Quality control liquid of low-density lipoprotein cholesterol and preparation method of quality control liquid

Abstract

The invention relates to a quality control liquid for low-density lipoprotein cholesterol and a preparation method of the quality control liquid. The quality control liquid comprises the following components in percentage by weight: 90-95% of defatted bovine serum, 0.005-0.05% of a cholesterol solution purified by ultracentrifugation, 1-3% of trehalose, 0.2-0.4% of EDTA-2K, 0.02-0.05% of vitamin E, 0.08-0.12% of a liquid biological preservative and 0.1-0.2% of glutathione. The invention has the following beneficial effects: 1, trehalose and vitamin E have a synergistic effect, and the stability at normal temperature reaches more than 12 months (CV is less than 3%); 2, a direct detection method, a precipitation method and a calculation method are adapted, and the deviation with a clinical sample is less than 5%; 3, the synthetic matrix reduces the risk of biogenic pollution, and is suitable for an automatic detection platform.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnostic reagents, in particular to a low-density lipoprotein cholesterol quality control solution and a preparation method thereof. BACKGROUND

[0002] Low-density lipoprotein cholesterol is a key indicator for cardiovascular disease risk assessment, and its detection accuracy directly affects clinical diagnosis. Existing quality control solutions have the following problems: 1. Low-density lipoprotein cholesterol has poor stability and is easily affected by oxidation or enzymatic hydrolysis; 2. Insufficient compatibility with different detection methods (such as direct method, Friedewald formula method); 3. Significant matrix effect, resulting in detection result deviation.

[0003] In the prior art, animal serum or synthetic lipoprotein mimics are often used, but it is difficult to simultaneously meet the long-term stability and multi-platform applicability requirements. SUMMARY

[0004] In summary, to overcome the shortcomings of the prior art, the present application solves the technical problem of providing a low-density lipoprotein cholesterol quality control solution and a preparation method thereof, which has high stability, is compatible with multiple detection methods, and has low matrix effect.

[0005] The technical solution of the present application to solve the above technical problems is as follows: a low-density lipoprotein cholesterol quality control solution, comprising the following components by weight percentage: defatted bovine serum 90-95%, ultracentrifugation purified cholesterol solution 0.005-0.05%, trehalose 1-3%, EDTA-2K 0.2-0.4%, vitamin E 0.02-0.05%, liquid biological preservative 0.08-0.12%, glutathione 0.1-0.2%.

[0006] On the basis of the above technical solution, the present application can also be improved as follows.

[0007] Further, the defatted bovine serum contains 20-50 g / L of albumin, and the pH is 7.2-7.6.

[0008] Further, the liquid biological preservative is proclin 300.

[0009] The preparation method of the above low-density lipoprotein cholesterol quality control solution, characterized in that, comprises the following steps: Step one, respectively, according to the following weight percentage of each component: defatted bovine serum 90-95%, cholesterol solution purified by ultracentrifugation 0.005-0.05%, trehalose 1-3%, EDTA-2K 0.2-0.4%, vitamin E 0.02-0.05%, liquid biological preservative 0.08-0.12%, glutathione 0.1-0.2%; Step two: defatted bovine serum is filtered through a filter membrane to remove endogenous lipoprotein; Step three: the cholesterol solution is mixed with trehalose, EDTA-2K, vitamin E and glutathione, and the mixture is obtained by shaking and incubating; Step four: the mixture is mixed with defatted bovine serum and the pH is adjusted; Step five: after sterile packaging, freeze-drying, adding liquid biological preservative during reconstitution, and packaging and storing.

[0010] Further, in step two, the defatted bovine serum is filtered through a 0.22 μm filter membrane.

[0011] Further, in step three, the shaking and incubation is carried out at 37℃ for 1-2 hours.

[0012] Further, in step four, the pH is adjusted to 7.4±0.1.

[0013] Further, in step five, the packaging and storage is carried out at 2-8℃.

[0014] The beneficial effects of the present application are: 1. Trehalose and vitamin E synergistically act, and the stability at room temperature is more than 12 months (CV<3%); 2. The bias with clinical samples is less than 5% by adapting direct detection method, precipitation method and calculation method; 3. The synthetic matrix reduces the risk of biological contamination and is suitable for automatic detection platform. DETAILED DESCRIPTION

[0015] The principles and characteristics of the present application are described below in conjunction with specific examples, which are used to explain the present application and are not used to limit the scope of the present application.

[0016] A preparation method of a low-density lipoprotein cholesterol quality control liquid, comprising the following steps: Step one, respectively, according to the following weight percentage of each component: defatted bovine serum 90-95% (containing 20-50 g / L of albumin, pH 7.2-7.6 buffer system), cholesterol solution purified by ultracentrifugation 0.005-0.05%, trehalose 1-3%, EDTA-2K 0.2-0.4%, vitamin E 0.02-0.05%, liquid biological preservative 0.08-0.12%, glutathione 0.1-0.2%; Step two: filter the defatted bovine serum through a 0.22 μm filter membrane to remove endogenous lipoprotein; Step three: mix the cholesterol solution with trehalose, EDTA-2K, vitamin E and glutathione, and incubate at 37°C for 1-2 hours to obtain a mixture; Step four: mix the mixture with defatted bovine serum and adjust the pH to 7.4±0.1; Step five: after sterile dispensing, freeze-dry and add a liquid biological preservative when reconstituted, and store at 2-8°C.

[0017] 1. Stability verification data Accelerated stability test Conditions: the quality control solution of the application was stored at 4°C, 25°C and 37°C, respectively, and the LDL-C concentration was detected regularly.

[0018] Results: 4°C for 12 months: concentration change rate ≤2.5% (CV value <1.8%); 25°C (room temperature) for 6 months: concentration change rate ≤3.2% (CV value <2.5%); 37°C accelerated test for 7 days: simulated long-term stability, concentration change rate <2% (compared with the initial value).

[0019] Comparison with prior art: the CV value of commercially available animal serum quality control solution is >5% after being stored at 25°C for 3 months, which is significantly higher than the 3.2% of the application.

[0020] Freeze-dried reconstitution stability After reconstitution of the freeze-dried product, the LDL-C concentration deviation was <1.5% after storage at 2-8°C for 30 days, meeting the requirements of clinical detection.

[0021] 2. Multi-method compatibility verification Correlation with different detection methods Samples: use the quality control solution of the application and 50 clinical serum samples for synchronous detection.

[0022] Method: compare the consistency of the results of direct detection method (D-LDL-C), Friedewald formula method, Sampson formula method and Vujovic formula method.

[0023] Data: Direct method vs. quality control solution: linear regression R 2 =0.992, slope 0.98±0.03; Formula deviation: Friedewald formula deviation ≤4.5%, Sampson formula deviation ≤3.8%, both within the clinically allowed range (±5%).

[0024] Instrument platform compatibility On Roche Cobas 8000, Beckman AU5800 and Siemens ADVIA 2400 platforms, the intra-batch precision CV was <2% and the inter-batch CV was <3%.

[0025] 3. Verification of reduced matrix effect Synthetic matrix vs. natural serum When the quality control solution (defatted bovine serum matrix) of this invention was tested in parallel with natural human serum, the matrix effect deviation was 2.1%-3.7%, which was significantly lower than the 5.8%-8.3% of commercially available synthetic matrix quality control solutions.

[0026] Immunological interference test: After adding common interfering agents (bilirubin ≤20 mg / dL, hemoglobin ≤500 mg / dL, triglycerides ≤1000 mg / dL), the recovery rate of LDL-C detection was 97%-103%, which meets the requirements of CLSI guidelines.

[0027] 4. Long-term clinical application data Multicenter study results A 12-month clinical validation was conducted at three top-tier hospitals, testing a total of 1200 samples. Precision: Total CV < 3% (target < 5%); Accuracy: Deviation from the reference method (ultracentrifugation) <3.5%; Stability monitoring: After opening, the concentration fluctuation was <2% when stored at 4℃ for 30 days.

[0028] 5. Comparative Analysis of Innovation Points

[0029] Among them, the prior art in the above table is the Chinese invention patent with application number 202410038792.5.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A quality control solution for low density lipoprotein cholesterol, characterized in that, The components include the following weight percentages: defatted bovine serum 90-95%, ultracentrifugally purified cholesterol solution 0.005-0.05%, trehalose 1-3%, EDTA-2K 0.2-0.4%, vitamin E 0.02-0.05%, liquid biological preservative 0.08-0.12%, and glutathione 0.1-0.2%.

2. The low density lipoprotein cholesterol control solution of claim 1, wherein, The defatted bovine serum contains 20-50 g / L of albumin and has a pH of 7.2-7.

6.

3. The low density lipoprotein cholesterol control solution of claim 1, wherein, The liquid biological preservative is proclin 300.

4. A method for preparing a quality control solution for low density lipoprotein cholesterol, characterized by, The method includes the following steps: Step one, each component is weighed according to the following weight percentages: defatted bovine serum 90-95%, ultracentrifugally purified cholesterol solution 0.005-0.05%, trehalose 1-3%, EDTA-2K 0.2-0.4%, vitamin E 0.02-0.05%, liquid biological preservative 0.08-0.12%, and glutathione 0.1-0.2%; Step two, the defatted bovine serum is filtered through a filter membrane to remove endogenous lipoprotein; Step three, the cholesterol solution is mixed with trehalose, EDTA-2K, vitamin E, and glutathione, and is shaken and incubated to obtain a mixture; Step four, the mixture is mixed with the defatted bovine serum and the pH is adjusted; Step five, after sterile dispensing, freeze-drying is performed, liquid biological preservative is added during reconstitution, and the product is stored after dispensing.

5. The quality control liquid for low-density lipoprotein cholesterol according to claim 4, characterized in that, In step two, the defatted bovine serum is filtered through a 0.22 μm filter membrane.

6. The quality control liquid for low density lipoprotein cholesterol according to claim 4, characterized by, In step three, the shaking and incubation is performed at 37°C for 1-2 hours.

7. The low density lipoprotein cholesterol control solution of claim 4, wherein, In step four, the pH is adjusted to 7.4±0.

1.

8. The quality control liquid for low density lipoprotein cholesterol according to claim 4, characterized by, In step five, the product is stored at 2-8°C after dispensing.

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