CN117969842A - Magnetic particle detection HBsAg chemiluminescence kit, and preparation method and application thereof - Google Patents
Magnetic particle detection HBsAg chemiluminescence kit, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a magnetic particle detection HBsAg chemiluminescence kit, which comprises: reagent R1 prepared from antibody-magnetic particles, reagent R2 prepared from biotin label-goat polyclonal antibody, reagent R3 prepared from neutravidin-alkaline phosphatase, and a calibration product formed by diluting a positive sample. The magnetic particle detection HBsAg chemiluminescence kit obtained by the technical scheme of the invention has the advantages of good detection specificity, high sensitivity and good application prospect.
Description
Technical Field
The invention relates to the field of biological diagnostic reagents, in particular to a chemiluminescent kit for detecting HBsAg by magnetic particles, a preparation method and application thereof.
Background
HBsAg exists in the outer shell of hepatitis B virus (HEPATITIS B VIRUS, HBV) particles and in small spherical particles and tubular particles, and the molecular weight of HBsAg protein is about 24 kilodaltons. The HBsAg main protein consists of 226 amino acids. HBsAg itself is not infectious but plays an important role in the viral infection of hepatocytes. HBsAg appears in serum 4-7 days after HBV infection in humans, and is often accompanied by HBV, and thus can be used as a marker of HBV infection. HBsAg is detected in serum from patients suffering from immune tolerance, chronic hepatitis B (immune clearance), inactive portable state and reactivation, HBV infection and partial liver cirrhosis and liver cancer. In addition, HBsAg can be detected in the late latent and acute phases of HBV infection.
On an enzymatic chemiluminescence detection platform, a direct-labeled double-antibody sandwich method is generally adopted to detect the HBsAg, the specific mode is that a capture antibody is connected to a magnetic bead, a labeled antibody is connected to an enzyme, and common enzymes are HRP, ALP and the like, and the specific detection process is as follows: after mixing the sample, anti-HBs coated magnetic beads and enzyme-labeled anti-HBs, incubation and washing are performed. If the sample contains HBsAg, the HBsAg is captured by the antibody to form a magnetic bead-antibody, antigen and antibody-enzyme sandwich complex. After magnetic attraction and separation, excitation liquid or a luminescent substrate is added, a generated chemiluminescent signal is used for judging the content of HBsAg in a sample through the luminescent signal, and the purpose of detecting the HBsAg is achieved.
The method for detecting HBsAg in the prior art mainly has the following defects: because the molecular weight of the enzyme is 110kd or 44kd, the molecular weight is equivalent to that of the antibody, the volume ratio is large, and the steric hindrance is large after the enzyme is covalently bound with the antibody, so that the binding between the labeled antibody and the HBsAg can be influenced, and the sensitivity of the reagent is influenced. In order to avoid the influence of steric hindrance of the enzyme, a labeled antibody is generally labeled with biotin having a smaller molecular weight, and the biotin-labeled antibody is bound to the HBsAg and then bound to the SA-enzyme through biotin on the antibody. The method can greatly reduce the sensitivity reduction caused by the steric hindrance of the enzyme, and improve the binding efficiency of the labeled antibody, thereby improving the sensitivity of the reagent; however, if SA-tagged enzyme is used, the result of false positive is caused by strong nonspecific adsorption of the magnetic beads and the SA-enzyme, and the reagent performance is affected.
Therefore, there is a need in the art for a novel chemiluminescent kit for detecting HBsAg to increase the sensitivity of the reagent.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention adopts the following technical scheme:
in a first aspect the invention provides a magnetic particle detection HBsAg chemiluminescent kit comprising: reagent R1 prepared from antibody-magnetic particles, reagent R2 prepared from biotin label-sheep polyclonal antibody, reagent R3 prepared from neutravidin-alkaline phosphatase, and calibration product formed by diluting positive samples;
further, the reagent R1 prepared from the antibody-magnetic particles comprises the following steps:
S1: cleaning toluene sulfonyl magnetic particles with boric acid solution with the concentration of 0.05-0.2 mol/L, pH and the value of 9.45-9.55, adding HBs monoclonal antibody, rotating at the temperature of 35-40 ℃ for reaction for 16-24 hours to enable the anti-HBs monoclonal antibody to coat the toluene sulfonyl magnetic particles, cleaning, sealing and storing the HBsAg capture antibody-magnetic particles with an R1 buffer solution to enable the concentration of the HBsAg capture antibody-magnetic particles to be 8-12 mg/mL, and diluting the HBsAg capture antibody-magnetic particles to be 0.1-0.2 mg/mL with the R1 buffer solution to serve as a reagent R1; wherein the average particle diameter of the tosyl magnetic particles is 1-3 mu m, and HBs monoclonal antibody: the mass ratio of the toluene sulfonyl magnetic particles is 20-40 mug: 1mg, calculating the mass of the added HBs monoclonal antibody and the volume of the boric acid solution according to the concentration of the HBsAg capture antibody-magnetic particles, the mass of the crosslinked tosyl magnetic particles and the crosslinking ratio; further, the R1 buffer solution is phosphate buffer solution containing 0.5-5 wt% of bovine serum albumin, 0.1-1.0 wt% of surfactant and 0.5-2 wt% of ProClin and having a concentration of 0.01-0.03 mol/L, pH value of 7.15-7.25; preferably, the R1 buffer solution is phosphate buffer solution containing 1wt% of bovine serum albumin, 0.5 wt% of surfactant and 1wt% of ProClin, wherein the concentration of the phosphate buffer solution is 0.02mol/L, pH and the value of the phosphate buffer solution is 7.15-7.25;
Further, the preparation method of the reagent R2 prepared from the biotin-labeled goat polyclonal antibody comprises the following steps:
S2: replacing a sheep polyclonal antibody storage solution for identifying a plurality of sites of HBsAg with a phosphate buffer solution, and adding a biotin solution, wherein the molar ratio of the sheep polyclonal antibody for identifying the plurality of sites of HBsAg to the biotin is 1:8-12, reacting for 40-80 min at room temperature, and removing unreacted biotin after the reaction is finished to obtain the biotin-marked sheep polyclonal antibody; preferably, the molar ratio of the sheep polyclonal antibody recognizing the HBsAg to the biotin is 1:10;
S3: diluting the biotin-marked sheep polyclonal antibody to 3-10 mug/mL by using an R2 buffer solution to obtain a reagent R2 prepared from the biotin-marked sheep polyclonal antibody; further, the R2 buffer solution is a Tris buffer solution containing 3-6wt% of bovine serum albumin, 0.1-1.0wt% of casein, 0.05-2wt% of tween 20 and 0.01-0.03wt% of sodium azide, and the concentration is 0.05-0.2 mol/L, pH value of 7-8;
Further, the preparation method of the neutravidin-alkaline phosphatase comprises the following steps:
S4 neutravidin activation: weighing a certain amount of neutravidin, dissolving the neutravidin with a buffer solution without amino groups, and preparing the neutravidin according to the following steps: 2-iminothiolane (2-IT) molar ratio 1: 5-200 adding 2-IT solution, reacting at room temperature for 10-120 min, removing unreacted 2-IT to obtain activated neutravidin
S5: alkaline phosphatase (ALP) and succinimide-dPEG-maleimide (SM (PEG) 12) in a molar ratio of 1: 5-200, reacting at room temperature for 10-120 min, removing unreacted SM (PEG) 12, and obtaining activated alkaline phosphatase;
s6: activating neutral avidin: mixing the activated alkaline phosphatase according to a molar ratio of 1:5-5:1, and reacting overnight at 0-10 ℃ to obtain coupled neutral avidin and the activated alkaline phosphatase;
S7: taking a certain amount of N-ethyl maleimide (NEM), diluting to 50-200 mM by using a buffer solution without amino, adding the NEM solution into the coupled neutral avidin and activated alkaline phosphatase according to the same molar amount as 2-IT, reacting for 5-20 min at room temperature, removing unreacted NEM, purifying to obtain NA-AP, testing the NA-AP concentration, and diluting the NA-AP to 0.5-2 mug/mL by using an R3 buffer solution to serve as a reagent R3;
Further, the buffer solution without amino is selected from any one or more of phosphate buffer solution, 3-morpholinopropanesulfonic acid buffer solution and 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution;
Further, the R3 buffer solution is a Tris buffer solution containing 0.005-0.02 mol/L of magnesium chloride, 0.05-0.2 mmol/L of zinc chloride, 8-12 wt% of bovine serum, 0.2-1.0 wt% of gelatin, 0.05-0.2 wt% of tween 20 and 0.01-0.03 wt% of sodium azide, and having a concentration of 0.05-0.2 mol/L, pH of 7-8;
Further, the preparation method of the HBsAg calibration product comprises the following steps: diluting the HBsAg positive sample with a standard substance diluent to a concentration of 0.3-0.6 IU/mL, subpackaging, and storing at 2-8deg.C; further, the calibration product diluent is phosphate buffer solution containing 1-3wt% of bovine serum albumin, 15-30wt% of glycerol and 0.01-0.03wt% of sodium azide, and the concentration is 0.05-0.2 mol/L, pH value 7-8;
The second aspect of the invention provides a preparation method of a chemiluminescent kit for detecting HBsAg by magnetic particles, which comprises the following steps: the reagent R1, the reagent R2, the reagent R3 and the calibration product are subjected to subpackaging, sealing and packaging to obtain the reagent;
Advantageous effects
By adopting the technical scheme of the invention, the obtained magnetic particle detection HBsAg chemiluminescence kit has good detection specificity and high sensitivity.
Detailed Description
Example 1
The invention provides a reagent kit for detecting HBsAg magnetic particles by chemiluminescence, which comprises a reagent R1 prepared from antibody-magnetic particles, a reagent R2 prepared from biotin label-goat polyclonal antibody, a reagent R3 prepared from neutral avidin-alkaline phosphatase and a calibration product formed by diluting a positive sample.
The kit provided by the invention is prepared according to the following steps:
(1) Preparation of reagent R1 from antibody-magnetic microparticles: washing the tosyl magnetic particles 2 times by using boric acid solution with the concentration of 0.1mol/L, pH and the value of 9.45-9.55, then adding HBs monoclonal antibody, carrying out rotary reaction at the temperature of 37 ℃ for 20 hours to enable the anti-HBs monoclonal antibody to coat the tosyl magnetic particles, washing for 2 times by using R1 buffer solution, sealing and preserving the HBsAg capture antibody-magnetic particles, enabling the concentration of the HBsAg capture antibody-magnetic particles to be 10mg/mL, and then diluting the HBsAg capture antibody-magnetic particles to be 0.15mg/mL by using R1 buffer solution to serve as a reagent R1; wherein the average particle diameter of the tosyl magnetic particles is 1-3 mu m, and HBs monoclonal antibody: the mass ratio of the tosyl magnetic particles to the cross-linking is 30 mug: 1mg, calculating the mass of the added HBs monoclonal antibody and the volume of the boric acid solution according to the concentration of the HBsAg capture antibody-magnetic particles, the mass of the crosslinked tosyl magnetic particles and the crosslinking ratio;
Further, the R1 buffer solution is a phosphate buffer solution containing 1wt% of bovine serum albumin, 0.5 wt% of surfactant and 1 per mill ProClin of 300, wherein the concentration of the phosphate buffer solution is 0.02mol/L, pH and the concentration is 7.15-7.25;
(2) Preparation of R2 prepared from biotin-labeled goat polyclonal antibody
Preparation of biotin-labeled sheep polyclonal antibody: preparing a biotin solution with the concentration of 0.01 mol/L; after the sheep polyclonal antibody storage solution for recognizing a plurality of sites of HBsAg is replaced by phosphate buffer, biotin solution is added, and the molar ratio of biotin is 1:10; reacting for 60min at room temperature; after the reaction is finished, unreacted biotin is removed by adopting a desalting column, so that biotin-marked sheep polyclonal antibody is obtained; testing the protein concentration, and diluting the protein concentration to 5 mug/mL by using an R2 buffer solution to serve as a reagent R2; the R2 buffer is a Tris buffer with a concentration of 0.1mol/L, pH and a value of 7.4, and contains 5wt% of bovine serum albumin, 0.5wt% of casein, 0.1wt% of surfactant and 0.02wt% of sodium azide;
(3) Preparation of neutravidin-alkaline phosphatase:
(3-1) neutravidin activation: a certain amount of neutravidin is weighed and dissolved by phosphate buffer. According to neutral avidin: 2-IT molar ratio 1:100 adding a 2-IT solution, and reacting for 60min at room temperature; removing unreacted 2-IT by a desalting column (other desalting modes);
(3-2) alkaline phosphatase activation: alkaline phosphatase (ALP) and SM (PEG) 12 were prepared according to ALP: SM (PEG) 12 solution is added according to the molar ratio of SM (PEG) 12 (1:100), and the reaction is carried out for 10-120min at room temperature; unreacted SM (PEG) 12 was removed with a desalting column;
(3-3) coupling of streptavidin and alkaline phosphatase after activation: according to neutral avidin: ALP molar ratio (1:1) was mixed and reacted overnight at 4 ℃. An amount of NEM was taken and dissolved to 100mM in a buffer containing no amino group. According to NEM: NEM solution is added in the molar ratio of 2-IT (1:1), and the reaction is carried out for 10min at room temperature, and unreacted NEM is removed by a desalting column; purifying to obtain NA-AP, testing NA-AP concentration, and diluting to 1 mug/mL by using R3 buffer solution as a reagent R3; the R3 buffer is a Tris buffer with a concentration of 0.1mol/L, pH and a value of 7.4, and contains 0.01mol/L magnesium chloride, 0.1mmol/L zinc chloride, 10wt% bovine serum, 0.5wt% gelatin, 0.1wt% surfactant and 0.02wt% sodium azide;
(4) Preparation of HBsAg calibration: diluting the HBsAg positive sample with a standard dilution to a concentration of 0.5IU/mL; subpackaging, and storing at 2-8 ℃;
The standard product diluent is phosphate buffer solution with the concentration of 0.1mol/L, pH and the value of 7.4, and contains 2 weight percent of bovine serum albumin, 20 weight percent of glycerol and 0.02 weight percent of sodium azide;
(5) And subpackaging, sealing and packaging the reagent R1, the reagent R2, the reagent R3 and the calibration product to obtain the chemiluminescent kit.
Experimental example 1
1. The detection process using the chemiluminescent kit of the invention comprises the following steps:
Scaling: the full-automatic chemiluminescence analyzer is used as a detection tool, the reaction mode is a two-step method, namely, 75 mu L of a calibration product, 50 mu L of a reagent R2 (biotin-labeled sheep polyclonal antibody and R2 buffer) and 30 mu L of a reagent R1 (HBsAg capture antibody-magnetic particles and R1 buffer) are sequentially added into the instrument, the reaction is carried out for 20min, 100 mu L of a reagent R3 (neutral avidin-alkaline phosphatase and R3 buffer or streptavidin-alkaline phosphatase and R3 buffer) is added after magnetic separation and cleaning, the reaction is carried out for 10min, 100 mu L of a substrate is added after magnetic separation and cleaning, the luminous intensity is read, the HBsAg calibration product is tested for 3 times, the luminous value is obtained, and the average value/100 of the luminous value is used as the cutoff value.
Test sample: the full-automatic luminescence analyzer is used as a detection tool, the reaction mode is a two-step method, namely, the instrument sequentially adds 75 mu L of sample, 50 mu L of reagent R2 and 30 mu L of reagent R1, the reaction is carried out for 20min, 100 mu L of reagent R3 is added after magnetic separation and cleaning, the reaction is carried out for 10min, 100 mu L of substrate is added after magnetic separation and cleaning, the luminescence value is read after incubation for 5min, and the sample test result S/CO=luminescence value/cut off.
2. The reference kit 1 is a one-step reaction mode, and the detection steps are as follows:
Scaling: the full-automatic chemiluminescence analyzer is used as a detection tool, the reaction mode is a one-step method, i.e. the instrument is sequentially added
75 Mu L of blood sample, 50 mu L of reagent R2 (goat polyclonal antibody-alkaline phosphatase and R2 buffer), 30 mu L of reagent R1 (HBsAg capture antibody-magnetic particles and R1 buffer), reacting for 20min, adding 100 mu L of substrate after magnetic separation and washing, reading luminous intensity, testing HBsAg calibration, and taking luminous value/100 as cut off value.
Test sample: the full-automatic chemiluminescence analyzer is used as a detection tool, the reaction mode is a one-step method, i.e. the instrument is sequentially added
75 Mu L of blood sample, 50 mu L of reagent R2 (goat polyclonal antibody-alkaline phosphatase and R2 buffer), 30 mu L of reagent R1 (HBsAg capture antibody-magnetic particles and R1 buffer), reaction for 20min, magnetic separation and washing, adding 100 mu L of substrate, incubating for 5min, reading a luminescence value, and testing a sample to obtain a result of S/CO=luminescence value/cut off.
3. The reference kit 2 is a two-step reaction mode, and the detection steps are as follows:
The full-automatic chemiluminescence analyzer is used as a detection tool, and the reaction mode is a two-step method, namely, the instrument is sequentially added
75. Mu.L of blood sample, 50. Mu.L of reagent R2 (biotin-labeled sheep polyclonal antibody and R2 buffer), 30. Mu.L of reagent R1 (HBsAg capture antibody-magnetic particle and R1 buffer), reaction for 20min, magnetic separation washing, addition of 100. Mu.L of reagent R3 (neutravidin-alkaline phosphatase and R3 buffer or streptavidin-alkaline phosphatase and R3 buffer), reaction for 10min, magnetic separation washing, addition of 100. Mu.L of substrate, reading luminescence intensity, testing of HBsAg standard, and using luminescence value/100 as cut off value.
Test sample: the full-automatic luminescence analyzer is used as a detection tool, the reaction mode is a two-step method, namely, the instrument sequentially adds 75 mu L of sample, 50 mu L of reagent R2 and 30 mu L of reagent R1, the reaction is carried out for 20min, 100 mu L of reagent R3 is added after magnetic separation and cleaning, the reaction is carried out for 10min, 100 mu L of substrate is added after magnetic separation and cleaning, the luminescence value is read after incubation for 5min, and the sample test result S/CO=luminescence value/cut off.
Experimental example 2
1. Clinical sample testing: 40 samples are collected, the samples are tested by using the chemiluminescent kit, the reference kit 1 and the reference kit 2, the test results are shown in the table 1, the detection result is judged by a critical value, the critical value (Cut Off value, CO value) =quality control serum average luminescent value/100, the sample to be tested is judged to be negative when the detection luminescent value/critical value (S/CO) is less than 1.00, and the sample to be tested is judged to be positive when the S/CO is more than or equal to 1.00. As shown in table 1.
TABLE 1
Testing the same sample, wherein the positive coincidence rate of the invention is 100 percent consistent with that of the reference kit 1 and the reference kit 2, and the negative coincidence rate of the invention is 100 percent greater than that of the reference kit 1 (83 percent) and the reference kit 2 (87 percent), which indicate that the specificity of the kit is good; the same calibration product is tested, the luminous value of the kit is higher than the luminous value 302% of the reference kit 1, and the luminous value is consistent with the reference kit 2, so that the kit has high sensitivity, and the sensitivity of the two-step method is better than that of the one-step method. Among the 3 different sample types, the negative coincidence rate of the serum, EDTA plasma and heparin plasma of the kit is 100 percent, which is higher than that of the reference kit 1 (100 percent, 70 percent and 90 percent) and the reference kit 2 (100 percent, 70 percent and 90 percent), which indicates that the kit has strong capability of resisting different types of anticoagulant interference and good specificity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that the invention is not limited thereto, but various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the present invention.
Claims (9)
1. A magnetic particle detection HBsAg chemiluminescent kit comprising: reagent R1 prepared from antibody-magnetic particles, reagent R2 prepared from biotin label-goat polyclonal antibody, reagent R3 prepared from neutravidin-alkaline phosphatase, and a calibration product formed by diluting a positive sample.
2. The magnetic particle detection HBsAg chemiluminescent kit of claim 1 wherein the antibody-magnetic particle-made reagent R1 comprises the steps of:
Cleaning toluene sulfonyl magnetic particles with boric acid solution with the concentration of 0.05-0.2 mol/L, pH and the value of 9.45-9.55, adding HBs monoclonal antibody, rotating at the temperature of 35-40 ℃ for reaction for 16-24 hours to enable the anti-HBs monoclonal antibody to coat the toluene sulfonyl magnetic particles, cleaning, sealing and storing the HBsAg capture antibody-magnetic particles with an R1 buffer solution to enable the concentration of the HBsAg capture antibody-magnetic particles to be 8-12 mg/mL, and diluting the HBsAg capture antibody-magnetic particles to be 0.1-0.2 mg/mL with the R1 buffer solution to serve as a reagent R1; wherein the average particle diameter of the tosyl magnetic particles is 1-3 mu m, and HBs monoclonal antibody: the mass ratio of the toluene sulfonyl magnetic particles is 20-40 mug: 1mg, the mass of HBsAb added and the volume of boric acid solution were calculated from the HBsAg capture antibody-magnetic particle concentration, the mass of crosslinked tosyl magnetic particles and the crosslinking ratio.
3. The magnetic particle detection HBsAg chemiluminescence kit of claim 2, wherein the R1 buffer solution is phosphate buffer solution containing 0.5-5 wt% of bovine serum albumin, 0.1-1.0 wt% of surfactant, 0.5-2 wt% of ProClin and 0.01-0.03 mol/L, pH value of 7.15-7.25.
4. The magnetic particle detection HBsAg chemiluminescence kit of claim 1, wherein the preparation method of the reagent R2 prepared from the biotin-labeled sheep polyclonal antibody comprises the following steps:
S2: replacing a sheep polyclonal antibody storage solution for identifying a plurality of sites of HBsAg with a phosphate buffer solution, and adding a biotin solution, wherein the molar ratio of the sheep polyclonal antibody for identifying the plurality of sites of HBsAg to the biotin is 1:8-12, reacting for 40-80 min at room temperature, and removing unreacted biotin after the reaction is finished to obtain the biotin-marked sheep polyclonal antibody; preferably, the molar ratio of the sheep polyclonal antibody recognizing the HBsAg to the biotin is 1:10;
S3: diluting the biotin-marked sheep polyclonal antibody to 3-10 mug/mL by using an R2 buffer solution to obtain a reagent R2 prepared from the biotin-marked sheep polyclonal antibody.
5. The magnetic particle detection HBsAg chemiluminescent kit of claim 4 wherein the R2 buffer is a Tris buffer containing 3-6 wt.% bovine serum albumin, 0.1-1.0 wt.% casein, 0.05-2 wt.% Tween 20, and 0.01-0.03 wt.% sodium azide at a concentration of 0.05-0.2 mol/L, pH to 7-8.
6. The magnetic particle detection HBsAg chemiluminescent kit of claim 1 wherein the method of preparation of neutravidin-alkaline phosphatase comprises the steps of:
S4 neutravidin activation: weighing a certain amount of neutravidin, dissolving the neutravidin with a buffer solution without amino groups, and preparing the neutravidin according to the following steps: 2-iminothiolane (2-IT) molar ratio 1: adding 2-IT solution into 5-200, reacting for 10-120 min at room temperature, removing unreacted 2-IT to obtain activated neutral avidin;
s5: alkaline phosphatase (ALP) and succinimide-dPEG-maleimide (SM (PEG) 12) in a molar ratio of 1: 5-200, reacting at room temperature for 10-120 min, removing unreacted SM (PEG) 12, and obtaining activated alkaline phosphatase;
s6: activating neutral avidin: mixing the activated alkaline phosphatase according to a molar ratio of 1:5-5:1, and reacting overnight at 0-10 ℃ to obtain coupled neutral avidin and the activated alkaline phosphatase;
S7: taking a certain amount of N-ethyl maleimide (NEM), diluting to 50-200 mM by using a buffer solution without amino, adding the NEM solution into the coupled neutral avidin and activated alkaline phosphatase according to the same molar amount as 2-IT, reacting for 5-20 min at room temperature, removing unreacted NEM, purifying to obtain NA-AP, testing the NA-AP concentration, and diluting the NA-AP to 0.5-2 mug/mL by using an R3 buffer solution to serve as a reagent R3.
7. The magnetic particle detection HBsAg chemiluminescent kit of claim 6 wherein the buffer solution free of amino groups is selected from any one or more of phosphate buffer solution, 3-morpholinopropanesulfonic acid buffer solution, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.
8. The magnetic particle detection HBsAg chemiluminescent kit of claim 6 wherein the R3 buffer is a Tris buffer containing 0.005-0.02 mol/L magnesium chloride, 0.05-0.2 mmol/L zinc chloride, 8-12 wt.% bovine serum, 0.2-1.0 wt.% gelatin, 0.05-0.2 wt.% Tween 20, and 0.01-0.03 wt.% sodium azide at a concentration of 0.05-0.2 mol/L, pH to 7-8.
9. The magnetic particle detection HBsAg chemiluminescent kit of claim 6 wherein the preparation method of the HBsAg marker comprises the steps of: diluting the HBsAg positive sample with a standard substance diluent to a concentration of 0.3-0.6 IU/mL, subpackaging, and storing at 2-8deg.C; further, the standard dilution is phosphate buffer solution containing 1-3wt% of bovine serum albumin, 15-30wt% of glycerol and 0.01-0.03wt% of sodium azide, and the concentration is 0.05-0.2 mol/L, pH value 7-8.
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