Apolipoprotein E determination kit and preparation method thereof

Abstract

The invention provides an apolipoprotein E determination kit and a preparation method thereof, the apolipoprotein E determination kit comprises a reagent 1, a reagent 2 and a calibrator / quality control product, the reagent 1 comprises the following components: a PIPES buffer solution, an ionic electrolyte, a polymer accelerator, a reaction accelerator, a surfactant, a stabilizer and a preservative; and the reagent 2 comprises the following components: an anti-ApoE antibody solution, a PIPES buffer solution, glycerol, an ionic electrolyte, a polymer accelerator, a reaction accelerator, a surfactant, a stabilizer and a preservative. The kit is optimized by adding EDTA2Na and mannitol, so that the opening test is more stable. And the preparation process of the reagent 2 and the PEG 6000 sample adding amount are adjusted. The kit is quick in reaction, high in accuracy and lower in blank absorbance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to an apolipoprotein E determination kit and a preparation method thereof. BACKGROUND

[0002] Apolipoprotein E (ApoE) is one of the main apolipoproteins in plasma, is a ligand for the binding of lipoproteins and receptors, and plays an important role in plasma lipid metabolism. ApoE is mainly synthesized by the liver, accounting for about 2 / 3-3 / 4 of the plasma content, and the liver parenchymal cells are the main site for the synthesis and secretion of ApoE and the main site for the secretion and metabolism of ApoE. As a cholesterol transport protein, ApoE plays an extremely important role in the formation of high-density lipoprotein, intermediate-density lipoprotein, very low-density lipoprotein and chylomicron and the maintenance of their stability. The determination of apolipoprotein E is used for evaluating and diagnosing lipid metabolism disorders, and has important significance for exploring the pathogenesis and clinical diagnosis of coronary heart disease and atherosclerosis.

[0003] At present, the methods for determining ApoE include chromatography, electrophoresis, chemiluminescence immunoassay and immunoturbidimetry. The detection principle of immunoturbidimetry is that, under the action of a dissociation agent R1, ApoE is separated from lipoprotein and then reacts with corresponding apolipoprotein antibody R2 to form an antigen-antibody complex, a certain turbidity is generated, and the turbidity is directly proportional to the content of the antigen when a certain amount of antibody exists. Immunoturbidimetry is welcomed due to its small sample amount, direct large-batch rapid detection and analysis on a full-automatic biochemical analyzer, and simple operation. The immunoturbidimetry reagent usually uses a polyethylene glycol (PEG) accelerator to accelerate the formation of the antigen-antibody complex, but too much PEG can cause a non-specific reaction, resulting in a too high blank absorbance; too little PEG cannot accelerate the reaction, and the test result is inaccurate. In clinical use of the reagent kit, a set of reagent cannot be used up in a short time, the reagent is placed in the automatic biochemical analyzer bin, and the oxidation reaction is easy to occur due to a long opening time, thereby resulting in an inaccurate test result. The determination of the ApoE content in the sample by immunoturbidimetry has a high blank absorbance and poor opening stability in the use process, which needs to be improved. SUMMARY

[0004] In view of the deficiencies in the prior art, the application aims to provide an apolipoprotein E determination kit with good opening stability and a lower blank absorbance.

[0005] In one aspect, the application provides an apolipoprotein E assay kit, which comprises reagent 1, reagent 2, a calibrator and a quality control, wherein the reagent 1 mainly comprises: PIPES buffer (pH 7.30) 15-25 mmol / L, ionic electrolyte, high molecular accelerator, reaction promoter, surfactant, stabilizer and preservative.

[0006] Further, the ionic electrolyte in the reagent 1 and the reagent 2 is NaCl, and the concentration is 1-10 g / L.

[0007] Further, the high molecular accelerator in the reagent 1 and the reagent 2 is PEG 6000, and the concentration is 10-30 g / L.

[0008] Further, the reaction promoter in the reagent 1 is polybrene, and the concentration is 1-10 mg / L.

[0009] Further, the surfactant in the reagent 1 and the reagent 2 is polyoxyethylene lauryl ether 0.5-2 g / L and Triton X-100 0.5-2 ml / L.

[0010] Further, the stabilizer in the reagent 1 is EDTA 2Na 2 g / L and mannitol 1 g / L.

[0011] Further, the preservative in the reagent 1 and the reagent 2 is proclin-300 0.1-0.5 ml / L and p-hydroxybenzoic acid 0.1-0.3 g / L.

[0012] Further, the concentration of PIPES buffer (pH 7.30) in the reagent 1 and the reagent 2 is 10-30 mmol / L.

[0013] Further, the concentration of goat anti-human ApoE antibody in the reagent 2 is 5-20%, and the concentration of glycerol is 5-15 g / L.

[0014] The application has the following beneficial effects: 1. The performance indicators of the kit are better than the requirements of the industry standard.

[0015] 2. By adding stabilizers EDTA 2Na and mannitol in the reagent, the main components in the reagent are effectively protected, so that the opening stability of the reagent is better.

[0016] 3. By optimizing the preparation process, adjusting the amount of high molecular accelerator PEG6000, accelerating the formation of antigen-antibody complex, without producing non-specific reaction. The blank absorbance of the prepared kit is reduced, and the reaction is rapid. DETAILED DESCRIPTION

[0017] The application will be further described below by way of examples, but the application is not limited to the examples.

[0018] Example 1 The application provides an ApoE detection kit, and the main components are as follows: Reagent 1: Piperazine-N,N'-di(2-ethanesulfonic acid) (PIPES) 25 mmol / L, EDTA 2Na 2 g / L, NaCl 8.766 g / L, pH value is adjusted to 7.30 by sodium hydroxide, PEG6000 60 g / L, polybrene 10 mg / L, mannitol 1 g / L, p-hydroxybenzoic acid 0.1 g / L, polyoxyethylene lauryl ether 1 g / L, gelatin 2 g / L, Triton X-100 1 ml / L, proclin 300 0.3 ml / L, and purified water is added to 1 L.

[0019] Reagent 2: First prepare 2 times the concentration of reagent 2, including piperazine-N,N'-di(2-ethanesulfonic acid) (PIPES) 50 mmol / L, NaCl 17.532 g / L, pH value is adjusted to 7.30 by sodium hydroxide, PEG6000 40 g / L, glycerol 20.2 g / L, p-hydroxybenzoic acid 0.2 g / L, polyoxyethylene lauryl ether 2 g / L, gelatin 4 g / L, Triton X-100 2 ml / L, proclin 300 0.8 ml / L, and goat anti-human ApoE antiserum 40%, and purified water is added to 1 L. After being placed in a 4°C refrigerator for 24 h, centrifugation is performed at 20°C and 10,000 r / min for 20 min. The supernatant is mixed with purified water at a ratio of 1:1 to obtain the required reagent 2.

[0020] After reagent 1 and reagent 2 are prepared, calibration, determination repeatability, analysis sensitivity, intra-assay precision, inter-assay precision, recovery rate and linearity are performed.

[0021] (1) Calibration results Calibration: five calibrators with concentrations of 0 mg / L, 15.75 mg / L, 31.5 mg / L, 63 mg / L and 126 mg / L are used to calibrate the kit of example 1. The determination results are as follows: (2) Repeatability: After the kit was calibrated, the quality control sample with a concentration of 40.00 mg / L was repeatedly determined for 5 times to analyze the repeatability.

[0022] Substance QC sample measured value (mg / L) Target value 40.00 Measurement 1 41.03 Measurement 2 40.19 Measurement 3 40.13 Measurement 4 39.76 Measurement 5 40.68 Average 40.36 Deviation (%) 0.89 (3) Intra-batch precision: After the kit of Example 1 was calibrated, the samples with low, medium and high concentrations of 15.0±3.0 (mg / L), 35.0±7.0 (mg / L) and 80.0±16.0 (mg / L) were determined for 20 times, respectively, to calculate the intra-batch precision of the kit.

[0023] (4) Inter-day precision: After the kit of Example 1 was calibrated, the samples with low, medium and high concentrations of 15.0±3.0 (mg / L), 35.0±7.0 (mg / L) and 80.0±16.0 (mg / L) were determined in the morning and afternoon from day 1 to day 10, respectively, to calculate the inter-day precision of the kit.

[0024] (5) Recovery rate determination: After Example 1 was calibrated, the human serum sample and the sample added with standard solution were determined to calculate the recovery rate of the kit.

[0025] (6) Linearity It is found in the present study that the ApoE immunoturbidimetry reagent has good performance. The relative deviation is 0.89% when the quality control sample with a concentration of 40.00 mg / L is determined. The intra-batch precision is within the range of 15.0 mg / L-80.0 mg / L, and the CV value is between 1.15%-3.00%. The inter-day precision is within 10 days, and the CV value is between 2.11%-4.03%. The recovery rate is 101.79%-104.43% when the standard solution with a concentration of 127.55 mg / L is added, and the recovery rate is 99.44%-106.08% when the standard solution with a concentration of 40.52 mg / L is added. The kit is within the linear range of 10.4 mg / L-123.0 mg / L, and the linear correlation coefficient r 2 =0.9999. The performance indicators of the kit are all better than the industry standard requirements.

[0026] A comparative example 1 is set up to investigate whether the opening stability of the kit of Example 1 is improved.

[0027] Comparative Example 1: The present application provides an apolipoprotein E detection kit, which mainly comprises the following components: The embodiment is different from example 1 in that EDTA2Na and mannitol are not added in reagent 1, and example 1 and comparative example 1 are calibrated. The calibration is performed at 0, 5, 10, 15, 20, 23, 26, 29, 32, 35, 38, and 41 days, respectively, and no recalibration is performed during the period. The results are compared, and the results are as follows: The test results show that for the reagent 1 kit, the low and medium value quality control test values deviate by more than 10% from the first day, and the target value deviation is also more than 10% after 20 days of opening. The low value quality control test value deviation of the reagent kit of example 1 is more than 10% after 35 days of opening, and the medium and high value quality control deviations are still controlled within 10%. The time delay of example 1 compared with comparative example 1 is 15 days. Therefore, the addition of EDTA2Na and mannitol in reagent 1 is beneficial to improve the opening stability of the reagent.

[0028] Comparative example 2 is set up to compare whether the absorbance of the reagent kit of example 1 is reduced.

[0029] The commonly used transmission immunoturbidimetric assay reagent currently uses polyethylene glycol (PEG 6000) accelerator to accelerate the formation of antigen-antibody complex, but too much PEG can cause non-specific reaction, resulting in high blank limit and inaccurate test results. The reagent components of comparative example 2 are the same as those of example 1, and the difference lies in the reagent 2 preparation process.

[0030] The reagent 2 preparation process is as follows: piperazine-N,N'-di(2-ethanesulfonic acid) (PIPES) 25 mmol / L, NaCl 8.766 g / L, pH value is adjusted to 7.30 by sodium hydroxide, PEG6000 20 g / L, glycerol 10.1 g / L, p-hydroxybenzoic acid 0.1 g / L, polyoxyethylene lauryl ether 1 g / L, gelatin 2 g / L, Triton X-100 1 ml / L, proclin 300 0.4 ml / L, goat anti-human ApoE antiserum 20%, and purified water is added to 1 L.

[0031] Example 1 and comparative example 2 are calibrated, test control products are tested, and blank absorbance is recorded. The results are compared, and the results are as follows: (1) Calibration results (2) Quality control results 　 Comparative Example 2 Example 1 Comparative Example 2 Example Target value (mg / L) 40.00 40.00 68.45 68.45 Measurement 1 43.60 41.03 72.11 69.03 Measurement 2 43.10 40.19 71.12 68.19 Measurement 3 51.49 40.13 72.21 68.43 Average 43.10 40.45 71.81 68.55 Deviation (%) 7.75% 1.12% 4.91% 0.15% (3) Blank absorbance Example Measurement 1 Measurement 2 Measurement 3 Average Comparative Example 2 0.0598 0.0504 0.0525 0.0542 Example 1 0.0168 0.0201 0.0195 0.0188 The experimental results show that the deviation of Comparative Example 2 is 7.75% when measuring the target value of 40 mg / L quality control sample, and the deviation is 4.91% when measuring the target value of 68.45 mg / L quality control sample, both of which are higher than the measured value of Example 1. The reagent blank absorbance of Comparative Example 2 is 0.0542, which is much higher than the reagent blank absorbance value of Example 1 kit. Therefore, the measured value deviation of the kit obtained in Example 1 is smaller, the blank absorbance is lower, and the performance is better than that of Comparative Example 2.

[0032] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An apolipoprotein E assay kit and a preparation method thereof, the kit comprising reagent 1, reagent 2, a calibrator and a quality control, the reagent 1 mainly comprising: PIPES buffer (pH 7.30) 15-25 mmol / L, ionic electrolyte, high molecular accelerator, reaction promoter, surfactant, stabilizer and preservative; the reagent 2 mainly comprising: PIPES buffer (pH 7.30), goat anti-human ApoE antibody, glycerol, ionic electrolyte, high molecular accelerator, surfactant and preservative; the calibrator and the quality control mainly comprising: phosphate buffered saline (PBS) (pH 7.40), ApoE antigen, stabilizer and preservative, and being subjected to freeze-drying treatment after preparation.

2. An apolipoprotein E assay kit and a method for preparing the same according to claim 1, wherein The ionic electrolyte in the reagent 1 and the reagent 2 is Nacl, and the concentration is 1-10 g / L.

3. An apolipoprotein E assay kit and a method for preparing the same according to claim 1, wherein the apolipoprotein E assay kit comprises an antibody against apolipoprotein E and a buffer solution. The high molecular accelerator in the reagent 1 and the reagent 2 is PEG 6000, and the concentration is 10-60 g / L.

4. An apolipoprotein E assay kit and a method of preparing the same according to claim 1, wherein the apolipoprotein E assay kit comprises an antibody against apolipoprotein E and a buffer solution. The reaction promoter in the reagent 1 is polybrene, and the concentration is 1-10 mg / L.

5. The apolipoprotein E assay kit and its preparation method as described in claim 1, characterized in that, The surfactant in the reagent 1 and the reagent 2 is polyoxyethylene lauryl ether 0.5-2 g / L and Triton X-100 0.5-2 ml / L.

6. An apolipoprotein E assay kit and a method for preparing the same according to claim 1, wherein the apolipoprotein E assay kit comprises an antibody against apolipoprotein E and a buffer solution. The stabilizer in the reagent 1 is EDTA 2Na 2 g / L and mannitol 1 g / L.

7. An apolipoprotein E assay kit and a method of preparing the same according to claim 1, wherein the apolipoprotein E assay kit comprises an antibody against apolipoprotein E and a buffer solution. The preservative in the reagent 1 and the reagent 2 is proclin-300 0.1-0.5 ml / L and p-hydroxybenzoic acid 0.1-0.3 g / L.

8. An apolipoprotein E assay kit and a method for preparing the same according to claim 1, wherein The PIPES buffer (pH 7.30) in the reagent 1 and the reagent 2 has a concentration of 10-30 mmol / L.

9. An apolipoprotein E assay kit and a method for preparing the same according to claim 1, wherein, The concentration of the goat anti-human ApoE antibody in the reagent 2 is 5-20%, and the concentration of glycerol is 5-15 g / L.

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