Stable free fatty acid determination kit

文档序号：1948482 发布日期：2021-12-10 浏览：12次中文

阅读说明：本技术 一种稳定的游离脂肪酸测定试剂盒 (Stable free fatty acid determination kit ) 是由黄孟于 2021-09-30 设计创作，主要内容包括：本发明公开了一种稳定的游离脂肪酸测定试剂盒,属于医疗设备领域。所述试剂盒包括试剂R1和试剂R2,试剂R1中含有以下成分：缓冲液、辅酶A、ATP、酰基辅酶A合成酶、牛乳清白蛋白、酪蛋白钠盐、正辛酸钠、Trinder底物、表面活性剂、酶保护剂；试剂R2中含有以下成分：缓冲液、黄素腺嘌呤二核苷酸、4-氨基安替比林、过氧化物酶、酰基辅酶A氧化酶、表面活性剂、防腐剂、复合巯基试剂、色原、十二烷基聚乙二醇醚、浓盐。本发明在试剂盒中添加复合巯基试剂、牛乳清白蛋白、酪蛋白钠盐,其中复合巯基试剂可以与辅酶A的巯基反应而去除辅酶A对色原的干扰,酪蛋白钠盐能够与牛乳清白蛋白结合,提高试剂盒的稳定性。(The invention discloses a stable free fatty acid determination kit, and belongs to the field of medical equipment. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: buffer solution, coenzyme A, ATP, acyl coenzyme A synthetase, bovine whey albumin, casein sodium salt, sodium n-caprylate, Trinder substrate, surfactant and enzyme protectant; the reagent R2 contains the following components: buffer solution, flavin adenine dinucleotide, 4-aminoantipyrine, peroxidase, acyl coenzyme A oxidase, surfactant, preservative, composite thiol reagent, chromogen, dodecyl polyglycol ether and concentrated salt. According to the kit, the composite sulfhydryl reagent, the bovine whey albumin and the casein sodium salt are added into the kit, wherein the composite sulfhydryl reagent can react with the sulfhydryl of the coenzyme A to remove the interference of the coenzyme A on the chromogen, and the casein sodium salt can be combined with the bovine whey albumin to improve the stability of the kit.)

1. A stable free fatty acid determination kit is characterized by comprising a reagent R1 and a reagent R2,

the reagent R1 contains the following components: buffer solution 20-80mmol/L, coenzyme A1-10 mmol/I, ATP 1-4mmol/L, acyl coenzyme A synthetase 0.4-1KU/L, bovine whey albumin 10-20g/L, casein sodium salt 5-15g/L, sodium n-caprylate 5-15g/L, Trinder substrate 0.8-1.5g/L, surfactant 5-80ml/L, and enzyme protectant 2-10 ml/L;

the reagent R2 contains the following components: 20-80mmol/L of buffer solution, 1-4mmol/L of flavin adenine dinucleotide, 2-8mmol/L of 4-aminoantipyrine, 20-35KU/L of peroxidase, 5-10KU/L of acyl coenzyme A oxidase, 5-80ml/L of surfactant, 1-3ml/L of preservative, 100ml/L of composite sulfhydryl reagent, 0.1-1mmol/L of chromogen, 1-1.5g/L of dodecyl polyglycol ether and 1-5ml/L of concentrated hydrochloric acid.

2. The stable free fatty acid assay kit as claimed in claim 1, wherein the reagent R1 contains the following components: 50mmol/L buffer solution, 6mmol/I, ATP3mmol/L coenzyme A, 0.8KU/L acyl coenzyme A synthetase, 15g/L bovine whey albumin, 10g/L casein sodium salt, 10g/L, Trinder g/L sodium n-caprylate substrate, 1.2g/L surfactant, and 6ml/L enzyme protectant;

the reagent R2 contains the following components: 60mmol/L of buffer solution, 2mmol/L of flavin adenine dinucleotide, 6mmol/L of 4-aminoantipyrine, 28KU/L of peroxidase, 7KU/L of acyl coenzyme A oxidase, 35ml/L of surfactant, 2ml/L of preservative, 150ml/L of composite sulfhydryl reagent, 0.5mmol/L of chromogen, 1.3g/L of dodecyl polyglycol ether and 3ml/L of concentrated hydrochloric acid.

3. The stable free fatty acid assay kit of claim 2, wherein the buffer is one of sodium dihydrogen phosphate or glycine.

4. The stable free fatty acid assay kit of claim 1, wherein reagent R1 is formulated as follows: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to the pH value of 7.2 by NaOH, and sequentially added with ATP, acyl coenzyme A synthetase, bovine whey albumin, casein sodium salt, sodium n-caprylate, Trinder substrate, surfactant and enzyme protective agent.

5. The stable free fatty acid assay kit of claim 4, wherein reagent R2 is formulated as follows: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to 7.2 by NaOH, and then added with flavin adenine dinucleotide, 4-aminoantipyrine, peroxidase, acyl coenzyme A oxidase, a surfactant, a preservative, a composite sulfhydryl reagent, a chromogen, dodecyl polyglycol ether and concentrated hydrochloric acid in sequence.

6. The stable free fatty acid assay kit of claim 1, wherein the surfactant is one of Tween 20, EMULGEN a-60, EMULGEN a-90.

7. The stable free fatty acid assay kit of claim 1, wherein the chromogen is a mixture of TOPS and DAOS.

8. The stable free fatty acid assay kit of claim 7, wherein the TOPS and DAOS are present in a 1:1 ratio.

9. The stable free fatty acid assay kit of claim 1, wherein the preservative is one of sodium azide, potassium sorbate, and sodium benzoate.

10. The stable free fatty acid assay kit of claim 1, wherein the enzyme protectant is a 1:1 mixture of dipotassium ethylenediaminetetraacetate and magnesium sulfate.

Technical Field

The invention belongs to the field of medical equipment, and particularly relates to a stable free fatty acid determination kit.

Background

Free Fatty Acid (FFA), also known as non-esterified fatty acid, is a fatty acid generally referred to clinically as C10 or higher, which is an intermediate product of fat metabolism, and is also one of the donors for synthesizing cell membrane lipid structure and prostaglandin and important energy substances for energy metabolism of the body. Lipid substances are the major components of cell membranes and have a variety of important biological functions. Lipids present in the human body are roughly classified into 4 types, such as cholesterol, neutral fat (triglyceride), phospholipid, and free fatty acid. Free fatty acids are substances into which neutral fats are broken down. When liver cheese, which is the energy source required for muscle activity, is exhausted, the adipose tissue decomposes neutral fat into free fatty acids to serve as an energy source. Therefore, free fatty acids can be said to be substances required for long-lasting activities. Free fatty acids in normal serum mainly include oleic acid (oleic acid accounts for 54%), palmitic acid (palmitic acid accounts for 34%), stearic acid (stearic acid accounts for 6%), and the like, which are transported in blood in combination with albumin. By detecting the level of fatty acids in plasma, the relationship of certain important diseases to lipids and fatty acids can be studied. In particular, long-chain unsaturated fatty acids are important because they are a raw material for synthesizing prostaglandins and promote the conversion and excretion of cholesterol, thereby reducing the concentration of cholesterol in blood.

Although various FFA detection kits are available on the market, detection principles and methods are different, indexes such as sensitivity, specificity and the like for disease prediction are different, evaluation on various detection methods and systematic methodology and diagnosis efficiency among different reagents is still lacked, and the determination method is not unified, so that research and evaluation on the rapid and accurate characteristics of the existing detection method are needed, which is beneficial to development of clinical work. The determination method of serum FFA mainly comprises a titration method, a colorimetric method, a chemiluminescence method, a gas chromatography and an enzyme method, and the enzyme method has the reaction principle that free fatty acid generates acyl coenzyme A under the action of acyl coenzyme A synthetase in the presence of ATP and coenzyme A, the acyl coenzyme A is oxidized by acyl coenzyme A oxidase to generate 2, 3-trans-phthalein coenzyme A and hydrogen peroxide, the generated hydrogen peroxide generates a colored substance with Trinder chromogen and 4-aminoantipyrine in the presence of peroxidase, and the concentration of the free fatty acid in a sample can be obtained by measuring the absorbance of the colored substance.

Chinese patent document "a free fatty acid measurement kit and a preparation method thereof (patent application No. CN 201610831420.3)" discloses a free fatty acid measurement kit, which contains a reagent R1 and a reagent R2, wherein the reagent R1 contains the following components: 50mmol/L of sodium dihydrogen phosphate with pH of 7.0, 0.05mmol/L of coenzyme A, 3mmol/L of adenosine triphosphate, 0.4KU/L, MgCl 22 mmol/L, Trinder of acyl coenzyme A synthetase substrate, 0.5g/L of Tween 20<1mL/L, and pH of 7.2; the reagent R2 contains the following components: 60mmol/L of sodium dihydrogen phosphate with pH of 7.0, 2mmol/L of flavin adenine dinucleotide, 10mmol/L of 4-aminoantipyrine, 40KU/L of peroxidase, 30KU/L of acyl coenzyme A oxidase, 20<1mL/L of Tween, 20mL/L-300mL/L, brij35 (dodecyl polyglycol ether) 1-1.5g/L of glycerol, 1g/L of disodium ethylene diamine tetraacetate, and pH of 7.2. The invention improves the viscosity of the reagent, reduces the viscosity of the reagent, so that the solution is easy to filter quickly, but still has the problem that the stability of the kit is to be improved.

Disclosure of Invention

The invention aims to provide a stable free fatty acid determination kit, and aims to solve the problem of how to optimize components, dosage, process and the like on the basis of the disclosure of 'a free fatty acid determination kit and a preparation method thereof (the patent application number is CN 201610831420.3)' in Chinese patent literature, so that the stability of the stable free fatty acid determination kit is improved.

In order to solve the technical problems, the invention adopts the following technical scheme:

a stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

the reagent R1 contains the following components: buffer solution 20-80mmol/L, coenzyme A1-10 mmol/L, ATP 1-4mmol/L, acyl coenzyme A synthetase 0.4-1KU/L, bovine whey albumin 10-20g/L, casein sodium salt 5-15g/L, sodium n-caprylate 5-15g/L, Trinder substrate 0.8-1.5g/L, surfactant 5-80ml/L, and enzyme protectant 2-10 ml/L;

Preferably, the reagent R1 contains the following components: 50mmol/L buffer solution, 6mmol/L coenzyme A, 3mmol/L ATP, 0.8KU/L acyl coenzyme A synthetase, 15g/L bovine whey albumin, 10g/L casein sodium salt, 10g/L, Trinder sodium n-caprylate substrate 1.2g/L surfactant 45ml/L, and 6ml/L enzyme protective agent;

Preferably, the buffer is one of sodium dihydrogen phosphate or glycine.

Preferably, the reagent R1 is prepared by the following method: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to the pH value of 7.2 by NaOH, and sequentially added with ATP, acyl coenzyme A synthetase, bovine whey albumin, casein sodium salt, sodium n-caprylate, Trinder substrate, surfactant and enzyme protective agent.

Preferably, the reagent R2 is prepared by the following method: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to 7.2 by NaOH, and then added with flavin adenine dinucleotide, 4-aminoantipyrine, peroxidase, acyl coenzyme A oxidase, a surfactant, a preservative, a composite sulfhydryl reagent, a chromogen, dodecyl polyglycol ether and concentrated hydrochloric acid in sequence.

Preferably, the surfactant is one of Tween 20, EMULGEN A-60, and EMULGEN A-90.

Preferably, the chromogen is a mixture of TOPS and DAOS.

Preferably, the TOPS and DAOS are used in a 1:1 ratio.

Preferably, the preservative is one of sodium azide, potassium sorbate and sodium benzoate.

Preferably, the enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate in a concentration ratio of 1: 1.

The invention has the following beneficial effects:

(1) as can be seen from the data of example 1 and comparative examples 1-4, the free fatty acid assay kit is added with a composite sulfhydryl reagent, bovine whey albumin and casein sodium salt, wherein the composite sulfhydryl reagent can react with sulfhydryl of coenzyme A to remove interference of the coenzyme A on chromogen, and a substance with absorption at 546nm is formed, and the main reaction of the kit has maximum absorption in the range of 500-700 nm. On the basis of adopting a mixture chromogen of TOPS and DAOS, the interference caused by coenzyme A can be completely eliminated, the stability of the kit is greatly improved, and the kit can be stabilized for 1 year at the temperature of 2-8 ℃. The bovine whey albumin is an enzyme activator and an enzyme stabilizer, 34 disulfide bonds and one sulfydryl group are arranged in the structure, the chemical reaction of the sulfydryl groups is very active, and the disulfide bonds have the effect of oxidation reduction resistance, so that the bovine whey albumin can be combined with various cations, anions and small molecules, the sodium caseinate can be combined with the bovine whey albumin, and metal ions can be introduced in the process of bovine whey albumin chelation enzyme, so that the inhibition effect of the metal ions on the enzyme is relieved, and the stability of the kit is improved. In addition, the active center of the coenzyme A contains a large amount of sulfhydryl reduced amino acid, the reaction activity is high, the coenzyme A is easy to oxidize, and the bovine whey albumin can improve the catalytic efficiency of the coenzyme A, so that the stability of the enzyme is improved.

(2) Although the invention adds different combinations of protein protective agents, brij35 (dodecyl polyglycol ether), glycerol, tween 20 and EDTA-2Na (disodium ethylene diamine tetraacetate), to prolong the enzyme activity stability, and prolong the stability of the enzyme at 37 ℃ from 7 days to 14 days, the invention still has the problem that the stability is to be improved under low temperature environment, based on solving the technical problems, the invention further optimizes and improves the formula and the process, and through a plurality of experimental researches, when the free fatty acid determination kit is added with a composite sulfhydryl reagent, bovine whey albumin and casein sodium salt, the composite sulfhydryl reagent can react with the sulfhydryl of coenzyme A to remove the interference of the coenzyme A on the chromogen, the sodium caseinate can be combined with the bovine whey albumin, and metal ions can be introduced in the process of bovine whey albumin chelatase, so that the inhibition of the metal ions on the enzyme is relieved, and the stability of the kit is improved; the technical problems appearing in the background art documents can be solved, and unexpected effects are produced.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate, but not to limit the scope of the present invention.

A stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

The preparation method of the reagent R1 comprises the following steps: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to the pH value of 7.2 by NaOH, and sequentially added with ATP, acyl coenzyme A synthetase, bovine whey albumin, casein sodium salt, sodium n-caprylate, Trinder substrate, surfactant and enzyme protective agent.

The preparation method of the reagent R2 comprises the following steps: 900mL of water is taken, added with a buffer solution with the pH value of 7.0, fully dissolved, adjusted to 7.2 by NaOH, and then added with flavin adenine dinucleotide, 4-aminoantipyrine, peroxidase, acyl coenzyme A oxidase, a surfactant, a preservative, a composite sulfhydryl reagent, a chromogen, dodecyl polyglycol ether and concentrated hydrochloric acid in sequence.

The buffer solution is one of sodium dihydrogen phosphate or glycine.

The surfactant is one of Tween 20, EMULGEN A-60 and EMULGEN A-90.

The chromogen is a mixture of TOPS and DAOS with the dosage ratio of 1: 1.

The preservative is one of sodium azide, potassium sorbate and sodium benzoate.

The enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate with the concentration ratio of 1: 1.

The following is a more specific example.

Example 1

A stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

the reagent R1 contains the following components: 50mmol/L sodium dihydrogen phosphate, 6mmol/L coenzyme A, 3mmol/L ATP, 0.8KU/L acyl coenzyme A synthetase, 15g/L bovine whey albumin, 10g/L casein sodium salt, 10g/L, Trinder sodium n-caprylate substrate 1.2g/L, Tween 2045 ml/L and 6ml/L enzyme protective agent; the enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate with the concentration ratio of 1: 1;

the reagent R2 contains the following components: 60mmol/L of sodium dihydrogen phosphate, 2mmol/L of flavin adenine dinucleotide, 6mmol/L of 4-aminoantipyrine, 28KU/L of peroxidase, 7KU/L, Tween 2035 ml/L of acyl coenzyme A oxidase sodium azide, 2ml/L of composite sulfhydryl reagent, 0.5mmol/L of chromogen, 1.3g/L of dodecyl polyglycol ether and 3ml/L of concentrated hydrochloric acid. The chromogen is a mixture of TOPS and DAOS with the dosage ratio of 1: 1.

Example 2

A stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

the reagent R1 contains the following components: 40mmol/L glycine, 1mmol/L coenzyme A, 1mmol/L ATP, 1KU/L acyl coenzyme A synthetase, 15g/L bovine whey albumin, 15g/L casein sodium salt, 15g/L, Trinder g/L sodium n-caprylate substrate, 1.1g/L EMULGEN A-6055 ml/L, and 10ml/L enzyme protectant; the enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate with the concentration ratio of 1: 1;

the reagent R2 contains the following components: 40mmol/L of glycine, 1mmol/L of flavin adenine dinucleotide, 6mmol/L of 4-aminoantipyrine, 35KU/L of peroxidase, 9KU/L of acyl coenzyme A oxidase, 1ml/L of EMULGEN A-605 ml/L of potassium sorbate, 145ml/L of composite sulfhydryl reagent, 0.1mmol/L of chromogen, 1.5g/L of dodecyl polyglycol ether and 3ml/L of concentrated hydrochloric acid. The chromogen is a mixture of TOPS and DAOS with the dosage ratio of 1: 1.

Example 3

A stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

the reagent R1 contains the following components: 80mmol/L sodium dihydrogen phosphate, 7mmol/L coenzyme A, 3mmol/L ATP, 0.4KU/L acyl coenzyme A synthetase, 20g/L bovine whey albumin, 5g/L casein sodium salt, 5g/L, Trinder sodium n-caprylate substrate 1.5g/L EMULGEN A-9080 ml/L enzyme protective agent 2 ml/L; the enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate with the concentration ratio of 1: 1;

the reagent R2 contains the following components: 80mmol/L of sodium dihydrogen phosphate, 2mmol/L of flavin adenine dinucleotide, 8mmol/L of 4-aminoantipyrine, 20KU/L of peroxidase, 10KU/L of acyl coenzyme A oxidase, 2ml/L of sodium benzoate, 200ml/L of composite sulfhydryl reagent, 0.7mmol/L of chromogen, 1g/L of dodecyl polyglycol ether and 5ml/L of concentrated hydrochloric acid. The chromogen is a mixture of TOPS and DAOS with the dosage ratio of 1: 1.

Example 4

A stable free fatty acid determination kit comprises a reagent R1 and a reagent R2,

the reagent R1 contains the following components: 20mmol/L of glycine, 10mmol/L of coenzyme A, 4mmol/L of ATP, 0.7KU/L of acyl coenzyme A synthetase, 10g/L of bovine whey albumin, 11g/L of casein sodium salt, 10g/L, Trinder g/0.8 g/L, Tween 205 ml/L of sodium n-caprylate substrate and 6ml/L of enzyme protective agent; the enzyme protective agent is a mixture of ethylene diamine tetraacetic acid dipotassium and magnesium sulfate with the concentration ratio of 1: 1.

The reagent R2 contains the following components: 20mmol/L of glycine, 4mmol/L of flavin adenine dinucleotide, 2mmol/L of 4-aminoantipyrine, 28KU/L of peroxidase, 5KU/L, Tween 2080 ml/L of acyl coenzyme A oxidase, 2080 ml/L of potassium sorbate, 100ml/L of composite sulfhydryl reagent, 1mmol/L of chromogen, 1.2g/L of dodecyl polyglycol ether and 1ml/L of concentrated hydrochloric acid. The chromogen is a mixture of TOPS and DAOS with the dosage ratio of 1: 1.

Comparative example 1

The procedure of example 1 was followed except that the stable free fatty acid assay kit was prepared without adding a complex thiol reagent, bovine whey albumin, and casein sodium salt.

Comparative example 2

The preparation method was substantially the same as that of example 1, except that a complex thiol reagent was not added in the preparation of a stable free fatty acid assay kit.

Comparative example 3

The procedure was substantially the same as in example 1, except that bovine whey albumin was not added to the preparation of the stable free fatty acid assay kit.

Comparative example 4

The preparation method was substantially the same as that of example 1, except that the stable free fatty acid assay kit was prepared without adding sodium caseinate.

Comparative example 5

The method described in example 1 in the chinese patent document "a free fatty acid assay kit and a method for preparing the same (patent application No. CN 201610831420.3)" was used to prepare a free fatty acid assay kit.

The stability was examined according to the free fatty acid measurement kits prepared in examples 1 to 4 and comparative examples 1 to 5,

placing the reagent at the temperature of 2-8 ℃ for 12 months, and evaluating the character, blank absorbance and accuracy of the reagent every 3 months;

wherein the character of the reagent is as follows: mainly judging whether the reagent is precipitated or not;

evaluation of blank absorbance: measuring the absorbance of the R1 reagent and the R2 reagent at the wavelength of 546nm and under the optical path of 10mm, and taking the average value as blank absorbance, wherein the blank absorbance is less than or equal to 1.0000;

evaluation of accuracy: and measuring a third party quality control product as a target value, and calculating the deviation value of the measured average value and the target value of the kit disclosed by the invention as the accuracy, wherein the accuracy is less than or equal to 10% and meets the requirement.

The results are shown in the following table.

From the above table, it can be seen that: (1) as can be seen from the data of examples 1 to 4 and comparative example 5, the stability of the free fatty acid assay kit prepared in examples 1 to 4 was significantly higher than that of the free fatty acid assay kit prepared in comparative example 5, and example 1 was the most preferable example.

(2) As can be seen from the data of example 1 and comparative examples 1-4, the free fatty acid assay kit is added with a composite sulfhydryl reagent, bovine whey albumin and casein sodium salt, wherein the composite sulfhydryl reagent can react with sulfhydryl of coenzyme A to remove interference of the coenzyme A on chromogen, and a substance with absorption at 546nm is formed, and the main reaction of the kit has maximum absorption in the range of 500-700 nm. On the basis of adopting a mixture chromogen of TOPS and DAOS, the interference caused by coenzyme A can be completely eliminated, the stability of the kit is greatly improved, and the kit can be stabilized for 1 year at the temperature of 2-8 ℃.

The bovine whey albumin is an enzyme activator and an enzyme stabilizer, 34 disulfide bonds and one sulfydryl group are arranged in the structure, the chemical reaction of the sulfydryl groups is very active, and the disulfide bonds have the effect of oxidation reduction resistance, so that the bovine whey albumin can be combined with various cations, anions and small molecules, the sodium caseinate can be combined with the bovine whey albumin, and metal ions can be introduced in the process of bovine whey albumin chelation enzyme, so that the inhibition effect of the metal ions on the enzyme is relieved, and the stability of the kit is improved. In addition, the active center of the coenzyme A contains a large amount of sulfhydryl reduced amino acid, the reaction activity is high, the coenzyme A is easy to oxidize, and the bovine whey albumin can improve the catalytic efficiency of the coenzyme A, so that the stability of the enzyme is improved.

The above description should not be taken as limiting the invention to the embodiments, but rather, as will be apparent to those skilled in the art to which the invention pertains, numerous simplifications or substitutions may be made without departing from the spirit of the invention, which shall be deemed to fall within the scope of the invention as defined by the claims appended hereto.

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Stable free fatty acid determination kit

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