Apolipoprotein B detection kit and use method thereof

Abstract

The invention provides an apolipoprotein B detection kit and a use method thereof, the apolipoprotein B detection kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises the following components: 40-80 mmol / L of an HEPES buffer solution, and 1-5 g / L of polyethylene glycol 6000; naCl is 5 g / L to 9 g / L; the preservative is 1g / L to 5g / L, and the surfactant is 1g / L to 5g / L; the pH value of the reagent R1 is 7.0 to 7.5; the pH value is 7.0-7.5 in the reagent R1, so that the pH value is below the isoelectric point of apolipoprotein B, and after the apolipoprotein B and the reagent R1 are mixed, protein in a detection sample can present weak negative charge property; accordingly, hyaluronic acid is added into the reagent R2, the hyaluronic acid and the anti-human APOB antibody in the reagent can form an integrated substance with weak covalent bond combination, and the hyaluronic acid has positive charges, so that apolipoprotein B can be rapidly combined with the anti-human APOB antibody through the attraction of the charges, the problem of long-time incubation is avoided, and the detection accuracy is improved. The detection time is shortened and the accuracy of the detection result is ensured.

Description

Technical Field

[0001] The present invention relates to biological detection technology, and in particular to an apolipoprotein B detection kit and a use method thereof. Background Art

[0002] Apolipoprotein B (ApoB) is a key lipoprotein that is mainly synthesized in the liver and intestines and is responsible for the transport and metabolism of lipid substances in the body, including cholesterol and triglycerides.

[0003] There are many methods for the detection of apolipoprotein B, among which enzyme-linked immunosorbent assay (ELISA) is a very common one. Enzyme-linked immunosorbent assay (ELISA) is used to quantitatively determine the concentration of ApoB. This is a commonly used laboratory technique with high sensitivity and specificity.

[0004] However, in the existing kits for the detection of apolipoprotein B, turbidity is usually formed after the combination of apolipoprotein B and anti-human APOB antibodies, and the concentration is determined by the instrument; however, the binding process of apolipoprotein B and anti-human APOB antibodies depends on the inherent characteristics of the protein and is not accelerated, which results in a slow or incomplete protein binding process, which can easily cause distortion of the test results and a slow detection process. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned technologies, the present invention provides a technology for improving the binding ability and rate of apolipoprotein B and APOB antibody, so as to improve the detection effect.

[0006] The present invention first provides an apolipoprotein B detection kit, which includes R1 reagent and R2 reagent, wherein the R1 reagent includes the following components: HEPES buffer 40-80 mmol / L, polyethylene glycol 6000 1 g / L to 5 g / L; NaCl 5 g / L to 9 g / L; preservative 1 g / L to 5 g / L, surfactant 1 g / L to 5 g / L; pH of the R1 reagent is 7.0 to 7.5;

[0007] In addition, the R2 reagent includes the following components: 20 mg / L to 30 mg / L of anti-human APOB antibody, 1 g / L to 5 g / L of polyethylene glycol 6000, 1 g / L to 5 g / L of surfactant, and 1 g / L to 5 g / L of hyaluronic acid.

[0008] Preferably, the preservative is one or more of sodium azide, proclin 300, MIT and sodium benzoate.

[0009] Preferably, the surfactant is selected from one or more of Tween-20, Triton X-100 and EMULGEN 709, a polyoxyethylene alkyl ether.

[0010] Preferably, the volume ratio of the reagent R1 to the reagent R2 is 3:1.

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

[0012] Step 1: Determine the absorbance of R1 reagent as A1 blank, mix R1 reagent and serum sample, incubate at 37°C for 2 minutes, and determine the absorbance as A1 sample, mix R1 reagent and calibrator of known concentration, mix well, and determine the absorbance as A1 calibration;

[0013] Step 2: Determine the absorbance of R2 reagent as A2 blank, mix R1 reagent, R2 reagent and serum sample, incubate at 37°C for 2 minutes after mixing evenly, and determine the absorbance as A2 sample, mix R1 reagent, R2 reagent and calibrator with known apolipoprotein B concentration evenly, and determine its absorbance as A2 calibration;

[0014] Step 3: The content of apolipoprotein B in the serum sample is ΔA0 / ΔAs*Cs, wherein: the absorbance of the sample tube with the absorbance of the blank tube as the reference ΔA0=(A2 sample-A1 sample)-(A2 blank-A1 blank), the absorbance of the calibrator with the absorbance of the blank tube as the reference ΔAs=(A2 calibration-A1 calibration)-(A2 blank-A1 blank); Cs: the concentration of apolipoprotein B in the calibrator.

[0015] The beneficial effects of the present invention are as follows: in the present invention, the pH value in the R1 reagent is 7.0 to 7.5, so that the pH value is below the isoelectric point of apolipoprotein B, so that when the two are mixed, the protein in the test sample can present a weak positive charge property; accordingly, hyaluronic acid is added to the R2 reagent, and the hyaluronic acid can form a binding substance with the anti-human APOB antibody in the reagent; and the hyaluronic acid has a negative charge, so through the attraction of electrostatic charge, the apolipoprotein B can be quickly combined with the anti-human APOB antibody, thus avoiding the problem of long-term incubation, shortening the detection time, and ensuring the accuracy of the detection result.

[0016] In addition, hyaluronic acid itself has antiseptic properties, so there is no need to add additional preservatives to the R2 reagent, which saves costs and ensures the antiseptic properties of the reagent, avoiding storage deterioration problems.

[0017] The main mechanism of hyaluronic acid binding to anti-human APOB antibody in R2 reagent is based on spatial structure and binding sites, that is, the long chain structure of hyaluronic acid can provide multiple binding sites in space, thereby allowing it to partially bind to anti-human APOB antibody molecules. Hyaluronic acid has the ability to adsorb and bind to two proteins, anti-human APOB antibody and apolipoprotein B, so it will accelerate the speed of their combination, thereby improving the detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a detection concentration data diagram obtained after the kit of the embodiment of the present invention and the kit of the reference example detect different concentrations of apolipoprotein B. DETAILED DESCRIPTION

[0019] The present invention first provides an apolipoprotein B detection kit, which includes R1 reagent and R2 reagent, wherein the R1 reagent includes the following components: HEPES buffer 40-80 mmol / L, polyethylene glycol 6000 1 g / L to 5 g / L; NaCl 5 g / L to 9 g / L; preservative 1 g / L to 5 g / L, surfactant 1 g / L to 5 g / L; pH of the R1 reagent is 7.0 to 7.5;

[0020] In addition, the R2 reagent includes the following components: 20 mg / L to 30 mg / L of anti-human APOB antibody, 1 g / L to 5 g / L of polyethylene glycol 6000, 1 g / L to 5 g / L of surfactant, and 1 g / L to 5 g / L of hyaluronic acid.

[0021] Preferably, the preservative is one or more of sodium azide, proclin 300, MIT and sodium benzoate.

[0022] Preferably, the surfactant is selected from one or more of Tween-20, Triton X-100 and EMULGEN 709, a polyoxyethylene alkyl ether.

[0023] Preferably, the volume ratio of the reagent R1 to the reagent R2 is 3:1.

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

[0025] Step 1: Determine the absorbance of R1 reagent as A1 blank, mix R1 reagent and serum sample, incubate at 37°C for 2 minutes, and determine the absorbance as A1 sample, mix R1 reagent and calibrator of known concentration, mix well, and determine the absorbance as A1 calibration;

[0026] Step 2: Determine the absorbance of R2 reagent as A2 blank, mix R1 reagent, R2 reagent and serum sample, incubate at 37°C for 2 minutes after mixing evenly, and determine the absorbance as A2 sample, mix R1 reagent, R2 reagent and calibrator with known apolipoprotein B concentration evenly, and determine its absorbance as A2 calibration;

[0027] Step 3: The content of apolipoprotein B in the serum sample is ΔA0 / ΔAs*Cs, wherein: the absorbance of the sample tube with the absorbance of the blank tube as the reference ΔA0=(A2 sample-A1 sample)-(A2 blank-A1 blank), the absorbance of the calibrator with the absorbance of the blank tube as the reference ΔAs=(A2 calibration-A1 calibration)-(A2 blank-A1 blank); Cs: the concentration of apolipoprotein B in the calibrator.

[0028] Example:

[0029] The apolipoprotein B detection kit of this embodiment includes R1 reagent and R2 reagent, wherein the components of R1 reagent are: HEPES buffer 60mmol / L, polyethylene glycol 6000 5g / L; NaCl 7g / L; preservative 5g / L, surfactant 5g / L; pH of R1 reagent is 7.0;

[0030] In addition, the R2 reagent includes the following components: 30 mg / L anti-human APOB antibody, 5 g / L polyethylene glycol 6000, 5 g / L surfactant, and 1 g / L to 5 g / L hyaluronic acid.

[0031] The preservative is sodium azide. The surfactant is selected from Tween-20.

[0032] The volume ratio of the reagent R1 to the reagent R2 is 3:1.

[0033] Table 1, the test results analysis and shelf life data analysis obtained after the sample concentration test in the embodiment and the control example, and the incubation time is 2 minutes.

[0034]

[0035] In the above case, different amounts of hyaluronic acid were added to the R2 reagent to observe the test results. In the test method, after the R1 reagent or R2 reagent and the serum sample are mixed, they are incubated at 37°C for 2 minutes. In the prior art, the incubation time is 5 minutes or 10 minutes or longer. Based on the incubation time here, the entire test process is shortened.

[0036] The solution of the present invention shortens the incubation time to 2 minutes, and shortens it to 2 minutes in both step 1 and step 2, thus shortening the test time as a whole; and the test results show that accurate test values ​​are obtained in the results of Examples 1 to 3; while in Control Example 1, no hyaluronic acid is added, and the test results have a certain error, with an accuracy of only about 90%, which shows that the incubation time is too short and causes distortion of the test results. Furthermore, in Control Example 2, since the amount of hyaluronic acid added is too small, only 0.1 g / L, the test results are still distorted. Accordingly, through the solution provided by the embodiments of the present invention, based on the principle of electrostatic binding and the adsorption and binding ability of hyaluronic acid itself for apolipoprotein B and antibodies, the negatively charged anti-human APOB antibody in the R2 reagent can bind to apolipoprotein B in the sample more quickly;

[0037] Furthermore, the characteristics of hyaluronic acid itself enable it to attach or depend on protein molecules. This mechanism is also conducive to the rapid binding of anti-human APOB antibodies to apolipoprotein B, thereby speeding up the time required for testing.

[0038] In the above Table 1, a shelf life analysis was also conducted, that is, the existing shelf life detection method was used to measure the performance stability of the detection kit, that is, the performance changes at different time points such as 10 days, 20 days, 30 days, and 2 months were measured. It was found that the shelf life of the reagent after adding hyaluronic acid can still meet the needs of long-term storage, and it still maintains stability at 18 months. The shelf life of the R2 reagent without hyaluronic acid is relatively short, and it will deteriorate in a few months. The mechanism is that hyaluronic acid can easily form a hydration film to isolate bacteria and achieve a preservation effect.

[0039] exist Figure 1 In the test, samples of apolipoprotein B with different concentrations (horizontally) were tested, where Series 1 is the data tested in Example 2; Series 2 is the data tested in Control Example 2. The test results show that the test concentration in Series 1 is very close to the actual concentration value, with good test result accuracy and a good linear relationship. In Series 2, since the amount of hyaluronic acid added is significantly less, the binding rate between the protein and the antibody is not enough, which results in ineffective binding and distorted results. It can be seen that the data at multiple test points are all less than the actual concentration, which further illustrates that the method kit of the embodiment of the present invention can complete accurate testing under shorter incubation conditions, reduce test waiting time, and has a good application effect.

Claims

1. An apolipoprotein B detection kit, characterized in that: The invention comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises the following components: HEPES buffer 40-80 mmol / L, polyethylene glycol 6000 1 g / L to 5 g / L; NaCl 5 g / L to 9 g / L; preservative 1 g / L to 5 g / L, surfactant 1 g / L to 5 g / L; pH value of the R1 reagent is 7.0 to 7.5; In addition, the R2 reagent includes the following components: 20 mg / L to 30 mg / L of anti-human APOB antibody, 1 g / L to 5 g / L of polyethylene glycol 6000, 1 g / L to 5 g / L of surfactant, and 1 g / L to 5 g / L of hyaluronic acid.

2. The apolipoprotein B detection kit according to claim 1, characterized in that: The preservative is one or more of sodium azide, proclin 300, MIT and sodium benzoate.

3. The apolipoprotein B detection kit according to claim 1, characterized in that: The surfactant is selected from one or more of Tween-20, Triton X-100 and EMULGEN 709, a polyoxyethylene alkyl ether.

4. The apolipoprotein B detection kit according to claim 1, characterized in that: The volume ratio of the reagent R1 to the reagent R2 is 3:

1.

5. The method for using the apolipoprotein B detection kit according to any one of claims 1 to 4, characterized in that: The following steps are included: Step 1: Determine the absorbance of R1 reagent as A1 blank, mix R1 reagent and serum sample, incubate at 37°C for 2 minutes, and determine the absorbance as A1 sample, mix R1 reagent and calibrator of known concentration, mix well, and determine the absorbance as A1 calibration; Step 2: Determine the absorbance of R2 reagent as A2 blank, mix R1 reagent, R2 reagent and serum sample, incubate at 37°C for 2 minutes after mixing evenly, and determine the absorbance as A2 sample, mix R1 reagent, R2 reagent and calibrator with known apolipoprotein B concentration evenly, and determine its absorbance as A2 calibration; Step 3: The content of apolipoprotein B in the serum sample is ΔA0 / ΔAs*Cs, wherein: the absorbance of the sample tube with the absorbance of the blank tube as the reference ΔA0=(A2 sample-A1 sample)-(A2 blank-A1 blank), the absorbance of the calibrator with the absorbance of the blank tube as the reference ΔAs=(A2 calibration-A1 calibration)-(A2 blank-A1 blank); Cs: the concentration of apolipoprotein B in the calibrator.

Images