in the R2 reagent is mercaptoethanol and mannitol with the mass ratio of 1: 3.

Comparative example 1

This comparative example differs from example 1 in that: the R2 reagent does not contain dilauryl glycerol sulfate and Triton X-114, and only uses ethyl acetate as a cosolvent to dissolve a substrate 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester.

Comparative example 2

This comparative example differs from example 1 in that: the reagent R2 has no Triton X-114, and only ethyl acetate and dilauryl glycerol sulfate are used as cosolvent to dissolve the substrate.

Comparative example 3

This comparative example differs from example 1 in that: the content of dilauryl glycerol sulfate in the R2 reagent is 0.03g/L, the content of ethyl acetate is 2ml/L, and the mass percent of Triton X-114 is 0.08%.

Comparative example 4

This comparative example differs from example 1 in that: the content of dilauryl glycerol sulfate in the R2 reagent is 2.5g/L, the content of ethyl acetate is 12ml/L, and the mass percent of Triton X-114 is 1.3%.

Application example

The 8 sets of kits obtained in the above examples 1 to 4 and comparative examples 1 to 4 were subjected to the following performance evaluations, respectively:

(1) the reagent properties are as follows: observing whether the reagent is clear or not and whether a precipitate exists or not;

(2) accuracy: testing the third-party quality control product, wherein the relative deviation is not more than 10%;

(3) precision: repeating the measurement 20 times by using the same fixed value serum, wherein the coefficient of variation (CV%) of the measured value is not more than 6.0%;

(4) linear range: determining a sample with the lipase concentration of 2-1000U/L, and calculating a correlation coefficient, wherein the correlation coefficient is more than or equal to 0.99, so that the use requirement can be met;

(5) evaluation of reagent batch-to-batch difference: three batches of reagents are used for measuring the serum with the same fixed value, and the difference between the batches is not more than 10%;

(6) evaluation of stability: a. long-term stability, the property, accuracy and precision of the reagent are observed after the reagent is placed at 2-8 ℃ for 12 months; b. and (4) opening the bottle to ensure stability, and observing the properties of the reagent after the reagent is placed at 2-8 ℃ for 31 days after the bottle is opened.

The results of the measurement of the current formulation are shown in Table 1, and the results of the measurement of the 2-8 ℃ after 12 months of storage and the 2-8 ℃ after 31 days of storage after opening the bottle are shown in Table 2.

TABLE 1 evaluation data for Performance of the groups of reagents

TABLE 2 Performance evaluation data for Long-term stability and decap stability

According to the measurement results in table 1, compared with examples 1 and 2, when the components in the cosolvent are reduced (comparative examples 1-2) or the content of the cosolvent is changed (comparative examples 3-4), the R2 reagent is in a turbid state, i.e. the substrate is not sufficiently dissolved, specifically referring to fig. 1, wherein 1-1 is the R2 reagent prepared by the method of comparative example 1, and 1-2 is the R2 reagent prepared by the method of example 1, it can be clearly observed that the reagents of fig. 1-2 are clearer than the reagents of fig. 1-1, i.e. the substrate is more sufficiently dissolved. The above results show that the dissolution of the substrate in the R2 reagent is significantly affected by the variation of the components or contents of the cosolvent, resulting in the difference between different batches of reagents, and thus significantly affecting the accuracy, precision and linearity of the reagents.

According to the determination results of table 2, compared to examples 1-2, comparative examples 1-4 stored at 2-8 ℃ for 12 months or placed at 2-8 ℃ for 31 days after being opened, the reagents all have turbidity and precipitates, and the accuracy, precision and linearity of the reagent detection all have significant changes, which cannot meet the use requirements, indicating that the change of the cosolvent component or content has significant influence on the long-term stability and the bottle opening stability of the reagents. In contrast, in examples 3-4, compared with examples 1-2, although the reagent became turbid and had a small amount of precipitates after being stored at 2-8 ℃ for 12 months or left in an open bottle at 2-8 ℃ for 31 days, the accuracy, precision and linearity of the reagent could still meet the use requirements.

According to the results, the ethyl acetate, the dilauryl glycerol sulfate and the Triton surfactant are compounded according to a certain concentration ratio, and when the compound is used as a cosolvent, the synergistic effect of the ethyl acetate, the dilauryl glycerol sulfate and the Triton surfactant can be exerted, so that the solubility and the dissolution efficiency of a substrate are greatly improved, the batch difference is reduced, the accuracy, the precision and the linearity of a reagent are improved, meanwhile, the mercaptoethanol and the mannitol with the mass ratio of 1:1-3 are used as a compound stabilizer, the long-term stability and the bottle opening stability of the reagent can be maintained, and the lipase determination kit with good stability, high reaction sensitivity and precision and the linearity range of 2-1000U/L is obtained.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A lipase determination kit comprises an R1 reagent and an R2 reagent, and is characterized in that the R1 reagent comprises a buffer solution, sodium deoxycholate, colipase, metal salt, a protective agent, a surfactant and a preservative; the R2 reagent comprises L-tartaric acid, sodium taurodeoxycholate, 1, 2-o-dilauryl-racemic-glycerol-3-pentanoic acid- (6-methyl resorufin) ester, a cosolvent, a stabilizer and a preservative, wherein the cosolvent comprises ethyl acetate, dilauryl glycerol sulfate and a Triton surfactant.

2. The lipase assay kit as claimed in claim 1, wherein the concentration of ethyl acetate in the cosolvent is 3-10 ml/L, the concentration of dilauryl glycerol sulfate is 0.5-2 g/L, and the mass percentage of Triton surfactant is 0.1-1%.

3. The lipase assay kit as claimed in claim 1, wherein the stabilizer in the R2 reagent is mercaptoethanol and mannitol in a mass ratio of 1: 1-3.

4. The lipase assay kit as claimed in claim 3, wherein the stabilizer in the R2 reagent is mercaptoethanol and mannitol in a mass ratio of 1: 2.

5. The lipase assay kit as claimed in claim 1, wherein the pH of the R1 reagent is 8.0, and the kit specifically comprises:

the pH value of the R2 reagent is 4.0, and the reagent specifically comprises

6. The lipase assay kit as claimed in claim 1, wherein the buffer in the R1 reagent is one or more of BICINE buffer, Tris buffer, PIPES buffer and HEPES buffer.

7. The lipase assay kit as claimed in claim 1, wherein the metal salt in the R1 reagent is one or more of sodium chloride, potassium chloride, manganese dichloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, zinc sulfate, sodium tetraprocate, potassium carbonate, disodium edetate and sodium tartrate.

8. The lipase assay kit as claimed in claim 1, wherein the protective agent in the R1 reagent is an amino acid protective agent comprising one or more of glycine, L-serine, oxalic acid, aspartic acid, bovine serum albumin, and sodium glutamate.

9. The lipase assay kit as claimed in claim 1, wherein the surfactant in the R1 reagent is: tween-20, tween-80, polyoxyethylene lauryl ether, polyethylene glycol monolauryl ether, glycerol, castor oil, PVP K40, GENAPOLX-080, NP series, polyethylene glycol series, and Emulgen series.

10. The method for preparing the lipase assay kit as set forth in any one of claims 1 to 9, characterized by comprising the steps of: (1) preparation of R1 reagent: weighing a proper amount of pure water, adding a buffer solution, adjusting the pH value to 8.0, then adding other components in proportion, and fixing the volume to obtain an R1 reagent;