A stable lipoprotein-associated phospholipase A2 assay kit

Abstract

The invention belongs to the technical field of clinical in vitro detection reagents, and discloses a stable lipoprotein-associated phospholipase A2 assay kit, comprising reagent R1, reagent R2 and a calibrator, wherein the components in reagent R1 include a buffer and a preservative, and the components in reagent R2 include a buffer, a lipoprotein-associated phospholipase A2 substrate, a preservative, a stabilizer and a surfactant; wherein the concentration of the stabilizer is 1-5 g / L, and the stabilizer is selected from one or more of amino acids, sugars, polyethylene glycol, hydroxyethyl starch and beta-cyclodextrin. The kit has good storage stability.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical in vitro detection reagents, and in particular relates to a stable lipoprotein-associated phospholipase A2 determination kit. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is secreted by macrophages, T cells and mast cells in the vascular intima. Lp-PLA2 expression is upregulated in atherosclerotic plaques and is strongly expressed in macrophages in the fibrous cap of vulnerable plaques.

[0004] Lp-PLA2 released into the blood circulation is mainly bound to lipoproteins rich in apolipoprotein (Apo) B. In patients with atherosclerotic diseases, Lp-PLA2 levels are positively correlated with LDL subfraction levels.

[0005] Commercially available lipoprotein-associated phospholipase A2 assay kits generally have the advantages of high precision and specificity analysis, and do not require expensive equipment for detection, and are widely promoted in clinical practice. However, existing lipoprotein-associated phospholipase A2 assay kits have the problem of poor stability and short storage time, which easily affects the accuracy of detection. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a stable lipoprotein-associated phospholipase A2 assay kit. Compared with conventional kits, the kit has better accuracy, repeatability and stability, and is more conducive to the clinical promotion of the kit.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A stable lipoprotein-associated phospholipase A2 assay kit comprises a reagent R1, a reagent R2 and a calibrator, wherein the components in the reagent R1 comprise a buffer and a preservative, and the components in the reagent R2 comprise a buffer, a lipoprotein-associated phospholipase A2 substrate, a preservative, a stabilizer and a surfactant;

[0009] Among them, the concentration of the stabilizer is 1-5g / L, and the stabilizer is selected from one or more of amino acids (glycine, arginine, L-proline, etc.), sugars (sucrose, trehalose, etc.), polyethylene glycol (PEG200, PEG400, PEG600, PEG800, PEG6000, PEG8000, etc.), Pluoronic F-68, hydroxyethyl starch and β-cyclodextrin.

[0010] The beneficial effects of the present invention are as follows:

[0011] In the above lipoprotein-associated phospholipase A2 detection kit, glycine and Pluoronic F-68 can reduce the conformation or colloidal stability of the enzyme protein, β-cyclodextrin can enhance the chemical modification of the protein and increase the half-life of the protein, and trehalose can protect the thermal stability of the enzyme protein;

[0012] By adding a stabilizer composed of one or more of casone, glycine, trehalose, Pluoronic F-68 and beta-cyclodextrin into the reagent, the stability of the lipoprotein-associated phospholipase A2 detection kit can be effectively improved and its storage time can be extended. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 is a correlation diagram of the test results of the accuracy verification experiment of Example 1 of the present invention and the comparative example;

[0015] Figure 2 This is a correlation diagram of the accuracy verification test results of Example 2 and the comparative example;

[0016] Figure 3 This is a correlation diagram of the accuracy verification test results of Example 3 and the comparative example;

[0017] Figure 4 This is a correlation diagram of the accuracy verification test results of Example 4 and the comparative example;

[0018] Figure 5 This is a correlation diagram of the accuracy verification test results of Example 5 and the comparative example;

[0019] Figure 6 This is a correlation diagram of the accuracy verification test results of Example 6 and the comparative example;

[0020] Figure 7 This is a correlation diagram of the accuracy verification test results of Example 7 and the comparative example;

[0021] Figure 8 This is a correlation diagram of the accuracy verification test results of Example 8 and the comparative example;

[0022] Fig. 9 This is a correlation diagram of the accuracy verification test results of Example 9 and the comparative example;

[0023] Fig.10 This is a correlation diagram of the accuracy verification test results of Example 10 and the comparative example;

[0024] Fig.11 This is a correlation diagram of the accuracy verification test results of Example 11 and the comparative example;

[0025] Fig.12 It is a correlation diagram of the accuracy verification experimental test results of Example 12 and the comparative example. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] A stable lipoprotein-associated phospholipase A2 assay kit comprises a reagent R1, a reagent R2 and a calibrator, wherein the components in the reagent R1 comprise a buffer and a preservative, and the components in the reagent R2 comprise a buffer, a lipoprotein-associated phospholipase A2 substrate, a preservative, a stabilizer and a surfactant;

[0028] The concentration of the stabilizer is 1-5 g / L, and the stabilizer is selected from one or more of amino acids, sugars, polyethylene glycol, Pluoronic F-68, hydroxyethyl starch and β-cyclodextrin.

[0029] In some embodiments, the amino acid is glycine, arginine, or L-proline;

[0030] The sugar is sucrose or trehalose;

[0031] The polyethylene glycol is PEG200, PEG400, PEG600, PEG800, PEG6000 or PEG8000.

[0032] In some embodiments, the stabilizer is a mixture of at least three of amino acids, sugars, polyethylene glycol, hydroxyethyl starch and β-cyclodextrin.

[0033] Preferably, the stabilizer is a mixture of at least four of amino acids, sugars, polyethylene glycol, hydroxyethyl starch, Pluoronic F-68 and β-cyclodextrin.

[0034] Further preferably, the stabilizer is a mixture of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin, and the concentrations of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin are 0.8-1.2 g / L, 0.8-1.2 g / L, 0.08-0.12 g / L and 0.8-1.2 g / L, respectively.

[0035] Glycine and Pluoronic F-68 can reduce the conformation or colloidal stability of enzyme proteins, β-cyclodextrin can enhance the chemical modification of proteins and increase the half-life of proteins, and trehalose can protect the thermal stability of enzyme proteins. When these four substances are combined in a specific proportion to prepare a stabilizer, the stability of the test kit can be effectively improved. The coefficient of variation of repeated tests of the prepared test kit is smaller and can be stored for a better time.

[0036] In some embodiments, the concentration of the buffer of reagent R1 is 0.1-0.2 mol / L, and the concentration of the buffer of reagent R2 is 0.05-0.1 mol / L; the pH values ​​of both buffers are 7.0.

[0037] Preferably, the buffer is selected from one or more of phosphate, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), boric acid, citric acid, carbonate, Tris and the like buffers.

[0038] In some embodiments, the preservative is selected from one or more of kasone, sodium azide, proclin 300, sodium benzoate or phenol.

[0039] Preferably, the concentration of the preservative in reagent R1 and reagent R2 is 1-5 g / L.

[0040] In some embodiments, the surfactant is Pluoronic F-68, Tweens or Triton;

[0041] Tweens is Tween20, Tween60 or Tween80;

[0042] The Triton is Triton X-100 or Triton X-405.

[0043] Preferably, the concentration of the surfactant is 0.1-0.5 g / L.

[0044] In some embodiments, the concentration of the lipoprotein-associated phospholipase A2 substrate is 10-30 mmol / L.

[0045] Example 1

[0046] A stable lipoprotein-associated phospholipase A2 detection kit comprises reagents R1 and R2.

[0047] The composition and content of reagent R1 are:

[0048] Phosphate (pH7.0) 0.1mol / L

[0049] Sodium azide 1g / L

[0050] The composition and content of reagent R2 are:

[0051]

[0052]

[0053] When in use, the determination method is to use an automatic biochemical analyzer, such as Hitachi 7180 automatic analyzer, and the operation is as follows:

[0054] Add 1.5 μl of physiological saline, sample or calibrator, then add 120 μl of reagent R1 and incubate for 5 min; add 40 μl of reagent R2, measure the initial absorbance A1 after 60 seconds, and then incubate for 150 seconds, read the absorbance A2, and calculate ΔA / min.

[0055] Example 2

[0056] A stable lipoprotein-associated phospholipase A2 detection kit comprises reagents R1 and R2.

[0057] The composition and content of reagent R1 are:

[0058] HEPES (pH 7.0) 0.15 mol / L

[0059] Kason 3g / L

[0060] The composition and content of reagent R2 are:

[0061]

[0062] The detection method is the same as in Example 1.

[0063] Example 3

[0064] A stable lipoprotein-associated phospholipase A2 detection kit comprises reagents R1 and R2.

[0065] The composition and content of reagent R1 are:

[0066] Tris (pH7.0) 0.2 mol / L

[0067] proclin 300 5g / L

[0068] The composition and content of reagent R2 are:

[0069]

[0070] The detection method is the same as in Example 1.

[0071] Example 4

[0072] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L, trehalose 1 g / L, Pluoronic F-68 0.1 g / L.

[0073] Example 5

[0074] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L, trehalose 1 g / L.

[0075] Example 6

[0076] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L.

[0077] Example 7

[0078] The difference from Example 1 is that the composition of the stabilizer is: trehalose 1 g / L.

[0079] Example 8

[0080] The difference from Example 1 is that the composition of the stabilizer is: 1 g / L of β-cyclodextrin.

[0081] Example 9

[0082] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L, sucrose 1 g / L.

[0083] Example 10

[0084] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L, sucrose 1 g / L, PEG200 3 g / L.

[0085] Embodiment 11

[0086] The difference from Example 1 is that the composition of the stabilizer is: L-proline 1 g / L, sucrose 1 g / L, PEG200 3 g / L, and hydroxyethyl starch 1 g / L.

[0087] Example 12

[0088] The difference from Example 1 is that the composition of the stabilizer is: glycine 1 g / L, trehalose 1 g / L, Pluoronic F-68 0.1 g / L, hydroxyethyl starch 1 g / L, and β-cyclodextrin 1 g / L.

[0089] Comparative Example

[0090] The comparative example is a lipoprotein-associated phospholipase A2 detection kit recognized in the market.

[0091] Reagent R1 is composed of:

[0092] HEPES buffer 200mmol / L

[0093] Reagent R2 consists of:

[0094] Citric acid buffer 20mmol / L

[0095] Lipoprotein-associated phospholipase A2 substrate 30mmol / L

[0096] The detection method is the same as in Example 1.

[0097] The analytical performance of the kits prepared in the above comparative examples and embodiments was verified by an accuracy experiment.

[0098] Accuracy verification experiment:

[0099] The kits of Examples 1-12 were used as the experimental group, and the lipoprotein-associated phospholipase A2 kit with excellent accuracy recognized in the market was used as the control group for comparative experiments. 40 samples were tested. The test results are as follows: Figure 1-Figure 12 .

[0100] pass Figure 1-Figure 12 The test data show that the linear correlation coefficient r between the test results of the test kits of Examples 1-12 and the test kits of the comparative example is 2 The correlation is good, indicating that the kit of the present invention is highly consistent with the commercially recognized lipoprotein-associated phospholipase A2 detection kit with excellent accuracy, which proves that the various other ingredients added to the kit of the present invention will not affect its accuracy, and the kit still maintains good accuracy.

[0101] Repeatability verification experiment

[0102] The comparative example was used as the control group, and the reagents of Examples 1-12 were tested against each other. The quality control product (target value was 335 U / L) was tested respectively, and the test was repeated 10 times. The coefficient of variation was calculated. The test results are shown in Tables 1-1, 1-2 and 1-3.

[0103] Table 1-1 Kit repeatability test results

[0104]

[0105]

[0106] Table 1-2 Kit repeatability test results

[0107]

[0108] Table 1-3 Kit repeatability test results

[0109]

[0110] It can be seen from Table 1-1, Table 1-2 and Table 1-3 that the repeatability CV values ​​of the reagents obtained in the above Examples 1-12 are all less than 2%, which is better than the reagents in the control group.

[0111] Stability verification experiment

[0112] The reagents were stored at 2°C to 8°C in a light-proof environment without corrosive gases, and the stability of the 12 reagents was tested. The same sample of the 12 reagents was selected for testing three times per month, and the average value was taken and compared with the test results of the fresh comparative example 1 reagent to determine the stability time of the reagent. The test data are shown in Table 2-1, Table 2-2 and Table 2-3.

[0113] Table 2-1 Stability verification test results

[0114]

[0115] Table 2-2 Stability verification test results

[0116]

[0117] Table 2-3 Stability verification test results

[0118]

[0119] The experimental results show that the reagent of Comparative Example 1 is stable when stored for 13 months in a light-proof environment at 2°C to 8°C without corrosive gases, while the reagents of Examples 1-12 are stable when stored for 18 months in a light-proof environment at 2°C to 8°C without corrosive gases, indicating that the addition of a stabilizer composed of one or more of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin to the reagent effectively increases the stability of the reagent.

[0120] Based on the above analysis, the lipoprotein-associated phospholipase A2 detection kit provided by the present invention can effectively improve the stability of the kit by adding a stabilizer composed of one or more of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin to reagent R2, and the accuracy and repeatability of the reagent are also good. Therefore, the lipoprotein-associated phospholipase A2 detection kit provided by the present invention is conducive to further promotion and use in the market.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A stable lipoprotein-associated phospholipase A2 assay kit, characterized in that: The method comprises a reagent R1, a reagent R2 and a calibrator, wherein the components in the reagent R1 include a buffer and a preservative, and the components in the reagent R2 include a buffer, a lipoprotein-associated phospholipase A2 substrate, a preservative, a stabilizer and a surfactant; Wherein, the concentration of the stabilizer is 1-5 g / L, and the stabilizer is selected from one or more of amino acids, sugars, polyethylene glycol, hydroxyethyl starch, Pluoronic F-68 and β-cyclodextrin; The concentration of the buffer of reagent R1 is 0.1-0.2 mol / L, and the concentration of the buffer of reagent R2 is 0.05-0.1 mol / L; the pH value of both buffers is 7.0; The surfactant is one of Pluoronic F-68, Tweens or Triton; Tweens is one of Tween20, Tween60 or Tween80; Triton is Triton X-100 or Triton X-405; The stabilizer is a mixture of glycine, trehalose, Pluoronic F-68, hydroxyethyl starch and β-cyclodextrin, and the concentrations of glycine, trehalose, Pluoronic F-68, hydroxyethyl starch and β-cyclodextrin are 0.8-1.2 g / L, 0.8-1.2 g / L, 0.08-0.12 g / L, 0.8-1.2 g / L and 0.8-1.2 g / L, respectively; Alternatively, the stabilizer is a mixture of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin, and the concentrations of glycine, trehalose, Pluoronic F-68 and β-cyclodextrin are 0.8-1.2 g / L, 0.8-1.2 g / L, 0.08-0.12 g / L and 0.8-1.2 g / L, respectively.

2. The stable lipoprotein-associated phospholipase A2 assay kit according to claim 1, characterized in that: The two buffers are both selected from one or more of phosphate, 4-hydroxyethylpiperazineethanesulfonic acid, boric acid, citric acid, carbonate, and Tris buffer.

3. The stable lipoprotein-associated phospholipase A2 assay kit according to claim 1, characterized in that: The preservative is selected from one or more of kasone, sodium azide, proclin 300, sodium benzoate or phenol.

4. The stable lipoprotein-associated phospholipase A2 assay kit according to claim 1, characterized in that: The concentration of lipoprotein-associated phospholipase A2 substrate is 10-30mmol / L.

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