阅读说明：本技术 一种游离脂肪酸的测定方法在制备试剂盒中的应用 (Application of free fatty acid determination method in preparation of kit ) 是由 潘利琴 于 2021-06-30 设计创作，主要内容包括：本发明提供了一种游离脂肪酸的测定方法在制备检测游离脂肪酸浓度的试剂和/或试剂盒中的应用。所述的测定方法中包括使用含有漆酶的试剂组合物消除样品中羟苯磺酸钙干扰,所述的含有漆酶的试剂直接与样本混合发生反应。所述的测定方法中还包括向含有漆酶的试剂直接与样本混合发生反应后的体系中添加与游离脂肪酸反应的试剂。本发明在测定游离脂肪酸时可特异性消除样本中羟苯磺酸钙的负干扰,提高了检测结果的准确度,并且可以应用于目前广泛使用的临床生化自动分析仪,从而达到大规模测定样本的要求。(The invention provides an application of a free fatty acid determination method in preparation of a reagent and/or a kit for detecting the concentration of free fatty acid. The determination method comprises the step of eliminating the interference of calcium dobesilate in a sample by using a laccase-containing reagent composition, wherein the laccase-containing reagent is directly mixed with the sample to react. The determination method also comprises the step of adding a reagent which reacts with the free fatty acid into a system after a reagent containing the laccase is directly mixed with the sample to react. The invention can specifically eliminate the negative interference of the calcium dobesilate in the sample when measuring the free fatty acid, improves the accuracy of the detection result, and can be applied to the clinical biochemical automatic analyzer which is widely used at present, thereby meeting the requirement of measuring the sample in a large scale.)

1. The application of a free fatty acid determination method in the preparation of a reagent and/or a kit for detecting the concentration of free fatty acid is characterized in that the determination method comprises the following steps: eliminating the interference of calcium dobesilate in a sample by using a reagent composition containing laccase, wherein the reagent composition containing laccase is directly mixed with a sample to react.

2. The use according to claim 1, wherein the concentration of laccase in the reagent composition is 0.5 to 15 KU/L.

3. The use of claim 2, wherein the reagent composition further comprises acetyl-coa synthetase at a concentration of 0.5-5KU/L, coa at a concentration of 0.3-1.0g/L, and adenosine triphosphate at a concentration of 2-8 mmoL/L.

4. The use of claim 3, wherein the reagent composition further comprises a Tris-HCl buffer at a pH of 7.20, wherein the concentration of the Tris-HCl buffer is 25 to 100 mmol/L.

5. The use according to claim 1, further comprising: adding a reagent which reacts with the free fatty acid, wherein the reagent which reacts with the free fatty acid is added into a system in which a reagent composition containing the laccase is directly mixed with a sample to react.

6. Use according to claim 5, wherein the reagent reactive with free fatty acids comprises: 10-100KU/L acetyl coenzyme A oxidase and 10-200KU/L peroxidase.

7. The use of claim 6, wherein the reagent for reacting with free fatty acid further comprises Tris-HCl buffer solution with pH 7.20, and the concentration of Tris-HCl buffer solution is 25-100 mmol/L.

8. The use of claim 7, wherein the reagent that reacts with free fatty acids further comprises 4-AAP, TritonX-100, N-ethylmaleimide, Proclin300 and trehalose.

9. Use according to claim 5, characterized in that it comprises the following steps:

s1, preparing a laccase reagent composition according to the proportion;

s2, mixing the reagent composition with the sample, and detecting a light absorption value as AU,1 after the reaction is finished;

s3, adding a reagent reacting with free fatty acid into the system after the reaction of S2, detecting the light absorption value as AU,2 after the reaction continues, and calculating the concentration of the free fatty acid in the sample according to the difference between AU,1 and AU, 2.

10. The use according to claim 9, wherein the dominant wavelength used for detecting the absorbance in steps S2 and S3 is 540-560 nm.

Technical Field

The invention belongs to the technical field of medical examination, and particularly relates to an application of a free fatty acid determination method in preparation of a free fatty acid determination kit.

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 in the prior art, the invention provides an application of a method for measuring free fatty acid capable of eliminating interference of calcium dobesilate in measurement of preparing free fatty acid. According to long-term research, the laccase can specifically oxidize a p-phenylene phenol ring in calcium dobesilate, so that negative interference on the determination of free fatty acid caused by the reaction of the p-phenylene phenol ring and hydrogen peroxide generated in the process is avoided, and the negative interference of the calcium dobesilate on the determination of the free fatty acid can be eliminated by adding the laccase in a reagent combination.

The terms:

4-APP: 4-aminoantipyrine;

TritonX-100: polyethylene glycol octyl phenyl ether;

TODB: n, N-bis (4-sulfobutyl) -3-methylaniline disodium salt.

In one aspect, the invention provides a method for determining free fatty acids.

The measuring method comprises the following steps: and (3) eliminating the interference of calcium dobesilate in the sample by using a reagent composition containing laccase, wherein the reagent containing laccase is directly mixed with the sample to react.

The concentration of the laccase in the reagent composition is 0.5-15 KU/L.

Preferably, the reagent composition also comprises acetyl coenzyme A synthetase with the concentration of 0.5-5KU/L, coenzyme A with the concentration of 0.3-1.0g/L and adenosine triphosphate with the concentration of 2-8 mmoL/L.

Preferably, the reagent composition also comprises a Tris-HCl buffer solution with the pH value of 7.20, and the concentration of the Tris-HCl buffer solution is 25-100 mmol/L.

Preferably, the reagent composition also comprises TODB, TritonX-100, Proclin300 and trehalose.

In some embodiments, the reagent composition includes: Tris-HCl buffer solution with pH 7.20 and concentration 30-100mmoL/L, laccase with concentration 10-50KU/L, acetyl coenzyme A synthetase with concentration 0.5-5KU/L, coenzyme A with concentration 0.3-1.0g/L, adenosine triphosphate with concentration 2-8mmoL/L, TODB with concentration 1-3mmoL/L, TritonX-100 with concentration 0.1-5mL/L, proclin300 with concentration 0.01-5mL/L, and trehalose with concentration 20-100 g/L.

As a preferred embodiment of the present invention, the reagent composition comprises Tris-HCl buffer solution with pH 7.20 and concentration of 50-80mmoL/L, laccase with concentration of 20-40KU/L, acetyl coenzyme A synthetase with concentration of 1.5-4KU/L, coenzyme A with concentration of 0.5-0.8g/L, adenosine triphosphate with concentration of 4-6mmoL, TODB with concentration of 1.5-2.5mmoL/L, TritonX-100 with concentration of 0.5-4.5mL/L, proclin300 with concentration of 0.02-2mL/L, and trehalose with concentration of 40-80 g/L.

The determination method further comprises the following steps: adding a reagent which reacts with free fatty acid into a system after a reagent containing laccase is directly mixed with a sample to react.

The reagent reacting with the free fatty acid comprises: 10-100KU/L acetyl coenzyme A oxidase and 10-200KU/L peroxidase.

Preferably, the reagent reacting with the free fatty acid also comprises Tris-HCl buffer solution with the pH value of 7.20, and the concentration of the Tris-HCl buffer solution is 25-100 mmol/L.

Preferably, the reagent reacting with free fatty acid also comprises 4-AAP, TritonX-100, N-ethylmaleimide, Proclin300 and trehalose.

Preferably, the reagent reacting with the free fatty acid comprises Tris-HCl buffer solution with the pH value of 7.20 and the concentration of 30-100mmol/L, acetyl coenzyme A oxidase with the concentration of 10-100KU/L, peroxidase with the concentration of 10-200KU/L, 4-AAP with the concentration of 1-5mmol/L, TritonX-100 with the concentration of 0.1-5mL/L, N-ethylmaleimide with the concentration of 0.1-1g/L, proclin300 with the concentration of 0.01-5mL/L and trehalose with the concentration of 20-100 g/L.

In some embodiments, the reagent reacting with free fatty acid comprises Tris-HCl buffer solution with pH 7.20 and concentration of 50-80mmol/L, acetyl coenzyme A oxidase with concentration of 30-80KU/L, peroxidase with concentration of 50-160KU/L, 4-AAP with concentration of 2-4mmol/L, TritonX-100 with concentration of 0.5-4.5mL/L, N-ethylmaleimide with concentration of 0.3-0.8g/L, proclin300 with concentration of 0.02-2mL/L, and trehalose with concentration of 40-80g g/L.

In a preferred embodiment of the present invention, the reagent reacting with free fatty acid comprises Tris-HCl buffer solution with pH 7.20 and concentration of 65mmol/L, acetyl coenzyme A oxidase with concentration of 55KU/L, peroxidase with concentration of 105KU/L, 4-AAP with concentration of 3mmol/L, TritonX-100 with concentration of 2.5mL/L, N-ethylmaleimide with concentration of 0.55g/L, proclin300 with concentration of 0.2mL/L, and trehalose with concentration of 60g g/L.

Preferably, the assay method comprises the steps of:

s1, preparing a laccase-containing reagent composition in proportion;

s2, mixing the reagent composition with the sample, and detecting a light absorption value as AU,1 after the reaction is finished;

s3, adding a reagent reacting with free fatty acid into the system after the reaction of S2, detecting the light absorption value as AU,2 after the reaction continues, and calculating the concentration of the free fatty acid in the sample according to the difference between AU,1 and AU, 2.

The dominant wavelength used for detecting the absorbance in the steps S2 and S3 is 540-560 nm.

Preferably, the absorbance detected in steps S2 and S3 is detected by a fully automatic biochemical analyzer.

Preferably, the main wavelength of the reaction carried out by the full-automatic biochemical analyzer is 540-560nm, and the secondary wavelength is 700 nm.

Preferably, the reaction temperature of the fully automatic biochemical analyzer is 37 ℃. + -. 1 ℃.

Preferably, the reaction time in the steps S2 and S3 is 3-5 min.

Preferably, the volume ratio of the sample to the laccase containing reagent composition is 1: 40-60.

Preferably, the volume ratio of sample to reagent that reacts with free fatty acid is 1: 10-15.

The principle of free fatty acid determination by adopting the reagent combination is as follows:

firstly, under the action of a laccase-containing reagent composition, free fatty acid in human serum and coenzyme A react under the action of acetyl coenzyme A synthetase to generate fatty acyl coenzyme A;

in the second step, fatty acyl-CoA is reacted with acetyl-CoA oxidase to form H₂O₂Then, a colored substance is generated by the Trinder's reaction under the action of peroxidase, and the concentration of free fatty acid is detected.

In another aspect, the invention provides the application of the aforementioned method for determining free fatty acid in the preparation of a reagent and/or a kit for detecting the concentration of free fatty acid.

The reagent is used for detecting the concentration of free fatty acid by the method.

The kit comprises the laccase-containing reagent composition.

The kit also comprises the reagent which reacts with the free fatty acid.

The invention has the beneficial effects that:

(1) the method for determining the free fatty acid capable of eliminating the interference of the calcium dobesilate has high accuracy of detection results.

(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 source information for each reagent in the examples is as follows:

example 1A method for measuring free fatty acid

The laccase containing reagent composition of this example is reagent composition (1); the reagent that reacts with the free fatty acid is reagent composition (2).

The reagent composition (1) comprises the following specific components in final concentration:

laccase with a concentration of 10KU/L, acetyl coenzyme A synthetase with a concentration of 0.5KU/L, coenzyme A with a concentration of 0.3g/L, adenosine triphosphate with a concentration of 2mmoL/L, TODB with a concentration of 1mmoL/L, TritonX-100 with a concentration of 0.1mL/L, proclin300 with a concentration of 0.01mL/L, trehalose with a concentration of 20g/L, Tris-HCl buffer with a concentration of 30mmoL/L and a pH of 7.20.

The reagent composition (2) comprises the following specific components in final concentration:

acetyl coenzyme A oxidase with the concentration of 10KU/L, peroxidase with the concentration of 10KU/L, 4-AAP with the concentration of 1mmol/L, TritonX-100 with the concentration of 0.1mL/L, N-ethylmaleimide with the concentration of 0.1g/L, proclin300 with the concentration of 0.01mL/L, trehalose with the concentration of 20g/L, and Tris-HCl buffer with the concentration of 30mmol/L and the pH value of 7.20.

The reagent combination is adopted for detection, an instrument adopted is a BECKMAN LX20 full-automatic biochemical analyzer, the reaction temperature is 37 ℃, the sample volume is 4 muL, the volume of the reagent (1) is 200 muL, the volume of the reagent (2) is 50 muL, the main/auxiliary wavelength of the measurement is 560/700nm, the measurement method is a two-point end point method, and the fitting mode of a calibration curve is linear fitting.

The detection steps are as follows:

s1, preparing a reagent composition (1) and a reagent composition (2) according to the proportion respectively;

mixing the S2 reagent composition (1) and a serum sample, reacting for 5 minutes at a measuring temperature, and respectively reading absorbance values AB,1, AS,1 and AU,1 of a blank tube, a standard tube and a serum tube;

and S3, adding the reagent composition (2), continuing to react for 5 minutes, reading the absorbance values AB,2, AS,2 and AU,2 of the blank tube, the standard tube and the serum tube respectively, and automatically calculating the concentration of the free fatty acid in the sample by the instrument. The concentrations of free fatty acid samples with low concentration (0.5mmol/L), medium concentration (1.0mmol/L) and high concentration (1.5mmol/L) were measured without and with different concentrations of calcium dobesilate, respectively, and the interference rate was calculated and the detection results are shown in Table 1 below.

Table 1 shows 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 1

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

Example 2 method for measuring free fatty acid

The laccase containing reagent composition of this example is reagent composition (1); the reagent that reacts with the free fatty acid is reagent composition (2).

The reagent composition (1) comprises the following specific components in final concentration:

50KU/L laccase, 5KU/L acetyl coenzyme A synthetase, 1.0g/L coenzyme A, 8mmoL/L adenosine triphosphate, 3mmoL/L TODB, 5mL/L TritonX-100, 5mL/L proclin300, 100g/L trehalose, and 30mmoL/L Tris-HCl buffer solution with pH of 7.20.

The reagent composition (2) comprises the following specific components in final concentration:

10KU/L of acetyl-CoA oxidase, 100KU/L of acetyl-CoA oxidase, 200KU/L of peroxidase, 5mmol/L of 4-AAP, 5mL/L of TritonX-100, 1g/L of N-ethylmaleimide, 5mL/L of proclin300, 100g/L of trehalose, 30mmol/L of Tris-HCl buffer solution with pH 7.20.

The reagent combination is adopted for detection, an instrument adopted is a BECKMANAU5400 full-automatic biochemical analyzer, the reaction temperature is 37 ℃, the sample volume is 4 mu L, the volume of the reagent (1) is 200 mu L, the volume of the reagent (2) is 50 mu L, the main/auxiliary wavelength of the measurement is 540/700nm, the measurement method is a two-point end point method, and the fitting mode of a calibration curve is linear fitting.

The procedure was as in example 1.

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 method for measuring free fatty acid

The laccase containing reagent composition of this example is reagent composition (1); the reagent that reacts with the free fatty acid is reagent composition (2).

The reagent composition (1) comprises the following specific components in final concentration:

30KU/L laccase, 2.75KU/L acetyl coenzyme A synthetase, 0.65g/L coenzyme A, 5mmoL/L adenosine triphosphate, 2mmoL/L TODB, 2.5mL/L TritonX-1000.2mL/L proclin300, 60g/L trehalose, and 30mmoL/L Tris-HCl buffer solution with pH of 7.20.

The reagent composition (2) comprises the following specific components in final concentration:

55KU/L of acetyl coenzyme A oxidase, 105KU/L of peroxidase, 3mmol/L of 4-AAP, 2.5mL/L of TritonX-100, 0.55g/L of N-ethylmaleimide, 0.2mL/L of proclin300, 60g g/L of trehalose, and 30mmol/L of Tris-HCl buffer solution with pH 7.20.

The instrument parameters and detection procedure were the same as in example 1.

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 kit for measuring free fatty acid

The kit of this example includes reagent composition (1) and reagent composition (2).

The reagent composition (1) comprises the following specific components in final concentration:

laccase at a concentration of 10KU/L, acetyl-CoA synthetase at a concentration of 0.5KU/L, coenzyme A at a concentration of 0.3g/L, adenosine triphosphate at a concentration of 2mmol/L, TODB at a concentration of 1mmol/L, TritonX-100 at a concentration of 0.1mL/L, proclin300 at a concentration of 0.01mL/L, trehalose at a concentration of 20g/L, Tris-HCl buffer at a concentration of 30mmol/L and a pH of 7.20.

The reagent composition (2) comprises the following specific components in final concentration:

acetyl coenzyme A oxidase with the concentration of 10KU/L, peroxidase with the concentration of 10KU/L, 4-AAP with the concentration of 1mmol/L, TritonX-100 with the concentration of 0.1mL/L, N-ethylmaleimide with the concentration of 0.1g/L, proclin300 with the concentration of 0.01mL/L, trehalose with the concentration of 20g/L, and Tris-HCl buffer with the concentration of 30mmol/L and the pH value of 7.20.

Comparative example

Based on the technical solutions of examples 1 to 3, the differences are only that laccase is not added in the reagent composition (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 concentrations of the isolated fatty acids and the blank serum at different calcium dobesilate concentrations measured in each pair of proportions are respectively shown in tables 4 to 6:

table 4 percent deviation (%) -of the concentrations of free fatty acids from the blank serum at different calcium dobesilate concentrations determined in comparative example 1

TABLE 5 percent deviation (%) -of the concentrations of free fatty acids from the blank serum at different calcium dobesilate concentrations determined in comparative example 2

TABLE 6 percent deviation (%) -of the concentrations of free fatty acids from the blank serum at different calcium dobesilate concentrations determined in comparative example 3

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

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.