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CN107478853B - A kind of apolipoprotein B detection kit and detection method - Google Patents

A kind of apolipoprotein B detection kit and detection method Download PDF

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Publication number
CN107478853B
CN107478853B CN201710678621.9A CN201710678621A CN107478853B CN 107478853 B CN107478853 B CN 107478853B CN 201710678621 A CN201710678621 A CN 201710678621A CN 107478853 B CN107478853 B CN 107478853B
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reagent
polyethylene glycol
apolipoprotein
buffer solution
preservative
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CN107478853A (en
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耿英利
罗湘宇
甘萍萍
黎明
龙腾镶
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Mike Biological Ltd By Share Ltd
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    • G01MEASURING; TESTING
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    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

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Abstract

The present invention provides a kind of apolipoprotein B detection kit, it includes reagent R1 and reagent R2, comprising including 0.9 52g/L of Nonidet P40,0.01 16g/L of magnesium salts, 0.01 20g/L of calcium acetate and antibody in Nonidet P40 0.9 52g/L, reagent R2 in wherein reagent R1.The kit antibody performance of the present invention is good, reproducible, reagent testing result is accurate, disclosure satisfy that requirement.

Description

Apolipoprotein B detection kit and detection method
Technical Field
The invention relates to the field of medical immunization in-vitro diagnosis, and in particular relates to an apolipoprotein B detection kit and a detection method.
Background
Turbidimetry is widely used in clinical testing. Immunoturbidimetry is now the most commonly used.
Most of the early immunoassay techniques analyze the presence or absence and content of specific proteins in a sample to be detected by observing the formation of precipitates, agglutination and hemolysis and measuring light scattering caused by aggregates, such as immunodiffusion, immunoelectrophoresis, direct and brief hemagglutination, passive hemagglutination, complement fixation experiments, etc., and these detection methods have the advantages of low cost, easy judgment of results, and easy technical mastery, and can be widely used for detecting various types of clinical samples. However, the above-mentioned methods tend to be eliminated due to their cumbersome operation, time-consuming and poor sensitivity and accuracy.
Immunoturbidimetry overcomes the above disadvantages and allows the development of automated instruments with precise quantitation in combination with clinical requirements. Therefore, for immunological detection, the immunoturbidimetry has the specificity of combining immunological antigen and antibody, has the characteristic of biochemical reaction, can detect a trace amount of substance to be detected in body fluid, particularly blood, on an automatic biochemical instrument, and is a practical clinical test technology with application prospect.
Immunoturbidimetry (Turbidimetric inhibition assay) is an antigen-antibody binding kinetic assay. The basic principle is as follows: when the antigen and antibody react in a special dilution system and in a suitable ratio (generally, an excess amount of antibody is specified), the formed soluble immune complex precipitates from the liquid phase under the action of the aggregation promoter in the dilution system to form microparticles, and turbidity appears in the reaction solution. When the antibody concentration is fixed, the amount of the immunocomplex formed increases with the increase in the amount of the antigen in the sample, and the turbidity of the reaction solution also increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction solution and comparing with a series of standard products.
Various detection instruments developed and developed based on the basic principle of immunoturbidimetry have been widely used in many aspects of clinical examination, which is the basic working principle of optical methods and immunological methods of blood coagulators; the method is a determination principle of apolipoprotein, hapten and other proteins in full-automatic biochemical determination; meanwhile, the method can also be applied to microorganism detection. By accurately quantifying a variety of substances, there is great clinical significance in the diagnosis, treatment and prognosis evaluation of many diseases.
The clinical commonly used immunoturbidimetry can directly analyze samples in batches on a full-automatic biochemical analyzer due to small sample consumption, and is simple to operate, but the currently established reagents and methods have some defects and shortcomings, which are mainly shown in that:
the antibody is easy to generate flocculent or flaky precipitation in the preservation process, so that the performance of the antibody is poor, the repeatability is poor, and the Coefficient of Variation (CV) is increased, the detection result of the reagent is inaccurate, a filtering step is added, the operation is complex, the antibody can be removed by filtering, the detection effect is influenced, a certain influence is caused on a patient, and the use requirement cannot be met.
Disclosure of Invention
In order to solve the problems, the invention discloses an apolipoprotein B detection kit and a detection method, and the kit has the advantages of good reagent stability, good uniformity, good detection result accuracy, convenience in operation and convenience in popularization and application.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides an apolipoprotein B detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 0.9-52g/L of ethylphenylpolyethylene glycol, and the reagent R2 comprises 0.9-52g/L of ethylphenylpolyethylene glycol, 0.01-16g/L of magnesium salt, 0.01-20g/L of calcium acetate and 10-1000mg/L of antibody.
An apolipoprotein B detection kit, wherein the ethyl phenyl polyethylene glycol in the reagent R1 is 5-40g/L, preferably 28 g/L; the ethyl phenyl polyethylene glycol in the reagent R2 is 4.5-41g/L, and preferably 30 g/L.
An apolipoprotein B detection kit, wherein the magnesium salt is 0.05-10g/L, preferably 6 g/L; the magnesium salt is preferably one or more of magnesium sulfate, magnesium chloride or magnesium acetate.
The detection kit for the apolipoprotein B comprises 0.05-16g/L of calcium acetate, and preferably 12g/L of calcium acetate.
Wherein,
the reagent R1 also comprises buffer solution, inorganic salt, preservative and aggregate; preferably, the reagent R1 further comprises buffer solution 20-150mmol/L, inorganic salt 1-30g/L, preservative 0.5-1g/L and aggregate 1-60 g/L.
The reagent R2 further comprises a buffer solution, an inorganic salt and a preservative, and preferably, the reagent R2 further comprises 20-100mmol/L of the buffer solution, 1-30g/L of the inorganic salt and 0.5-1g/L of the preservative.
An apolipoprotein B detection kit further comprises a calibrator, wherein the calibrator adopts multi-point calibration, and the calibrator comprises 20-100mmol/L of buffer solution, 1-30g/L of inorganic salt, 0.1-2g/L of preservative, 0.3-100g/L of glucan, 1-100g/L of trehalose, 1-100g/L of sucrose, 1-100g/L of bovine serum albumin and antigen.
An apolipoprotein B detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution 20-150mmol/L, an inorganic salt 1-30g/L, a preservative 0.5-1g/L, polyethylene glycol 60001-60g/L and ethylphenylpolyethylene glycol 0.9-52g/L, and the reagent R2 comprises a buffer solution 20-100mmol/L, an inorganic salt 1-30g/L, a preservative 0.5-1g/L, an antibody 10-1000mg/L, ethylphenylpolyethylene glycol 0.9-52g/L, magnesium sulfate 0.01-16g/L and calcium acetate 0.01-20 g/L;
preferably, the first and second electrodes are formed of a metal,
the reagent R1 comprises a buffer solution 20-150mmol/L, inorganic salt 1-30g/L, a preservative 0.5-1g/L, polyethylene glycol 60001-60g/L and ethyl phenyl polyethylene glycol 5-40g/L, and the reagent R2 comprises a buffer solution 20-100mmol/L, inorganic salt 1-30g/L, a preservative 0.5-1g/L, an antibody 10-1000mg/L, ethyl phenyl polyethylene glycol 4.5-41g/L, magnesium sulfate 0.05-10g/L and calcium acetate 0.05-16 g/L;
more preferably still, the first and second liquid crystal compositions are,
the reagent R1 comprises a buffer solution 20-150mmol/L, inorganic salt 1-30g/L, a preservative 0.5-1g/L, polyethylene glycol 60001-60g/L and ethyl phenyl polyethylene glycol 28g/L, and the reagent R2 comprises a buffer solution 20-100mmol/L, inorganic salt 1-30g/L, a preservative 0.5-1g/L, an antibody 10-1000mg/L, ethyl phenyl polyethylene glycol 30g/L, magnesium sulfate 6g/L and calcium acetate 12 g/L.
An apolipoprotein B detection kit, which comprises a kit body,
preferably, the antibody is a goat anti-human, rabbit anti-human, horse anti-human, mouse anti-human or other animal anti-human antibody;
preferably, the buffer solution is one or more of acetate buffer solution, ammonium chloride buffer solution, phosphate buffer solution, TRIS buffer solution, boric acid buffer solution, glycine buffer solution, CAPSO, MOPS or Hepes buffer solution;
preferably, the inorganic salt is one or two of sodium chloride and potassium chloride;
preferably, the preservative is one or more of sodium azide, phenol, p-hydroxybenzoic acid, ethyl p-hydroxybenzoate or ethylmercuric thiosulfate;
preferably, the aggregate is one or more of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000 or polyethylene glycol 8000;
the invention also provides a detection method using the apolipoprotein B detection kit, which comprises the following steps:
(1) adding the reagent R1 into a sample to be detected, and uniformly mixing, wherein the volume ratio of the sample to be detected to the reagent R1 is (1-9): 300, incubating at 37 ℃, and reading the absorbance A1 at a certain wavelength;
(2) adding a reagent R2 into the mixed solution obtained in the step (1), and uniformly mixing, wherein the volume ratio of the reagent R2 to the reagent R1 is 1: (1-6) adding, incubating at 37 ℃, and reading the absorbance A2 at a certain wavelength;
(3) obtaining absorbance △ A, wherein the absorbance △ A is A2-A1;
(4) and (3) fitting a standard curve on a full-automatic biochemical analyzer by using the calibrator through a built-in curve fitting mode, and automatically calculating the content of the apolipoprotein B in the sample to be detected according to the absorbance.
The aggregate referred to in the present invention is a substance that promotes aggregation of antigen and antibody in a reaction.
The invention provides an apolipoprotein B detection kit and a detection method, and relates to the following raw material sources:
due to the adoption of the technical scheme, the invention has the beneficial effects that:
the detection kit and the detection method for the apolipoprotein B provided by the invention have the advantages of good reagent stability, good uniformity, stable storage of an antibody, no precipitation phenomenon, high antibody titer, good performance, no filtration step, convenience in operation, low cost, accurate detection result, good repeatability and wider universality.
Detailed description of the preferred embodiments
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. Example 1 detection kit for Apolipoprotein B
Reagent R1:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Polyethylene glycol 6000 1g/L
Ethyl phenyl polyethylene glycol 0.9g/L
Reagent R2:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Sheep anti-human apolipoprotein B antibody 10mg/L
Ethyl phenyl polyethylene glycol 0.9g/L
Magnesium sulfate 0.01g/L
Calcium acetate 0.01g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (20mmol/L phosphate buffer, 2g/L sodium chloride, 0.1g/L sodium azide, 0.4g/L dextran, 1g/L trehalose, 1g/L sucrose, 2g/L bovine serum albumin), assayed with commercially available control reagents and adjusted to 120mg/L, stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 10mg/L, 20mg/L, 50mg/L and 70 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃. Example 2 detection kit for Apolipoprotein B
Reagent R1:
TRIS buffer 60mmol/L
Potassium chloride 6g/L
Phenol and its preparation 0.7g/L
Polyethylene glycol 6000 25g/L
Ethyl phenyl polyethylene glycol 52g/L
Reagent R2:
TRIS buffer 60mmol/L
Potassium chloride 6g/L
Phenol and its preparation 0.7g/L
Sheep anti-human apolipoprotein B antibody 50mg/L
Ethyl phenyl polyethylene glycol 52g/L
Magnesium sulfate 16g/L
Calcium acetate 20g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (40mmol/LTRIS buffer, 10g/L sodium chloride, 0.1g/L sodium azide, 50g/L dextran, 25g/L trehalose, 30g/L sucrose, 20g/L bovine serum albumin), detected with commercially available control reagents and adjusted to 100mg/L, stored in aliquots at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 10mg/L, 30mg/L, 50mg/L and 70 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Example 3 detection kit for Apolipoprotein B
Reagent R1:
acetate buffer solution 80mmol/L
Potassium chloride 20g/L
Sodium azide 0.8g/L
Polyethylene glycol 6000 50g/L
Ethyl phenyl polyethylene glycol 5g/L
Reagent R2:
acetate buffer solution 80mmol/L
Potassium chloride 20g/L
Sodium azide 0.8g/L
Sheep anti-human apolipoprotein B antibody 300mg/L
Ethyl phenyl polyethylene glycol 4.5g/L
Magnesium sulfate 0.05g/L
Calcium acetate 0.05g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (80mmol/L acetate buffer, 20g/L sodium chloride, 0.8g/L sodium azide, 70g/L dextran, 60g/L trehalose, 60g/L sucrose, 70g/L bovine serum albumin), assayed with commercially available control reagents and adjusted to 200mg/L, stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 2mg/L, 10mg/L, 40mg/L, 60mg/L and 80 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Example 4 detection kit for Apolipoprotein B
Reagent R1:
MOPS buffer solution 120mmol/L
Sodium chloride 20g/L
Sodium azide 1g/L
Polyethylene glycol 6000 60g/L
Ethyl phenyl polyethylene glycol 40g/L
Reagent R2:
MOPS buffer solution 100mmol/L
Sodium chloride 20g/L
Sodium azide 1g/L
Sheep anti-human apolipoprotein B antibody 600mg/L
Ethyl phenyl polyethylene glycol 41g/L
Magnesium sulfate 10g/L
Calcium acetate 16g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (100mmol/L MOPS buffer, 25g/L sodium chloride, 2g/L sodium azide, 20g/L dextran, 100g/L trehalose, 100g/L sucrose, 100g/L bovine serum albumin), detected with commercial control reagents and adjusted to 160mg/L, and stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 20mg/L, 40mg/L, 70mg/L and 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.45 mu m, and storing at the temperature of 2-8 ℃.
Example 5 detection kit for Apolipoprotein B
Reagent R1:
MOPS buffer solution 150mmol/L
Sodium chloride 30g/L
Sodium azide 1g/L
Polyethylene glycol 6000 60g/L
Ethyl phenyl polyethylene glycol 28g/L
Reagent R2:
MOPS buffer solution 100mmol/L
Sodium chloride 30g/L
Sodium azide 1g/L
Sheep anti-human apolipoprotein B antibody 1000mg/L
Ethyl phenyl polyethylene glycol 30g/L
Magnesium sulfate 6g/L
Calcium acetate 12g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (100mmol/L MOPS buffer, 30g/L sodium chloride, 1g/L sodium azide, 90g/L dextran, 100g/L trehalose, 100g/L sucrose, 100g/L bovine serum albumin), detected with commercially available control reagents and adjusted to 120mg/L, and stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 20mg/L, 50mg/L, 80mg/L and 100 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Example 6 detection kit for Apolipoprotein B
Reagent R1:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Polyethylene glycol 6000 1g/L
Ethyl phenyl polyethylene glycol 0.9g/L
Reagent R2:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Sheep anti-human apolipoprotein B antibody 10mg/L
Ethyl phenyl polyethylene glycol 0.9g/L
Magnesium sulfate 0.01g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (20mmol/L phosphate buffer, 2g/L sodium chloride, 0.1g/L sodium azide, 0.4g/L dextran, 1g/L trehalose, 1g/L sucrose, 2g/L bovine serum albumin), assayed with commercially available control reagents and adjusted to 120mg/L, stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 10mg/L, 20mg/L, 50mg/L and 70 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Example 7 detection kit for Apolipoprotein B
Reagent R1:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Polyethylene glycol 6000 1g/L
Ethyl phenyl polyethylene glycol 0.9g/L
Reagent R2:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Sheep anti-human apolipoprotein B antibody 10mg/L
Ethyl phenyl polyethylene glycol 0.9g/L
Calcium acetate 0.01g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (20mmol/L phosphate buffer, 2g/L sodium chloride, 0.1g/L sodium azide, 0.4g/L dextran, 1g/L trehalose, 1g/L sucrose, 2g/L bovine serum albumin), assayed with commercially available control reagents and adjusted to 120mg/L, stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 10mg/L, 20mg/L, 50mg/L and 70 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Example 8 detection kit for Apolipoprotein B
Reagent R1:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Polyethylene glycol 6000 1g/L
Ethyl phenyl polyethylene glycol 0.9g/L
Reagent R2:
sulfate buffer 20mmol/L
Sodium chloride 1g/L
Sodium azide 0.5g/L
Sheep anti-human apolipoprotein B antibody 10mg/L
Tween 20 0.9g/L
Calibration products:
ApoB antigen was dissolved in standard dilutions (20mmol/L phosphate buffer, 2g/L sodium chloride, 0.1g/L sodium azide, 0.4g/L dextran, 1g/L trehalose, 1g/L sucrose, 2g/L bovine serum albumin), assayed with commercially available control reagents and adjusted to 120mg/L, stored at-20 ℃. Before use, the sample is taken out and diluted into ApoB standard substances with different concentrations by standard substance diluent (ApoB antigen concentration: 0mg/L, 10mg/L, 20mg/L, 50mg/L and 70 mg/L). Then filtering and sterilizing by using a filter membrane with the diameter of 0.65 mu m, and storing at the temperature of 2-8 ℃.
Comparing the detection results of different embodiments, wherein the CV value is STDEV (1-7)/mean value.
1. A sample with an apolipoprotein B concentration of 40mg/L was prepared, and the test was repeated 7 times using the kit of example 1, with the test results shown in Table 1.
Table 1: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 1, the apolipoprotein B concentrations measured in example 1 in 1 month, 3 months and 12 months are all close to the true values, and the measured variation coefficients (CV values)) are all less than 2%, which indicates that the kit provided by the invention has good repeatability, stable performance and accurate measurement.
2. A sample with an apolipoprotein B concentration of 1mg/L was prepared, and the test was repeated 7 times using the kit of example 2, with the test results shown in Table 2.
Table 2: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 2, the apolipoprotein B concentrations measured in 1 month, 3 months and 12 months in example 2 are all close to the true values, and the measured variation coefficients are all less than 2%, which indicates that the kit provided by the invention has good repeatability, stable performance and accurate measurement.
3. A sample with an apolipoprotein B concentration of 25mg/L was prepared, and the assay was repeated 7 times using the kit of example 3, with the assay results shown in Table 3.
Table 3: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 3, the apolipoprotein B concentrations measured in 1 month, 3 months and 12 months in example 3 are all close to the true values, and the measured variation coefficients are all less than 1%, which indicates that the kit of the invention has good repeatability, stable performance and accurate measurement.
4. A sample with an apolipoprotein B concentration of 70mg/L was prepared, and the assay was repeated 7 times using the kit of example 4, with the assay results shown in Table 4.
Table 4: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 4, the apolipoprotein B concentrations measured in 1 month, 3 months and 12 months in example 4 are all close to the true values, and the measured variation coefficients are all less than 1%, which indicates that the kit provided by the invention has good repeatability, stable performance and accurate measurement.
5. A sample with an apolipoprotein B concentration of 25mg/L was prepared, and the assay was repeated 7 times using the kit of example 5, with the assay results shown in Table 5.
Table 5: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 5, the apolipoprotein B concentrations measured in 1 month, 3 months and 12 months in example 5 are all true values, and the measured variation coefficients are all less than 0.05%, which indicates that the kit provided by the invention has good repeatability, stable performance and accurate measurement.
6. A sample with an apolipoprotein B concentration of 40mg/L was prepared, and the test was repeated 7 times using the kit of example 6, with the test results shown in Table 6.
Table 6: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 6, the apolipoprotein B concentrations measured in example 6 in 1 month, 3 months and 12 months all deviate from the true values, and the measured variation coefficients are all more than 4%, which indicates that the kit has poor repeatability, unstable performance and inaccurate measurement.
7. A sample having an apolipoprotein B concentration of 40mg/L was prepared, and the sample was repeatedly tested 7 times using the kit of example 7, with the test results shown in Table 7.
Table 7: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 7, the apolipoprotein B concentrations measured in example 7 in 1 month, 3 months and 12 months all deviate from the true values, and the measured variation coefficients are all more than 4%, which indicates that the kit has poor repeatability, unstable performance and inaccurate measurement.
8. A sample with an apolipoprotein B concentration of 40mg/L was prepared, and the test was repeated 7 times using the kit of example 8, with the test results shown in Table 8.
Table 8: the measurement result of the storage at 2-8 ℃ for 1 month, 3 months and 12 months
As can be seen from Table 8, the apolipoprotein B concentrations measured in example 8 in 1 month, 3 months and 12 months all deviate from the true values, and the measured variation coefficients are all more than 5%, which indicates that the kit has poor repeatability, unstable performance and inaccurate measurement.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (15)

1. An apolipoprotein B detection kit, which is characterized in that: the kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 0.9-52g/L of ethylphenyl polyethylene glycol, and the reagent R2 comprises 0.9-52g/L of ethylphenyl polyethylene glycol, 0.01-16g/L of magnesium salt, 0.01-20g/L of calcium acetate and 10-1000mg/L of antibody; the reagent R1 further comprises a buffer solution, an inorganic salt, a preservative and aggregates, and the reagent R2 further comprises a buffer solution, an inorganic salt and a preservative, wherein the inorganic salt in R2 is one or two of sodium chloride and potassium chloride; and wherein
The antibody is an animal anti-human apolipoprotein B antibody,
the aggregate is one or more of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000 or polyethylene glycol 8000.
2. The apolipoprotein B detection kit according to claim 1, wherein: the ethyl phenyl polyethylene glycol in the reagent R1 is 5-40 g/L; the ethyl phenyl polyethylene glycol in the reagent R2 is 4.5-41 g/L.
3. The apolipoprotein B detection kit according to claim 2, wherein: the ethyl phenyl polyethylene glycol in the reagent R1 is 28 g/L; the ethyl phenyl polyethylene glycol in the reagent R2 is 30 g/L.
4. The apolipoprotein B detection kit according to claim 1, wherein: the magnesium salt is 0.05-10 g/; the magnesium salt is one or more of magnesium sulfate, magnesium chloride or magnesium acetate.
5. The apolipoprotein B detection kit according to claim 4, wherein: the magnesium salt is 6 g/L.
6. The apolipoprotein B detection kit according to claim 1, wherein: the calcium acetate is 0.05-16 g/L.
7. The apolipoprotein B detection kit according to claim 6, wherein: the calcium acetate is 12 g/L.
8. The apolipoprotein B detection kit according to any one of claims 1-7, wherein: the reagent R1 also comprises buffer solution 20-150mmol/L, inorganic salt 1-30g/L, preservative 0.5-1g/L and aggregate 1-60 g/L.
9. The apolipoprotein B detection kit according to any one of claims 1-7, wherein: the reagent R2 further comprises a buffer solution 20-100mmol/L, an inorganic salt 1-30g/L and a preservative 0.5-1g/L, wherein the inorganic salt in R2 is one or two of sodium chloride and potassium chloride.
10. The apolipoprotein B detection kit according to claim 1, wherein: the kit comprises a calibrator, wherein the calibrator is calibrated at multiple points, and the calibrator comprises 20-100mmol/L of buffer solution, 2-30g/L of inorganic salt, 0.1-2g/L of preservative, 0.4-90g/L of glucan, 1-100g/L of trehalose, 1-100g/L of sucrose, 2-100g/L of bovine serum albumin and human apolipoprotein B antigen.
11. An apolipoprotein B detection kit, which is characterized in that: the kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution of 20-150mmol/L, inorganic salt of 1-30g/L, a preservative of 0.5-1g/L, polyethylene glycol 60001-60g/L and ethyl phenyl polyethylene glycol of 0.9-52 g/L; the reagent R2 comprises buffer solution 20-100mmol/L, sodium chloride or potassium chloride 1-30g/L, preservative 0.5-1g/L, animal anti-human apolipoprotein B antibody 10-1000mg/L, ethyl phenyl polyethylene glycol 0.9-52g/L, magnesium sulfate 0.01-16g/L, and calcium acetate 0.01-20 g/L.
12. The kit according to claim 11, wherein the reagent R1 comprises a buffer solution of 20-150mmol/L, inorganic salts of 1-30g/L, a preservative of 0.5-1g/L, polyethylene glycol 60001-60g/L, ethyl phenyl polyethylene glycol of 5-40 g/L; the reagent R2 comprises 20-100mmol/L buffer solution, 1-30g/L sodium chloride or potassium chloride, 0.5-1g/L preservative, 10-1000mg/L animal anti-human apolipoprotein B antibody, 4.5-41g/L ethyl phenyl polyethylene glycol, 0.05-10g/L magnesium sulfate and 0.05-16g/L calcium acetate.
13. The kit according to claim 12, wherein the reagent R1 comprises 20-150mmol/L of buffer solution, 1-30g/L of inorganic salt, 0.5-1g/L of preservative, 60001-60g/L of polyethylene glycol, 28g/L of ethyl phenyl polyethylene glycol; the reagent R2 comprises 20-100mmol/L buffer solution, 1-30g/L sodium chloride or potassium chloride, 0.5-1g/L preservative, 10-1000mg/L animal anti-human apolipoprotein B antibody, 30g/L ethyl phenyl polyethylene glycol, 6g/L magnesium sulfate and 12g/L calcium acetate.
14. The apolipoprotein B detection kit according to claim 1 or 11, wherein:
the antibody is a sheep anti-human antibody, a rabbit anti-human antibody, a horse anti-human antibody, a mouse anti-human antibody or other animal anti-human antibody;
the buffer solution is one or more of acetate buffer solution, ammonium chloride buffer solution, phosphate buffer solution, TRIS buffer solution, boric acid buffer solution, glycine buffer solution, CAPSO, MOPS or Hepes buffer solution;
the inorganic salt is one or two of sodium chloride and potassium chloride;
the preservative is one or more of sodium azide, phenol, p-hydroxybenzoic acid, ethyl p-hydroxybenzoate or ethylmercuric thiosulfate;
the aggregate is one or more of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000 or polyethylene glycol 8000.
15. An assay method for non-disease diagnostic or therapeutic purposes using the apolipoprotein B detection kit of any one of claims 1 or 11, comprising the steps of:
(1) adding the reagent R1 into a sample to be detected, and uniformly mixing, wherein the volume ratio of the sample to be detected to the reagent R1 is (1-9): 300, incubating at 37 ℃, and reading the absorbance A1 at a certain wavelength;
(2) adding a reagent R2 into the mixed solution obtained in the step (1), and uniformly mixing, wherein the volume ratio of the reagent R2 to the reagent R1 is 1: (1-6) adding, incubating at 37 ℃, and reading the absorbance A2 at a certain wavelength;
(3) obtaining absorbance △ A, wherein the absorbance △ A is A2-A1;
(4) and (3) fitting a standard curve on a full-automatic biochemical analyzer by using the calibrator through a built-in curve fitting mode, and automatically calculating the content of the apolipoprotein B in the sample to be detected according to the absorbance.
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