CN111534568A - Serum high density lipoprotein cholesterol determination kit - Google Patents
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Abstract
The invention provides a high density lipoprotein cholesterol determination kit, the kit contains reagent R1 and reagent R2; the reagent R1 contains the following components: 3-morpholine propanesulfonic acid (MOPS) buffer solution, alpha-cyclic glucan sulfate, pullulan, magnesium chloride, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium, surfactant and preservative; reagent R2: 3-morpholine propanesulfonic acid (MOPS) buffer solution, Cholesterol Esterase (CEH), Cholesterol Oxidase (COD), Peroxidase (POD), 4-aminoantipyrine, flavin adenine dinucleotide disodium, surfactant, stabilizer and preservative. The invention also provides a preparation method and application of the kit, and the kit is a liquid kit with strong stability, strong anti-interference capability, high accuracy and good repeatability.
Description
Technical Field
The invention relates to the technical field of biochemical reagent determination, in particular to a high-density lipoprotein cholesterol determination kit, and also relates to a preparation method and application of the high-density lipoprotein cholesterol determination kit.
Background
High density lipoprotein cholesterol (HDL-C) is mainly synthesized in liver, is an anti-atherosclerotic lipoprotein, and can transport cholesterol from extrahepatic tissue to liver for metabolism, and is discharged from bile. The high density lipoprotein can take up cholesterol from cell membrane, and is catalyzed by lecithin cholesterol acyltransferase to form cholesterol ester, and then the carried cholesterol ester is transferred to very low density lipoprotein and low density lipoprotein. The content of cholesterol in the high-density lipoprotein is relatively fixed, and about 20-30% of the total cholesterol content of a human body is contained. The level of plasma high density lipoprotein cholesterol (HDL-C) is negatively correlated with the occurrence of coronary heart disease, and the anti-atherosclerosis is realized mainly through the mechanisms of promoting reverse cholesterol transport, resisting oxidation, resisting inflammation and the like.
The highest clinical value of the increase in high density lipoprotein cholesterol is the ability to transfer foam cells from atherosclerotic plaques to the liver for excretion from the body, which is seen in primary high HDL-emia, and many longevity of this family are found. They may also be elevated in patients receiving estrogen, insulin or certain drugs (e.g., niacin, vitamin E, heparin, etc.). The reduction of high density lipoprotein cholesterol is commonly seen in cerebrovascular disease coronary heart disease, hypertriglyceridemia, liver function damage such as acute and chronic hepatitis, liver cirrhosis, liver cancer, diabetes, smoking, lack of exercise, etc., and the reduction can be used as a risk index of coronary heart disease.
The method for detecting the high-density lipoprotein cholesterol is mainly a direct enzyme method in a biochemical reagent, has the advantages of high specificity, simple and quick operation, accuracy and safety, capability of automatic analysis and lower cost, but the domestic kit in the prior art has the defects of poor stability, poor interference resistance and the like, and the invention improves the defects of the existing high-density lipoprotein cholesterol kit so as to meet the requirements of clinical detection and chemical analysis.
Disclosure of Invention
In order to solve the problems, the invention provides a high-density lipoprotein cholesterol determination kit, a preparation method and application thereof.
The invention is realized by the following technical scheme:
a high density lipoprotein cholesterol determination kit, the kit contains reagent R1 and reagent R2;
the reagent R1 contains the following components:
50-120 mmol/L of 3-morpholine propanesulfonic acid buffer solution;
0.1-0.5 mmol/L of alpha-cyclic glucan sulfate;
0.1-1 g/L of dextran sulfate;
0.1-1 g/L of pullulan;
1-2 mmol/L magnesium chloride;
0.1-0.5g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium;
0.1% -1% of a surfactant;
0.5-1 ml/L of preservative;
the reagent R2 contains the following components:
50-120 mmol/L of 3-morpholine propanesulfonic acid buffer solution;
cholesterol esterase 100-300U/L;
1-5 KU/L of cholesterol oxidase;
peroxidase is 10-50 KU/L;
0.1-1.0 mmol/L of 4-aminoantipyrine;
0.1-0.5g/L of flavin adenine dinucleotide disodium;
0.1% -1% of a surfactant;
1-10 g/L of stabilizer;
0.5-1 ml/L of preservative;
wherein the percentages are by volume.
Preferably, the pH of the reagents R1 and R2 is 6.5-7.5.
Preferably, the surfactant in the reagents R1 and R2 is selected from one or two of tween series and triton series.
Preferably, the stabilizer in the reagent R2 is Bovine Serum Albumin (BSA) or trehalose.
Preferably, the preservative in the reagents R1 and R2 is one or more of sodium azide, proclin300, MIT, thimerosal and gentamicin sulfate.
Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1. More preferably, the volume ratio of reagent R1 to reagent R2 is 3: 1.
The preparation method of the high-density lipoprotein cholesterol determination kit comprises the following steps: adding 3-morpholine propanesulfonic acid buffer solution into the reagents R1 and R2, adjusting the pH value by using hydrochloric acid or sodium hydroxide, wherein the pH values of the reagents R1 and R2 are 6.5-7.5, and adding other substances according to the proportion for dissolving to prepare the high-density lipoprotein cholesterol determination kit.
The invention also discloses the application of the high density lipoprotein cholesterol determination kit, which is used for determining the concentration of the high density lipoprotein cholesterol in serum for the purposes of non-disease diagnosis and treatment.
The kit adopts a direct enzyme method, and the reaction principle is that reagent 1 contains polyanion and surfactant, the polyanion is selectively combined with VLDL and LDL, COD and CEH in reagent 2 are inhibited from acting on VLDL-CH and LDL-CH, and then selectively acts on HDL-CH, and the absorbance at 546nm is detected by the action of CEH-COD-POD and is in direct proportion to the concentration of HDL-C. The reaction has specificity, the full-automatic biochemical analyzer is used for detection, the operation is simple, the automation degree is high, the human error can be reduced, and the method is suitable for most clinical laboratories.
Advantageous effects
1) The stable high-density lipoprotein cholesterol determination kit is a liquid double reagent, does not need to be prepared by redissolution, and can be directly used after opening a bottle.
2) Bovine serum albumin and trehalose are added into the reagent R2 to form a composite stabilizer, and the components have synergistic effect, so that the stability of the enzyme in the reagent is effectively improved, and the stability of the reagent is excellent.
3) The pullulan and the dextran sulfate are added into the reagent R1 for synergistic interaction, so that the dosage of the dextran sulfate is reduced, the interference of low-density lipoprotein, chylomicron and the like can be effectively avoided, the anti-interference capability of the reagent is greatly enhanced, and the stability of the reagent is improved to a certain degree.
4) By adding flavin adenine dinucleotide disodium as an enzyme prosthetic group into the reagent R2, the reaction of the enzyme can be assisted, the stability of the enzyme is improved, and the anti-interference capability and the stability of the reagent are improved.
5) The reagent has excellent performance indexes such as accuracy, repeatability, linear range and the like, is low in price and convenient to use, and is favorable for further popularization in the market.
Drawings
FIG. 1 is a correlation curve for the reagents of example 1 and comparative example 1;
FIG. 2 is a correlation curve for the reagents of comparative example 1 and comparative example 2;
FIG. 3 is a linear correlation curve of example 1;
FIG. 4 is a graph showing the change in concentration of the high density lipoprotein cholesterol assay reagent provided in example 1 and comparative examples 1, 2, 3, 4, and 5, which were subjected to the stability test.
Detailed Description
The invention is further illustrated by the following specific examples:
when the kit of the embodiment is used, the determination method is to use a micheli 800 full-automatic biochemical analyzer with double reagent functions, and perform determination by using a rate method, wherein the main wavelength is 546nm and the sub-wavelength is 700nm, and the operation is as follows:
adding 3 muL of physiological saline, a sample or a calibrator, adding 225 muL of R1 reagent, pre-incubating for 5min, and recording an absorbance value A1;
and adding 75 muL of R2 reagent, mixing uniformly, reacting for 5min, recording an absorbance value A2, and calculating delta A.
High density lipoprotein cholesterol content (mmol/L) = (Δ a sample ÷ Δ a calibrator) × calibrator concentration.
Sample requirements:
1. insoluble blood serum.
2. Sample stability: the specimen can be stored stably for 3 days at the temperature of 2-8 ℃ and for 2 weeks at the temperature of-20 ℃.
Example 1
A conventional high density lipoprotein cholesterol assay kit comprises a reagent R1 and a reagent R2.
The reagent R1 contains the following components:
50mmol/L of 3-morpholine propanesulfonic acid buffer solution;
0.5mmol/L alpha-cyclic dextran sulfate;
dextran sulfate 0.5 g/L;
0.5g/L of pullulan;
2 mmol/L of magnesium chloride;
0.5mmol/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium;
triton-1001%;
proclin300 1ml/L；
the reagent R2 contains the following components:
50mmol/L of 3-morpholine propanesulfonic acid buffer solution;
cholesterol esterase 100U/L;
cholesterol oxidase 3 KU/L;
0.5mmol/L of 4-aminoantipyrine;
0.3g/L of flavin adenine dinucleotide disodium;
5g/L of bovine serum albumin;
5g/L of trehalose;
triton-1001%;
proclin300 1ml/L；
wherein the percentages are by volume.
In the reagent R1 and the reagent R2 of the present embodiment, a 3-morpholinopropanesulfonic acid buffer solution is prepared first, hydrochloric acid or sodium hydroxide is used for adjusting the pH value, the pH values of the reagents R1 and R2 are adjusted to 7.0, and then other substances are added according to the proportion for dissolution to prepare the reagent for measuring the high density lipoprotein cholesterol.
The ratio of the reagent R1 to the reagent R2 is 3: 1.
Comparative example 1
Commercially available imported Sigma high density lipoprotein cholesterol assay kit.
Comparative example 2
A commercial domestic high-density lipoprotein cholesterol determination kit.
Comparative example 3
The kit is the same as that of example 1 except that the reagent R1 does not contain pullulan and dextran sulfate is 1 g/L.
Comparative example 4
The kit is the same as that of example 1 except that the reagent R1 does not contain dextran sulfate, and the pullulan content is 1 g/L.
Comparative example 5
The reagent kit is different from the reagent kit for measuring high density lipoprotein cholesterol in example 1 only in that the reagent R2 does not contain flavin adenine dinucleotide disodium, and the other steps are the same as those in example 1.
Performance verification
Test No.)
Correlation experiments: the experimental scheme is as follows: example 1, comparative example 1 and comparative example 2, 40 clinical serum samples were tested simultaneously, correlation analysis was performed on the two sets of test results, and a correlation coefficient r was calculated; the relative deviation (r) of 40 pairs of data was calculated using the test results of comparative example 1 as control values, respectively. It is required that r is not less than 0.990 and the relative deviation is not more than. + -. 10%.
The results are shown in Table 1, and a correlation curve was obtained for the reagents of example 1 and comparative example 1 (as shown in FIG. 1), and for the reagents of comparative example 1 and comparative example 2 (as shown in FIG. 2).
TABLE 1 correlation comparative experiment results
Table 2 correlation coefficients of comparative example 1 with example 1 and comparative example 2, respectively
As can be seen from Table 1 and FIG. 1, the maximum value of the serum test deviation of the kits of example 1 and comparative example 1 is 4.88%, the correlation coefficient of the two reagents is 0.9938, and the detection results of example 1 and comparative example 1 are very close to each other, so that the detection reagent of example 1 provided by the invention has good correlation with the imported detection reagent, and can completely replace the imported reagent; as can be seen from Table 1 and FIG. 2, the test results of comparative example 2 and comparative example 1 have larger deviation, and the correlation coefficient is 0.9716, which shows that the accuracy of the kit of the present invention is better than that of comparative example 2.
Test No. two
And (3) repeatability experiment: the median quality control (target value 1.40 +/-0.10 mmol/L) is respectively detected in example 1, 20 times of detection are carried out on each quality control product, and the average value, the standard deviation and the coefficient of variation are calculated according to 20 times of detection results. The results are shown in Table 3, and it can be seen from Table 3 that the detection values in example 1 are close to the target values, and have small standard deviation, small coefficient of variation and good repeatability.
TABLE 3 quality control test results
Experiment three
Linear experiments: taking a high-value sample of the high-density lipoprotein cholesterol sodium as 5.20mmol/L, diluting, preparing 6 samples with different concentrations, sequentially measuring the samples with the concentrations of 5.20mmol/L, 2.60mmol/L, 1.30mmol/L, 0.65mmol/L, 0.325mmol/L and 0mmol/L for three times respectively at each concentration level, and respectively taking the average value. The assay was performed using the reagents of example 1. The detection results are shown in the table:
TABLE 4 results of the test of the linear correlation verification experiment
As can be seen from Table 4 and FIG. 3, the linear variation of example 1 of the present invention with the dilution concentration is achieved, the linear correlation coefficient reaches 0.9997 and is greater than 0.990, which indicates that the linear range of example 1 is better, the sample can meet the requirements of clinical cases, and the present invention has important significance for clinical examination.
Experiment four
Thermal stability experiments: the high density lipoprotein cholesterol assay reagents provided in example 1 and comparative examples 1, 2, 3, 4, and 5 were subjected to a stability test according to the following protocol: the reagents provided in example 1 and comparative examples 1, 2, 3, 4 and 5 were put together in a 37 ℃ water bath, a quality control having a target value of 1.40. + -. 0.10mmol/L was measured every day, and the change in the measured value of the quality control was monitored.
TABLE 5 verification of thermal stability of reagents
As can be seen from Table 5 and FIG. 4, the reagent of example 1 provided by the present invention has substantially no change in the water bath condition at 37 ℃ for 10 days, and has good stability; the reagents of comparative examples 1, 2, 3, 4 and 5 are obviously changed from those of example 1 in 10 days under the condition of 37 ℃ water bath. The stability of the kit of example 1 is superior to the stability of the kits of comparative examples 1.2, 3, 4, 5. The reagent of the comparative example 3 and the reagent of the comparative example 4 show that the pullulan and the dextran sulfate can respectively act, the two components are synergistic, the stability of the reagent is improved together, and the reagent of the comparative example 5, flavin adenine dinucleotide disodium, also improves the stability of the reagent to a certain extent. The stable buffer solution system and a plurality of components jointly improve the stability of the high-density lipoprotein cholesterol determination kit.
Experiment five
Interference experiments: a high-density lipoprotein cholesterol sample is taken and divided into 3 equal parts, and fat milk (without high-density lipoprotein cholesterol) with different concentrations is added to ensure that the concentration of the fat milk in serum reaches the requirement of table 6. Then, the content of HDL cholesterol in the sample was measured using the reagents of example 1, comparative example 3, comparative example 4 and comparative example 5, respectively, and the results of the measurement of each group after adding different interfering substances are shown in Table 6.
TABLE 6 anti-interference verification results of reagents
As can be seen from Table 6, the reagent of example 1 showed no significant interference with the results of the test at increasing fat milk concentrations. The interference of the reagents of comparative example 1, comparative example 3, comparative example 4 and comparative example 5 is obviously enhanced along with the increase of the concentration of the fat milk. Comparative example 3 and comparative example 4 show that although double concentrations of single components pullulan and dextran sulfate were added, the interference rejection was less than that of the simultaneous addition of the two components. The two components of the pullulan and the dextran sulfate can mutually cooperate, complement and act together on complex interference components, so that the interference of interference substances on detection results is reduced. The reagent of comparative example 5 shows that flavin adenine dinucleotide disodium also improves the stability of the reagent to some extent. This shows that the anti-interference performance of the reagent of example 1 is significantly improved by optimizing the reaction buffer system and adding anti-interference components and protective agents for enzymes, which is superior to the reagent of comparative example 1 and meets the clinical requirements.
In conclusion, the high-density lipoprotein cholesterol detection reagent has better correlation with the reagent of the comparative example 1 in the clinical sample detection process, but is superior to the reagent of the comparative example 1 in the aspects of stability and anti-interference capability, provides a good development space for the kit, and simultaneously enhances the market competitiveness of the kit.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (8)
1. A high density lipoprotein cholesterol assay kit, characterized in that, the kit contains reagent R1 and reagent R2;
the reagent R1 contains the following components:
50-120 mmol/L of 3-morpholine propanesulfonic acid buffer solution;
0.1-0.5 mmol/L of alpha-cyclic glucan sulfate;
0.1-1 g/L of dextran sulfate;
0.1-1 g/L of pullulan;
1-2 mmol/L magnesium chloride;
0.1-0.5g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium;
0.1% -1% of a surfactant;
0.5-1 ml/L of preservative;
the reagent R2 contains the following components:
50-120 mmol/L of 3-morpholine propanesulfonic acid buffer solution;
cholesterol esterase 100-300U/L;
1-5 KU/L of cholesterol oxidase;
peroxidase is 10-50 KU/L;
0.1-1.0 mmol/L of aminoantipyrine;
0.1-0.5g/L of flavin adenine dinucleotide disodium;
0.1% -1% of a surfactant;
1-10 g/L of stabilizer;
0.5-1 ml/L of preservative;
wherein the percentages are by volume.
2. The high-density lipoprotein cholesterol assay kit according to claim 1, wherein the pH of the reagents R1 and R2 is 6.5-7.5.
3. The high density lipoprotein cholesterol assay kit of claim 1 wherein the surfactant in the reagents R1 and R2 is selected from one or two of tween series and triton series.
4. The kit for measuring high density lipoprotein cholesterol as claimed in claim 1, wherein the stabilizer in the reagent R2 is one or two of bovine serum albumin and trehalose.
5. The high density lipoprotein cholesterol assay kit of claim 1 wherein the preservative in the reagents R1 and R2 is one or more of sodium azide, proclin300, MIT, thimerosal, and gentamicin sulfate.
6. The kit according to claim 1, wherein the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1.
7. A method for preparing the high density lipoprotein cholesterol assay kit according to any one of claims 1 to 6, comprising the steps of: adding 3-morpholine propanesulfonic acid buffer solution into the reagents R1 and R2, adjusting the pH value by using hydrochloric acid or sodium hydroxide, wherein the pH values of the reagents R1 and R2 are 6.5-7.5, and adding other substances according to the proportion for dissolving to prepare the high-density lipoprotein cholesterol determination kit.
8. Use of the high density lipoprotein cholesterol assay kit according to any one of claims 1 to 7 for determining serum high density lipoprotein cholesterol concentrations for non-disease diagnostic and therapeutic purposes.
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| CN112695071A (en) * | 2020-12-16 | 2021-04-23 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
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| CN112695071A (en) * | 2020-12-16 | 2021-04-23 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
| CN112695071B (en) * | 2020-12-16 | 2022-12-30 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
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