阅读说明：本技术 一种消除羟苯磺酸钙干扰的游离脂肪酸测定试剂盒 (Free fatty acid determination kit for eliminating interference of calcium dobesilate ) 是由 潘利琴 于 2021-06-30 设计创作，主要内容包括：本发明提供了一种消除羟苯磺酸钙干扰的游离脂肪酸测定试剂盒,包括：含有过氧化氢、TritonX-100的柠檬酸缓冲液；含乙酰辅酶A合成酶、过氧化氢酶、辅酶A、三磷酸腺苷、TODB、TritonX-100、proclin300、海藻糖的Tris缓冲液；含乙酰辅酶A氧化酶、过氧化物酶、4-AAP、TritonX-100、N-乙基顺丁烯二酰亚胺、5,5-二巯基-2,2-二硝基苯甲酸、2-甲基-4-异噻唑啉-3-酮、叠氮钠、海藻糖的Tris缓冲液。本发明的试剂盒准确度高,稳定性好,应用前景广阔。(The invention provides a free fatty acid determination kit for eliminating interference of calcium dobesilate, which comprises: a citric acid buffer solution containing hydrogen peroxide and TritonX-100; tris buffer solution containing acetyl coenzyme A synthetase, catalase, coenzyme A, adenosine triphosphate, TODB, TritonX-100, proclin300 and trehalose; tris buffer solution containing acetyl coenzyme A oxidase, peroxidase, 4-AAP, TritonX-100, N-ethylmaleimide, 5-dimercapto-2, 2-dinitrobenzoic acid, 2-methyl-4-isothiazoline-3-ketone, sodium azide and trehalose. The kit disclosed by the invention is high in accuracy, good in stability and wide in application prospect.)

1. A free fatty acid determination kit for eliminating interference of calcium dobesilate is characterized by comprising a reagent 1, a reagent 2 and a reagent 3;

the reagent 1 comprises a citric acid buffer solution containing hydrogen peroxide and TritonX-100, and the pH value of the citric acid buffer solution is 6.50;

the reagent 2 comprises a Tris buffer solution containing acetyl coenzyme A synthetase, catalase, coenzyme A, adenosine triphosphate, TODB, TritonX-100, proclin300 and trehalose, and the pH value of the Tris buffer solution is 7.2;

the reagent 3 comprises a Tris buffer solution containing acetyl coenzyme A oxidase, peroxidase, 4-AAP, TritonX-100, N-ethylmaleimide, 5-dimercapto-2, 2-dinitrobenzoic acid, 2-methyl-4-isothiazoline-3-ketone, sodium azide and trehalose, and the pH value of the Tris buffer solution is 7.2.

2. The free fatty acid assay kit according to claim 1, wherein in the reagent 1: the concentration of the hydrogen peroxide is 1.0-3.0mmol/L, TritonX-100 and the concentration of the citric acid buffer solution is 30-80 mmol/L.

3. The free fatty acid assay kit according to claim 1, wherein in the reagent 2: the concentration of acetyl coenzyme A synthetase is 1-5KU/L, the concentration of catalase is 600-1800KU/L, the concentration of coenzyme A is 0.2-0.8g/L, the concentration of adenosine triphosphate is 2-8mmoL/L, TODB, the concentration of adenosine triphosphate is 1-3mmoL/L, TritonX-100, the concentration of adenosine triphosphate is 1-3mL/L, proclin300, the concentration of trehalose is 20-100g/L, Tris, and the concentration of buffer solution is 50-100 mmoL/L.

4. The free fatty acid assay kit according to claim 1, wherein in the reagent 3: the concentration of acetyl coenzyme A oxidase is 20-120KU/L, the concentration of peroxidase is 20-200KU/L, the concentration of 4-AAP is 1-5mmol/L, TritonX-100, the concentration of 1-3mL/L, N-ethylmaleimide is 2-8mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 1-2mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 1-3mmol/L, the concentration of sodium azide is 3-5g/L, and the concentration of trehalose is 20-100g/L, Tris, and the concentration of buffer solution is 50-100 mmol/L.

5. The free fatty acid assay kit according to claim 2, wherein in the reagent 1: the concentration of the hydrogen peroxide is 1.5-2.5mmol/L, TritonX-100, the concentration is 1.5-2.5mL/L, and the concentration of the citric acid buffer solution is 50-60 mmol/L.

6. The free fatty acid assay kit according to claim 3, wherein in the reagent 2: the concentration of acetyl coenzyme A synthetase is 2-4KU/L, the concentration of catalase is 1000-1400KU/L, the concentration of coenzyme A is 0.4-0.6g/L, the concentration of adenosine triphosphate is 4-6mmol/L, TODB, the concentration of adenosine triphosphate is 1.5-2.5mmol/L, TritonX-100, the concentration of adenosine triphosphate is 1.5-2.5mL/L, proclin300, the concentration of trehalose is 0.1-1mL/L, and the concentration of trehalose is 40-80g/L, Tris, and the concentration of buffer solution is 60-90 mmol/L.

7. The free fatty acid assay kit according to claim 4, wherein in the reagent 3: the concentration of acetyl coenzyme A oxidase is 40-100KU/L, the concentration of peroxidase is 60-160KU/L, the concentration of 4-AAP is 2-4mmol/L, TritonX-100, the concentration of 1.5-2.5mL/L, N-ethylmaleimide is 4-6mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 1.5-2.5mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 1.5-2.5mmol/L, the concentration of sodium azide is 3.5-4.5g/L, and the concentration of trehalose is 40-80g/L, Tris, and the concentration of buffer solution is 60-90 mmol/L.

8. The free fatty acid assay kit according to claim 5, wherein in the reagent 1: the concentration of hydrogen peroxide is 2.0mmol/L, TritonX-100, the concentration is 2mL/L, and the concentration of citric acid buffer solution is 55 mmol/L.

9. The free fatty acid assay kit according to claim 6, wherein in the reagent 2: the concentration of acetyl coenzyme A synthetase is 3KU/L, the concentration of catalase is 1200KU/L, the concentration of coenzyme A is 0.5g/L, the concentration of adenosine triphosphate is 5mmoL/L, TODB, the concentration is 2mmoL/L, TritonX-100, the concentration is 2mL/L, proclin300, the concentration is 0.2mL/L, and the concentration of trehalose is 60g/L, Tris, and the concentration of buffer solution is 75 mmoL/L.

10. The free fatty acid assay kit according to claim 7, wherein in the reagent 3: the concentration of acetyl coenzyme A oxidase is 70KU/L, the concentration of peroxidase is 110KU/L, the concentration of 4-AAP is 3mmol/L, TritonX-100, the concentration of 2mL/L, N-ethylmaleimide is 5mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 2mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 2mmol/L, the concentration of sodium azide is 4g/L, and the concentration of trehalose is 60g/L, Tris, and the concentration of buffer solution is 75 mmol/L.

Technical Field

The invention belongs to the technical field of medical examination, and particularly relates to a free fatty acid determination kit for eliminating calcium dobesilate interference.

Background

Free Fatty acids (NEFA) refer to Fatty acids in serum that are not Esterified with glycerol, cholesterol, etc., and are also called Non-Esterified Fatty acids or Non-Esterified Fatty acids, with a half-life of 2-3 minutes in plasma, and normally, the content in plasma is very low. The NEFA in serum has extremely high metabolic activity, and is easily influenced by fat metabolism, carbohydrate metabolism and endocrine metabolism. With the intensive research and continuous technological progress, the relation between serum NEFA and diseases is gradually clarified, and NEFA has proved to be closely related to the occurrence and development of cardiovascular and cerebrovascular diseases, respiratory diseases, digestive diseases, endocrine diseases, immune diseases and other system diseases, and the energy metabolism of tumors, traumatic stress and the like.

The enzyme method determination method of acetyl coenzyme A synthetase (ACS) -acetyl coenzyme A oxidase (ACOD) coupling is the main method for clinically testing NEFA at present, and the detection principle is that free fatty acid in human serum and coenzyme A react under the action of acetyl coenzyme A synthetase to generate fatty acyl coenzyme A, and fatty acyl coenzyme A generates H under the action of acetyl coenzyme A oxidase₂O₂Then, a colored substance is generated by the action of peroxidase through a Trinder's reaction. The clinical application finds that the method is easily interfered by calcium dobesilate, so that the result has larger deviation and even wrong result is obtained^[1]The reason for this is probably that calcium dobesilate reduces H formed during the reaction₂O₂Causing negative interference to the assay. Calcium dobesilate is a common medicine clinically used for treating capillary vessel diseases caused by various reasons, such as diabetic retinopathy, varicosity, phlebitis, leg spasm, phlegm itching dermatitis and the like, and after the medicine is taken, a serum sample of a patient inevitably contains the medicine with higher concentration.

Therefore, the method and the kit for measuring the free fatty acid, which can eliminate the influence of the calcium dobesilate, have very important significance for clinical detection.

[1] Hollian, Guoxiu Zhi, Qiu Ling, et al, calcium dobesilate negatively interferes with enzymatic free fatty acid detection [ J ] test medicine 2016, (11). 936-.

Disclosure of Invention

In order to solve the problems, the invention provides a free fatty acid determination kit capable of eliminating interference of calcium dobesilate.

In one aspect, the invention provides a free fatty acid assay kit for eliminating calcium dobesilate interference.

The free fatty acid determination kit comprises a reagent 1; the reagent 1 comprises a citric acid buffer solution containing hydrogen peroxide and TritonX-100 and having pH of 6.50.

Preferably, in the reagent 1, the concentration of hydrogen peroxide is 1.0-3.0mmol/L, TritonX-100, the concentration is 1-3mL/L, and the concentration of citric acid buffer solution with pH6.50 is 30-80 mmol/L.

More preferably, in the reagent 1, the concentration of hydrogen peroxide is 1.5-2.5mmol/L, TritonX-100, the concentration is 1.5-2.5mL/L, and the concentration of citric acid buffer solution with pH value of 6.50 is 50-60 mmol/L.

Furthermore, in the reagent 1, the concentration of hydrogen peroxide is 2.0mmol/L, TritonX-100, the concentration is 2mL/L, and the concentration of citric acid buffer solution with pH6.50 is 55 mmol/L.

The free fatty acid determination kit also comprises a reagent 2; the reagent 2 comprises Tris buffer solution with pH7.2 containing acetyl coenzyme A synthetase, catalase, coenzyme A, adenosine triphosphate, TODB, TritonX-100, proclin300 and trehalose.

Preferably, in the reagent 2, the concentration of acetyl coenzyme A synthetase is 1-5KU/L, the concentration of catalase is 600-1800KU/L, the concentration of coenzyme A is 0.2-0.8g/L, the concentration of adenosine triphosphate is 2-8mmol/L, TODB, the concentration of triphosphate is 1-3mmol/L, TritonX-100, the concentration of triphosphate is 1-3mL/L, proclin300, the concentration of triphosphate is 0.02-2mL/L, the concentration of trehalose is 20-100g/L, and the concentration of Tris buffer solution with pH7.2 is 50-100 mmol/L.

More preferably, in the reagent 2, the concentration of acetyl coenzyme A synthetase is 2-4KU/L, the concentration of catalase is 1000-1400KU/L, the concentration of coenzyme A is 0.4-0.6g/L, the concentration of adenosine triphosphate is 4-6mmol/L, TODB, the concentration of adenosine is 1.5-2.5mmol/L, TritonX-100, the concentration of triphosphate is 1.5-2.5mL/L, proclin300, the concentration of trehalose is 40-80g/L, and the concentration of Tris buffer solution with pH7.2 is 60-90 mmol/L.

Furthermore, in the reagent 2, the concentration of acetyl coenzyme A synthetase is 3KU/L, the concentration of catalase is 1200KU/L, the concentration of coenzyme A is 0.5g/L, the concentration of adenosine triphosphate is 5mmoL/L, TODB, the concentration is 2mmoL/L, TritonX-100, the concentration is 2mL/L, proclin300, the concentration is 0.2mL/L, the concentration of trehalose is 60g/L, and the concentration of Tris buffer solution with pH7.2 is 75 mmoL/L.

The free fatty acid determination kit also comprises a reagent 3; the reagent 3 comprises Tris buffer solution with pH7.2 containing acetyl coenzyme A oxidase, peroxidase, 4-AAP, TritonX-100, N-ethylmaleimide, 5-dimercapto-2, 2-dinitrobenzoic acid, 2-methyl-4-isothiazoline-3-ketone, sodium azide and trehalose.

Preferably, in the reagent 3, the concentration of acetyl coenzyme A oxidase is 20-120KU/L, the concentration of peroxidase is 20-200KU/L, the concentration of 4-AAP is 1-5mmol/L, TritonX-100, the concentration of 1-3mL/L, N-ethylmaleimide is 2-8mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 1-3mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 1-3mmol/L, the concentration of sodium azide is 3-5g/L, the concentration of trehalose is 20-100g/L, and the concentration of a buffer solution with pH7.2 is 50-100 mmol/L.

More preferably, in the reagent 3, the concentration of acetyl coenzyme A oxidase is 40-100KU/L, the concentration of peroxidase is 60-160KU/L, the concentration of 4-AAP is 2-4mmol/L, TritonX-100, the concentration of 1.5-2.5mL/L, N-ethylmaleimide is 4-6mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 1.5-2.5mmol/L, the concentration of 2-methyl-4-isothiazoline-3-one is 1.5-2.5mmol/L, the concentration of sodium azide is 3.5-4.5g/L, the concentration of trehalose is 40-80g/L, and the concentration of Tris buffer solution with pH of 7.2 is 60-90 mmol/L.

Furthermore, in the reagent 3, the concentration of acetyl coenzyme A oxidase is 70KU/L, the concentration of peroxidase is 110KU/L, the concentration of 4-AAP is 3mmol/L, TritonX-100, the concentration of 2mL/L, N-ethylmaleimide is 5mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 2mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 2mmol/L, the concentration of sodium azide is 4g/L, the concentration of trehalose is 60g/L, and the concentration of Tris buffer solution with pH7.2 is 75 mmol/L.

In some embodiments, the free fatty acid assay kit comprises the following components:

reagent 1: the concentration of the hydrogen peroxide is 2.0mmol/L, TritonX-100, the concentration is 2mL/L, and the concentration of the citric acid buffer solution with pH6.50 is 55 mmol/L; reagent 2: the concentration of acetyl coenzyme A synthetase is 3KU/L, the concentration of catalase is 1200KU/L, the concentration of coenzyme A is 0.5g/L, the concentration of adenosine triphosphate is 5mmoL/L, TODB, the concentration is 2mmoL/L, TritonX-100, the concentration is 2mL/L, proclin300, the concentration is 0.2mL/L, the concentration of trehalose is 60g/L, and 75mmoL/L of Tris buffer solution with the pH value of 7.2; reagent 3: the concentration of acetyl coenzyme A oxidase is 70KU/L, the concentration of peroxidase is 110KU/L, the concentration of 4-AAP is 3mmol/L, TritonX-100, the concentration of 2mL/L, N-ethylmaleimide is 5mmol/L, the concentration of 5, 5-dimercapto-2, 2-dinitrobenzoic acid is 2mmol/L, the concentration of 2-methyl-4-isothiazoline-3-ketone is 2mmol/L, the concentration of sodium azide is 4g/L, the concentration of trehalose is 60g/L, and the concentration of Tris buffer solution with pH value of 7.2 is 75 mmol/L.

The determination principle of the kit provided by the invention is as follows:

firstly, preparing a citric acid buffer solution containing hydrogen peroxide and TritonX-100 with pH6.50 as a reagent 1, and adding a serum sample according to a certain proportion. Reducing substances such as calcium dobesilate that may be contained in the sample are oxidized by hydrogen peroxide to lose the reducing ability.

And secondly, preparing a Tris buffer solution with the pH value of 7.2 containing acetyl coenzyme A synthetase, catalase, coenzyme A, adenosine triphosphate, TODB, TritonX-100, proclin300 and trehalose as a reagent 2, and adding the reagent 2 into a mixed solution of the reagent 1 in the first step and the serum sample to be detected according to a certain proportion. The hydrogen peroxide remaining in reagent 1 is decomposed by catalase in reagent 2; reacting free fatty acid in serum with coenzyme A under the action of acetyl coenzyme A synthetase to generate fatty acyl coenzyme A, and detecting the absorbance value A1 of 560 nm.

Thirdly, preparing a Tris buffer solution with the pH of 7.2, which contains acetyl coenzyme A oxidase, peroxidase, 4-AAP, TritonX-100, N-ethylmaleimide, 5-dimercapto-2, 2-dinitrobenzoic acid, 2-methyl-4-isothiazoline-3-ketone, sodium azide and trehalose, as a reagent 3, and adding the reagent 3 into the mixed solution obtained in the second step according to a certain proportion. The sodium azide in the reagent 3 completely inhibits the activity of catalase in the mixed solution; and (2) generating hydrogen peroxide by using fatty acyl coenzyme A in the mixed solution under the action of acetyl coenzyme A oxidase, reacting the hydrogen peroxide with 4-aminoantipyrine and TODB under the action of peroxidase to generate a purple compound, causing the absorbance value at 560nm to rise, detecting the absorbance value A2 at 560nm, and calculating the concentration C of free fatty acid after comparing with the standard.

In another aspect, the invention also provides a use method of the free fatty acid determination kit.

Specifically, the method comprises the following steps:

(1) preparing a citric acid buffer solution containing hydrogen peroxide and TritonX-100 and having a pH value of 6.50 as a reagent 1, and adding a serum sample according to a certain proportion;

(2) preparing a Tris buffer solution with the pH value of 7.2 containing acetyl coenzyme A synthetase, catalase, coenzyme A, adenosine triphosphate, TODB, TritonX-100, proclin300 and trehalose as a reagent 2, adding the reagent 2 into a mixed solution of the reagent 1 in the first step and a serum sample to be detected according to a certain proportion, and detecting an absorbance value AU,1 at 560 nm;

(3) preparing a pH7.2 Tris buffer solution containing acetyl coenzyme A oxidase, peroxidase, 4-AAP, TritonX-100, N-ethylmaleimide, 5-dimercapto-2, 2-dinitrobenzoic acid, 2-methyl-4-isothiazoline-3-ketone, sodium azide and trehalose as a reagent 3, adding the reagent 3 into the mixed solution obtained by the reaction in the second step according to a certain proportion, and detecting an absorbance value AU,2 at 560 nm; the free fatty acid concentration C was calculated after comparison with a standard control.

Specifically, the reaction conditions in the step (1) are 180-300 seconds of reaction in an environment at 37 ℃.

Specifically, the volume ratio of the serum sample to the reagent 1 in the step (1) is vpre to vpre I4: 140.

Specifically, the reaction conditions in the step (2) are 180-300 seconds of reaction in an environment at 37 ℃.

Specifically, the volume ratio of the serum sample to the reagents 1 and 2 in the step (2) is vpre: vpre 1: vpre 2: 4:140: 60.

Specifically, the reaction conditions in the step (3) are 180-300 seconds of reaction in an environment at 37 ℃.

Specifically, the volume ratio of the serum sample to the reagents 1 and 2 in the step (3) is vpre: vpre 1: vpre 2: vpre 3: 4:140:60: 45.

Specifically, the calculation formula of the concentration of the free fatty acid in the step (3) is as follows:

c ═ [ (AU, 2-AB, 2)/(AS,2-AB,2) ] × C standard NEFA.

Wherein AU,2 is the absorbance value of the sample tube measured in the step (3), AS,2 is the absorbance value measured by replacing the sample with the standard solution with the same volume in the step (3), AB,2 is the absorbance change value measured by replacing the sample with the deionized water with the same volume in the step (3), and C standard NEFA is the concentration of the standard solution NEFA.

The invention has the beneficial effects that:

(1) the invention provides a free fatty acid determination kit capable of eliminating interference of calcium dobesilate, and accuracy of a detection result is improved.

(2) The invention has simple operation, and can be developed on the clinical biochemical automatic analyzer which is widely used at present, thereby meeting the requirement of large-scale sample determination.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

The information on the sources of the reagents used in the examples is as follows:

reagent	Purchasing manufacturer/brand	Goods number
			3% hydrogen peroxide	SHANDONG LIRCON MEDICAL TECHNOLOGY INCORPORATED Co.	344945
TritonX-100	VWR International LLC	0694
			acetyl-CoA synthetase	SUZHOU YACOO SCIENCE Co.,Ltd.	Y0844
Coenzyme A	Sigma-Aldrich	C4282
			Adenosine triphosphate	Roche	10000116103
TODB	SUZHOU YACOO SCIENCE Co.,Ltd.	S0089
			TritonX-100	VWR International LLC	0694
proclin300	Sigma-Aldrich	48918-U
			Trehalose	Shanghai-sourced bioscience technology Co., Ltd	C2072
acetyl-CoA oxidase	BEIJING AMBITION BIOTECHNOLOGY Co.,Ltd.	Abt-P-116
			Peroxidase enzymes	TOYOBO CO.,LTD.	PEO-302
4-AAP	Sigma-Aldrich	101788366
			N-ethylmaleimide	SUZHOU YACOO SCIENCE Co.,Ltd.	D0024
5, 5-dimercapto-2, 2-dinitrobenzoic acid	SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd.	S19139
			2-methyl-4-isothiazolin-3-one	Sigma-Aldrich	73569
Sodium azide solution 1%	Changzhou Beiyuan Xin biotechnologyLimited Co.	BYX1469H

Example 1 free fatty acid assay kit capable of eliminating interference of calcium dobesilate

Including reagent 1, reagent 2 and reagent 3.

Reagent 1 comprises hydrogen peroxide with the concentration of 1.0mmol/L, TritonX-100 with the concentration of 1mL/L and citric acid buffer solution with the concentration of 30mmol/L and pH 6.50.

The reagent 2 comprises acetyl coenzyme A synthetase with the concentration of 1KU/L, catalase with the concentration of 600KU/L, coenzyme A with the concentration of 0.2g/L, adenosine triphosphate with the concentration of 2mmol/L, TODB with the concentration of 1mmol/L, proclin300 with the concentration of 1mL/L TritonX-100, trehalose with the concentration of 20g/L and Tris-HCl buffer solution with the concentration of 50mmol/L and the pH value of 7.20.

The reagent 3 comprises acetyl coenzyme A oxidase with the concentration of 20KU/L, peroxidase with the concentration of 20KU/L, 4-AAP with the concentration of 1mmol/L, TritonX-100 with the concentration of 1mL/L, N-ethylmaleimide with the concentration of 2mmol/L, 5-dimercapto-2, 2-dinitrobenzoic acid with the concentration of 1mmol/L, 2-methyl-4-isothiazoline-3-ketone with the concentration of 3g/L, sodium azide with the concentration of 20g/L, trehalose with the concentration of 20g/L and Tris-HCl buffer solution with the concentration of 50mmol/L and the pH value of 7.20.

When the sample liquid is measured, the adopted instrument is a BECKMAN LX20 full-automatic biochemical analyzer, the reaction temperature is 37 ℃, the sample volume is 4 mu L, the volume of the reagent 1 is 140 mu L, the volume of the reagent 2 is 60 mu L, the volume of the reagent 3 is 45 mu L, and the measurement main/auxiliary wavelength is 560/800 nm. Mixing the reagent 1 and the sample, and reacting for 180 seconds at the measuring temperature; adding the reagent 2, uniformly mixing, reacting for 180 seconds, and respectively reading absorbance values AB,1, AS,1 and AU,1 of a blank tube, a standard tube and a serum tube; and continuously adding the reagent 3, reacting for 180 seconds, respectively reading absorbance values AB,2, AS,2, AU and 2 of the blank tube, the standard tube and the serum tube, and automatically calculating the concentration of the free fatty acid in the sample by an instrument. The fitting mode of the calibration curve is linear fitting.

The influence of the calcium dobesilate with different concentrations on the determination of free fatty acid samples with different concentrations is respectively determined by the method, the interference rate is calculated, and the detection results are shown in the following table 1.

Table 1 shows the percentage deviation (%) of the free fatty acid concentration from the blank serum at different calcium dobesilate concentrations determined by the kit of this example.

TABLE 1

As can be seen from table 1, all percent deviations (%) were less than 10%.

Example 2 free fatty acid assay kit capable of eliminating interference of calcium dobesilate

Including reagent 1, reagent 2 and reagent 3.

Reagent 1 comprises hydrogen peroxide with the concentration of 3.0mmol/L, TritonX-100 with the concentration of 3mL/L and citric acid buffer solution with the concentration of 80mmol/L and pH 6.50.

The reagent 2 comprises acetyl coenzyme A synthetase with the concentration of 5KU/L, catalase with the concentration of 1800KU/L, coenzyme A with the concentration of 0.8g/L, adenosine triphosphate with the concentration of 8mmol/L, TODB with the concentration of 3mmol/L, TritonX-100 with the concentration of 3mL/L, proclin300 with the concentration of 2mL/L, trehalose with the concentration of 100g/L and Tris-HCl buffer solution with the concentration of 100mmol/L and the pH value of 7.20.

The reagent 3 comprises acetyl coenzyme A oxidase with the concentration of 120KU/L, peroxidase with the concentration of 200KU/L, 4-AAP with the concentration of 5mmol/L, TritonX-100 with the concentration of 3mL/L, N-ethylmaleimide with the concentration of 8mmol/L, 5-dimercapto-2, 2-dinitrobenzoic acid with the concentration of 3mmol/L, 2-methyl-4-isothiazoline-3-ketone with the concentration of 5g/L, sodium azide with the concentration of 100g/L, trehalose with the concentration of 100g/L and Tris-HCl buffer solution with the concentration of 100mmol/L and the pH value of 7.20.

When the sample liquid is measured, a BECKMAN CX4 full-automatic biochemical analyzer is adopted as an instrument, the reaction temperature is 37 ℃, the sample volume is 4 mu L, the volume of the reagent 1 is 140 mu L, the volume of the reagent 2 is 60 mu L, the volume of the reagent 3 is 45 mu L, and the main/auxiliary wavelength of the measurement is 560/800 nm. The reagent 1 and the sample react for 240 seconds at the measurement temperature after being mixed; adding the reagent 2, uniformly mixing, reacting for 240 seconds, and respectively reading absorbance values AB,1, AS,1 and AU,1 of a blank tube, a standard tube and a serum tube; and (3) continuously adding the reagent 3, reacting for 240 seconds, respectively reading absorbance values AB,2, AS,2, AU and 2 of the blank tube, the standard tube and the serum tube, and automatically calculating the concentration of the free fatty acid in the sample by an instrument. The fitting mode of the calibration curve is linear fitting.

The method is used for respectively measuring the influence of the calcium dobesilate with different concentrations on the measurement of free fatty acid samples with different concentrations without and with different concentrations, and calculating the interference rate. Table 2 is the percent deviation (%) of the free fatty acid concentration from the blank serum at different calcium dobesilate concentrations determined by the method described in this example.

TABLE 2

As can be seen from table 2, all percent deviations (%) were less than 10%.

Example 3 free fatty acid assay kit capable of eliminating interference of calcium dobesilate

Including reagent 1, reagent 2 and reagent 3.

Reagent 1 comprises hydrogen peroxide with the concentration of 2.0mmol/L, TritonX-100 with the concentration of 2mL/L and citric acid buffer solution with the concentration of 55mmol/L and pH 6.50.

The reagent 2 comprises acetyl coenzyme A synthetase with the concentration of 3KU/L, catalase with the concentration of 1200KU/L, coenzyme A with the concentration of 0.5g/L, adenosine triphosphate with the concentration of 5mmol/L, TODB with the concentration of 2mmol/L, proclin300 with the concentration of 2mL/L, trehalose with the concentration of 60g/L and Tris-HCl buffer solution with the concentration of 75mmol/L and the pH value of 7.20.

The reagent 3 comprises acetyl coenzyme A oxidase with the concentration of 70KU/L, peroxidase with the concentration of 110KU/L, 4-AAP with the concentration of 3mmol/L, TritonX-100 with the concentration of 2mL/L, N-ethylmaleimide with the concentration of 5mmol/L, 5-dimercapto-2, 2-dinitrobenzoic acid with the concentration of 2mmol/L, 2-methyl-4-isothiazoline-3-ketone with the concentration of 4g/L, sodium azide with the concentration of 60g/L, trehalose with the concentration of 75mmol/L and Tris-HCl buffer solution with the concentration of 75mmol/L and the pH value of 7.20.

When the sample solution was measured, the apparatus used was a Japan Shimadzu UV2201 ultraviolet-visible spectrophotometer, the reaction temperature was 37 ℃, the sample volume was 4. mu.L, the reagent 1 volume was 140. mu.L, the reagent 2 volume was 60. mu.L, the reagent 3 volume was 45. mu.L, and the measurement primary/secondary wavelength was 560/800 nm. The reagent 1 and the sample are mixed and then react for 300 seconds at the measuring temperature; adding a reagent 2, uniformly mixing, reacting for 300 seconds, and respectively reading absorbance values AB,1, AS,1 and AU,1 of a blank tube, a standard tube and a serum tube; and (3) continuously adding the reagent for reaction for 300 seconds, respectively reading absorbance values AB,2, AS,2, AU and 2 of the blank tube, the standard tube and the serum tube, and calculating the concentration of the free fatty acid in the sample. The fitting mode of the calibration curve is linear fitting.

The method is used for respectively measuring the influence of the calcium dobesilate with different concentrations on the measurement of free fatty acid samples with different concentrations without and with different concentrations, and calculating the interference rate. Table 3 is the percent deviation (%) of the free fatty acid concentration from the blank serum at different calcium dobesilate concentrations determined by the method described in this example.

TABLE 3

As can be seen from table 3, all percent deviations (%) were less than 10%.

Example 4 precision examination of the kit

A low-concentration (0.5mmol/L), medium-concentration (1.0mmol/L) and high-concentration (1.5mmol/L) free fatty acid sample is taken, the measurement is repeated for 20 times in the same day by using the kit disclosed by the invention in the embodiment 1-3, the CV in the batch is calculated, then the measurement is continuously performed for 20 days by using the same sample, and the CV in the batch is calculated, and the results are shown in tables 4-6.

TABLE 4

TABLE 5

	Mean value (in batch)	Intra-lot CV (%)	Mean value (between batches)	Batch CV (%)
					Example 1	1.00	2.2	1.02	2.8
Example 2	0.99	2.3	1.01	2.9
					Example 3	1.00	2.4	1.04	2.9

TABLE 6

	Mean value (in batch)	Intra-lot CV (%)	Mean value (between batches)	Batch CV (%)
					Example 1	1.52	1.9	0.52	2.3
Example 2	1.51	1.8	0.52	2.2
					Example 3	1.54	2.0	0.51	2.4

Example 5 stability study of the kit

The kits prepared in examples 1 to 3 of the present invention were left at 4 ℃ in a refrigerator for 12 months, and then NEFA mean values and CV values were measured according to the method of example 4, wherein the samples were medium concentration (1.0mmol/L) free fatty acid samples, and the results are shown in table 7.

TABLE 7

	Mean value (in batch)	Intra-lot CV (%)	Mean value (between batches)	Batch CV (%)
					Example 1	1.04	3.1	1.02	3.7
Example 2	1.03	3.3	1.01	3.8
					Example 3	1.05	3.5	1.04	3.9

Comparative example

Based on the technical solutions of examples 1 to 3, the differences are only that no hydrogen peroxide is added into the reagent 1, the instrument parameters and the detection steps are the same as those of corresponding examples 1 to 3, so as to obtain corresponding comparative examples 1 to 3 (comparative example 1 corresponds to example 1, comparative example 2 corresponds to example 2, and comparative example 3 corresponds to example 3), and the percentage deviations (%) between the concentration of the isolated fatty acid and the blank serum at different concentrations of calcium dobesilate measured in each proportion are respectively shown in tables 8 to 10:

TABLE 8 measurement results of comparative example 1

TABLE 9 measurement results of comparative example 2

TABLE 10 measurement results of comparative example 3

It can be seen that the percent deviation (%) for each scale is much greater than that of the examples.