Apolipoprotein E detection kit and use method thereof

Abstract

The invention provides an apolipoprotein E detection kit and a use method thereof. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution, a surfactant and a preservative, and further comprises a block polymer; the reagent R2 further comprises an ApoE antibody, and the polystyrene is combined with the ApoE antibody in an adsorption manner so as to be used for specific recognition of the polyethylene glycol; the concentration of the ApoE antibody is 20 mg / L to 50 mg / L, and the concentration of the polyethylene glycol-polystyrene block polymer is 10 mg / L to 20 mg / L. A polystyrene and polyethylene glycol block polymer is added into a reagent R2, polystyrene and an ApoE antibody for detection have good binding and attaching properties, and polyethylene glycol and polystyrene are an integrated polymer, so that polyethylene glycol is close to an antigen, and the ApoE antibody for detection is detected. Therefore, the polyethylene glycol can directly act on the antigen and antibody combined molecular composition, the whole consumption is reduced through specific combination, and the inherent action effect of the polyethylene glycol is ensured.

Description

Technical Field

[0001] The invention relates to a kit for biochemical testing, and in particular to a technology for reducing the amount of polyethylene glycol concentration and maintaining the detection effect of an apolipoprotein E detection kit. Background Art

[0002] Apolipoprotein E (ApoE) is a soluble alkaline glycosylated secretory protein. It is the most important lipid transport protein in the body and the apolipoprotein with the greatest impact on lipid metabolism. ApoE is derived from a variety of tissues and organs and can participate in the synthesis of lipoproteins. It is often used in clinical practice to assess the risk of lipid metabolism disorders and to assist in the diagnosis of cardiovascular and cerebrovascular diseases.

[0003] Immunoturbidimetry is a common method for apolipoprotein E detection. The basic principle is: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally the antibody is in excess), the soluble immune complex formed will precipitate from the liquid phase under the action of the dilution system to form particles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of immune complex formed increases with the increase of the amount of antigen in the test sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standards, the content of the antigen in the test sample can be calculated.

[0004] Then, polyethylene glycol (PEG) is often added to the existing apolipoprotein E detection kit. The main purpose of adding this substance is to make it easier to expose the site so that the apolipoprotein can bind to the antibody in the kit; that is, PEG, as a high molecular weight hydrophilic substance, can improve the binding efficiency between the antibody and the antigen. Literature shows that in ELISA, PEG can shield unnecessary nonspecific binding and reduce background noise, thereby making the target antigen (such as ApoE) and the specific antibody binding more effective. For example, in the prior art, in the patent number 201410429303.5 of an apolipoprotein E detection kit, polyethylene glycol is added, and its concentration is 10-40g / L; in the patent number 201210291683.1 of an apolipoprotein E detection kit and its preparation method, the patent gives 5-10mmol / L polyethylene glycol, which is also a concentration greater than 20g / L when converted to mass;

[0005] Therefore, in the existing test kits, the amount of polyethylene glycol added is usually high, and the amount added is usually 10-100g / L. Such a concentration is too high. The reason is that no specific binding is formed. The polyethylene glycol is dispersed and does not specifically bind to the antibody position in the solution. This greatly reduces the efficacy and cannot improve the specificity. In addition, there are relevant records in the prior art about the binding of polystyrene to ApoE. For example, the description of ApoE binding to polypropylene, polystyrene, polyethylene and glass tubes is mentioned in the Chinese invention patent CN201780062562.0 on the detection method of apolipoprotein E4. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned technology, the present invention provides a method for adsorbing and fixing antibodies by introducing polystyrene, so that polyethylene glycol can specifically bind to the antibody in close proximity, thereby avoiding the problem of high consumption of the existing polyethylene glycol dispersed and added to the solution reagent.

[0007] An invention provided by the present invention provides an apolipoprotein E detection kit, which includes: reagent R1 and reagent R2; wherein reagent R1 includes a buffer, a surfactant and a preservative, and reagent R1 also includes a polyethylene glycol-polystyrene block polymer; reagent R2 includes a buffer, a surfactant and a preservative, and reagent R2 also includes an ApoE antibody; the concentration of the ApoE antibody is 20 mg / L to 50 mg / L, and the concentration of the polyethylene glycol-polystyrene block polymer is 10 mg / L to 20 mg / L.

[0008] Preferably, the molecular weight of polystyrene is 5,000 to 20,000, and the molecular weight of polyethylene glycol is 2,000 to 20,000.

[0009] Preferably, the buffer is a phosphate buffer, and the concentration of the buffer is 20-30 mmol / L; the surfactant is Tween 20, and the concentration of the surfactant is 0.05-0.1% by mass; the preservative is sodium azide, and the concentration of the preservative is 0.05% to 0.15% by mass.

[0010] Preferably, the polyethylene glycol-polystyrene block polymer is provided by Hangzhou Xinqiao Biotechnology or Sigma-Aldrich, and the PDI of the polymer material is less than 1.3.

[0011] The method for using the apolipoprotein E detection kit provided by the present invention comprises the following steps:

[0012] Step 1: Add reagent R1 to the sample to be tested and mix evenly. The volume ratio of the sample to be tested and reagent R1 is 1-10:300. Incubate at 37°C. The absorbance at a specific wavelength is A1.

[0013] Step 2: Add reagent R2 to the mixed solution in step 1 and mix well. Add reagent R2 and reagent R1 in a volume ratio of 1:1 to 6, incubate at 37°C, and the absorbance at a specific wavelength is A2;

[0014] Step 3: Obtain absorbance △A, △A=A2-A1;

[0015] Step 4: Use the calibrator to fit the standard curve on the fully automatic biochemical analyzer through the built-in curve fitting mode, and automatically calculate the content of apolipoprotein E in the sample to be tested based on the absorbance.

[0016] The beneficial effects of the present invention are as follows: polystyrene and polyethylene glycol block polymers are added to the detection reagent, polystyrene has good binding and attachment properties with the ApoE antibody used for detection, and polyethylene glycol and polystyrene are an integral polymer, so that the polyethylene glycol is close to the antigen, so that the polyethylene glycol can directly act on the molecular structure of the antigen and antibody combination, and the specific binding reduces the overall consumption and ensures the inherent effect of the polyethylene glycol.

[0017] The polystyrene and polyethylene glycol block polymers are in the R1 reagent. When the R1 reagent and the R2 reagent are mixed during detection, the polystyrene and polyethylene glycol block polymers quickly bind to the ApoE antibody to expose the site, which is beneficial to the detection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the combination of block polymers polystyrene and polyethylene glycol and ApoE antibody in the apolipoprotein E detection kit of the present invention.

[0019] Figure 2 This is a linear regression equation diagram after summarizing and analyzing the data after apolipoprotein E detection in Control Example 2. DETAILED DESCRIPTION

[0020] An invention provided by the present invention provides an apolipoprotein E detection kit, which includes: reagent R1 and reagent R2; wherein reagent R1 includes a buffer, a surfactant and a preservative, and reagent R1 also includes a polyethylene glycol-polystyrene block polymer; reagent R2 includes a buffer, a surfactant and a preservative, and reagent R2 also includes an ApoE antibody; the concentration of the ApoE antibody is 20 mg / L to 50 mg / L, and the concentration of the polyethylene glycol-polystyrene block polymer is 10 mg / L to 20 mg / L.

[0021] Preferably, the molecular weight of polystyrene is 5,000 to 20,000, and the molecular weight of polyethylene glycol is 2,000 to 20,000.

[0022] Preferably, the buffer is a phosphate buffer, and the concentration of the buffer is 20-30 mmol / L; the surfactant is Tween 20, and the concentration of the surfactant is 0.05-0.1% by mass; the preservative is sodium azide, and the concentration of the preservative is 0.05% to 0.15% by mass.

[0023] Preferably, the polyethylene glycol-polystyrene block polymer is provided by Hangzhou Xinqiao Biotechnology or Sigma-Aldrich, and the PDI of the polymer material is less than 1.3.

[0024] The method for using the apolipoprotein E detection kit provided by the present invention comprises the following steps:

[0025] Step 1: Add reagent R1 to the sample to be tested and mix evenly. The volume ratio of the sample to be tested and reagent R1 is 1-10:300. Incubate at 37°C. The absorbance at a specific wavelength is A1.

[0026] Step 2: Add reagent R2 to the mixed solution in step 1 and mix well. Add reagent R2 and reagent R1 in a volume ratio of 1:1 to 6, incubate at 37°C, and the absorbance at a specific wavelength is A2;

[0027] Step 3: Obtain absorbance △A, △A=A2-A1;

[0028] Step 4: Use the calibrator to fit the standard curve on the fully automatic biochemical analyzer through the built-in curve fitting mode, and automatically calculate the content of apolipoprotein E in the sample to be tested based on the absorbance.

[0029] In a specific embodiment, the following implementation cases are obtained.

[0030] Embodiment 1: An apolipoprotein E detection kit of this embodiment comprises: reagent R1 and reagent R2; wherein reagent R1 comprises a buffer, a surfactant and a preservative, and reagent R1 also comprises polyethylene glycol-polystyrene block polymer; reagent R2 comprises a buffer, a surfactant and a preservative, and reagent R2 also comprises an ApoE antibody; the concentration of the ApoE antibody is 50 mg / L, and the concentration of the polyethylene glycol-polystyrene block polymer is 10 mg / L. Among them, in the polyethylene glycol-polystyrene block polymer, the molecular weight of polystyrene is 5000, and the molecular weight of polyethylene glycol is 5000. The buffer is a phosphate buffer, and the concentration of the buffer is 20 mmol / L; the surfactant is Tween 20, and the concentration of the surfactant is 0.1% by mass; the preservative is sodium azide, and the concentration of the preservative is 0.05% by mass.

[0031] Example 2: An apolipoprotein E detection kit of this example includes: reagent R1 and reagent R2; wherein reagent R1 includes a buffer, a surfactant and a preservative, and reagent R1 also includes polyethylene glycol-polystyrene block polymer; reagent R2 includes a buffer, a surfactant and a preservative, and reagent R2 also includes an ApoE antibody; the concentration of the ApoE antibody is 50 mg / L, and the concentration of the polyethylene glycol-polystyrene block polymer is 20 mg / L. Wherein, the molecular weight of polystyrene is 5000, and the molecular weight of polyethylene glycol is 5000. The buffer is HEPES buffer, and the concentration of the buffer is 30 mmol / L; the surfactant is Tween 20, and the concentration of the surfactant is 0.05% by mass; the preservative is sodium azide, and the concentration of the preservative is 0.05% by mass.

[0032] Comparative Example 1: The difference from Example 1 is that polyethylene glycol-polystyrene block polymer is not used, but polyethylene glycol is added, and the concentration of polyethylene glycol 5000 is 10 g / L; that is, polyethylene glycol-polystyrene block polymer is replaced by polyethylene glycol;

[0033] Comparative Example 2: The difference from Example 2 is that polyethylene glycol-polystyrene block polymer is not used, but polyethylene glycol is added, and the concentration of polyethylene glycol 5000 is 10 mg / L; that is, polyethylene glycol-polystyrene block polymer is replaced by polyethylene glycol;

[0034] Comparative Example 3: The difference from Example 1 is that no polyethylene glycol-polystyrene block polymer is used and no polyethylene glycol is added.

[0035] Experimental Example 1: According to the above different examples 1 and control examples, statistics are made on whether block polymer and polyethylene glycol are added, and the R obtained after corresponding data detection is 2 .

[0036] Table 1:

[0037]

[0038]

[0039] The following Figure 2 First, different concentrations of apolipoprotein E (ApoE antigen) are prepared, and the horizontal axis corresponds to 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, and 80 mg / L; and the data on the vertical axis are the data and regression equation obtained when the detection kit of the implementation component of Control Example 2 is used for detection.

[0040] Accordingly, other Example 1, Control Example 2 and Control Example 3 are also tested according to the above data, and the corresponding data are summarized and regressed to obtain corresponding data, and the corresponding data are summarized in the above Table 1 for analysis.

[0041] As can be seen from Table 1 above, the R of the regression equations in Example 1 and Comparative Example 1 2 They are relatively close, all greater than 0.99, which indicates that the detection of this data has good linearity. And in Example 1, the improved detection is based on the block polymer, while the control example 1 is based on the existing detection kit, that is, the detection is performed by adding the corresponding polyethylene glycol. And in this application, by adding a very low concentration of block polymer, that is, the concentration of polyethylene glycol-polystyrene block polymer is 10 mg / L, the same level of detection effect can be achieved. This shows that by adding block polymers, the specific recognition of antibodies and exposure can be achieved to make the detection results more accurate.

[0042] The concentration of polyethylene glycol used in Control Example 2 was reduced, and the R in the regression equation of the corresponding detection kit was also affected to a certain extent. Furthermore, in the detection kit in Control Example 3, polyethylene glycol and block polymer were not used, which belongs to the traditional detection method. The data obtained by this method under the same conditions, R is lower than that of Example 1 and Control Example 2, which also shows that adding block polymer or polyethylene glycol has a corresponding effect.

[0043] exist Figure 1 The schematic diagram of the present application is given in the figure, that is, the PS in the block polymer PS (polystyrene)-polyethylene glycol (PEG) has good binding properties with the antibody, so that the polyethylene glycol is close to the antibody, so that the binding site of the antigen and the antibody is exposed, and the polyethylene glycol does not need to be dispersed in the entire reagent, and only a trace amount of the component is required to specifically recognize the antibody.

Claims

1. An apolipoprotein E detection kit, characterized in that: include: Reagent R1 and reagent R2; Reagent R1 includes a buffer, a surfactant and a preservative, and also includes polyethylene glycol-polystyrene block polymer; reagent R2 includes a buffer, a surfactant and a preservative, and also includes ApoE antibody; the concentration of ApoE antibody is 20 mg / L to 50 mg / L, and the concentration of polyethylene glycol-polystyrene block polymer is 10 mg / L to 20 mg / L.

2. The apolipoprotein E detection kit according to claim 1, characterized in that: The molecular weight of polystyrene is 5,000 to 20,000, and the molecular weight of polyethylene glycol is 2,000 to 20,000.

3. The apolipoprotein E detection kit according to claim 1, characterized in that: The buffer is a phosphate buffer or a HEPES buffer, and the concentration of the buffer is 20-30 mmol / L; the surfactant is Tween 20, and the concentration of the surfactant is 0.05-0.1% by mass; the preservative is sodium azide, and the concentration of the preservative is 0.05% to 0.15% by mass.

4. The apolipoprotein E detection kit according to claim 1, characterized in that: The polyethylene glycol-polystyrene block polymer is provided by Hangzhou Xinqiao Biotechnology or Sigma-Aldrich, and the PDI of the polymer material is less than 1.

3.

5. The method for using the apolipoprotein E detection kit according to any one of claims 1 to 4, characterized in that: The following steps are included: Step 1: Add reagent R1 to the sample to be tested and mix evenly. The volume ratio of the sample to be tested and reagent R1 is 1:300 to 10:

300. Incubate at 37°C. The absorbance at a specific wavelength is A1. Step 2: Add reagent R2 to the mixed solution in step 1 and mix well. Add reagent R2 and reagent R1 at a volume ratio of 1:1 to 1:6, incubate at 37°C, and measure the absorbance at a specific wavelength as A2; Step 3: Obtain absorbance △A, △A=A2-A1; Step 4: Use the calibrator to fit the standard curve on the fully automatic biochemical analyzer through the built-in curve fitting mode, and automatically calculate the content of apolipoprotein E in the sample to be tested based on the absorbance.

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