Magnetic particle for virus detection and preparation method and application thereof

Abstract

The invention relates to the technical field of immunodetection, in particular to magnetic particles for virus detection and a preparation method and application thereof. According to the magnetic particle for virus detection provided by the invention, the magnetic particle is firstly coated with the hepatitis B virus core antibody and then coated with the hepatitis B virus core antigen; therefore, the influence of various factors such as various mechanical shear forces and ion strength changes on the dynamic balance of virus particle assembly of the hepatitis B virus core antigen in the reaction process is reduced, and the detection precision is improved. By adopting the hepatitis B virus detection kit provided by the invention, through detection and analysis, the detection precision of the hepatitis B virus core antibody is remarkably improved, and the detection luminescence value CV value is reduced from 7.79%-33.39% to 1.74%-3.10%.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to magnetic particles for virus detection and a preparation method and application thereof. Background Art

[0002] Anti-HBc is the abbreviation of hepatitis B virus core antibody, which can be abbreviated as anti-HBc. It is produced by activating human T lymphocytes with HBVCoreAntigen (hepatitis B virus core antigen). HBV CoreAntigen is the core part of the hepatitis B virus, containing double-bonded DNA, specific DNAP and protein, and is a direct sign of the presence of hepatitis B virus. HBVCoreAntigen exists in the core of Dane (hepatitis B virus large spherical particle) particles, and is the structural protein viral nucleocapsid protein of HBV, which has important biological characteristics and clinical pathological significance.

[0003] After accidental contact with the blood and body fluids of HBV (hepatitis B virus) infected persons, HBV DNA, HBsAg (hepatitis B virus surface antigen), anti-HBs (hepatitis B virus surface antibody), HBeAg (hepatitis B virus e antigen), anti-HBe (hepatitis B virus e antibody), anti-HBc (hepatitis B virus core antibody), ALT (alanine aminotransferase) and aspartate aminotransferase (AST) should be tested immediately and rechecked within 3 and 6 months. When acute or chronic hepatitis B is diagnosed, it should be reported to the local Center for Disease Control and Prevention in accordance with regulations, and it is recommended that the patient's family members be tested for serum HBsAg, anti-HBc and anti-HBs, and susceptible persons (those with negative results for all three markers) should be vaccinated with hepatitis B vaccine.

[0004] High concentrations of anti-HBc (mainly IgM) indicate current infection, which often coexists with HBsAg, and often causes liver damage and abnormal liver enzymes. In follow-up of patients with high concentrations of anti-HBc, the decrease in anti-HBc concentrations coincides with the gradual recovery of liver enzymes. Low concentrations of anti-HBc (mainly IgG) indicate past infection, which often coexists with anti-HBs, and usually causes no liver damage or the liver damage has long been dormant.

[0005] Immunological detection of serum markers is an immune reaction between an antigen and the specific antibodies stimulated by it. There are many immunological detection methods, and the competitive method is a commonly used detection method in clinical practice. The detection principle of the present invention is to coat magnetic particles with hepatitis B core antibody / antigen and label the core antibody with horseradish peroxidase to prepare an enzyme conjugate. An antigen-enzyme-labeled antibody complex is formed through an immune reaction, and the complex catalyzes the luminescent substrate to emit photons, and the luminescence intensity is inversely proportional to the content of the core antibody.

[0006] The difficulty lies in that HBV CoreAntigen virus particles are formed by a series of assembly of C protein dimers. In the process of dimer assembly into particles, there are dynamic changes of self-assembly and disassembly, and the dynamic equilibrium of this process depends on the non-covalent bonding between domains. This force is very susceptible to mechanical shear force, ionic strength, temperature, pH, etc.

[0007] Since covalent bonds are sensitive to various mechanical forces in each reaction step of the fully automatic chemi-immunoassay analyzer, and the reaction process is accompanied by changes in ionic strength, the precision of repeated sample testing is very susceptible to interference when testing hepatitis B core antibodies. The common manifestation is an increase in the CV value of the precision indicator. The specific interference mechanism is as follows:

[0008] 1) The self-assembly and disassembly of dimers in HBV CoreAntigen occur simultaneously, and this mechanism will spontaneously maintain a certain number of free dimers to avoid assembly kinetic traps and maintain the dynamic balance of assembly kinetics;

[0009] 2) During different tests, the concentration of free dimers will deviate to a certain extent due to the relative imbalance of the self-assembly and disassembly rates of HBV CoreAntigen, resulting in different degrees of changes in the spatial structure and number of spatial epitopes of HBV CoreAntigen, thus leading to certain differences between different tests;

[0010] After HBV CoreAntigen particles self-assemble into virus-like particles in vitro, a certain number of free dimers will exist in themselves to avoid assembly kinetic traps. However, once the HBV CoreAntigen particles assembled by non-covalent interactions are subjected to mechanical shear force, ionic strength, and pH changes, the dynamic balance of particle self-assembly and disassembly processes will be destroyed, ultimately leading to varying degrees of changes in the spatial structure of the heart antigen and the number of spatial epitopes, which will have a certain impact on the precision of different test results. Summary of the invention

[0011] In view of this, the technical problem to be solved by the present invention is to provide magnetic microparticles for virus detection and their preparation method and application. The present invention can improve the precision of hepatitis B virus core antibody detection by first coating the magnetic microparticles with hepatitis B virus core antibodies and then coating the magnetic microparticles with hepatitis B virus core antigens.

[0012] The invention provides magnetic particles for virus detection, wherein the inner core of the magnetic particles is a magnetic particle, and the surface of the magnetic particles is coated with hepatitis B virus core antibody and hepatitis B virus core antigen in sequence.

[0013] The present invention provides a method for preparing magnetic particles for virus detection, comprising:

[0014] Step 1, taking magnetic particles for activation;

[0015] Step 2, mixing, reacting and blocking the activated magnetic particles with hepatitis B virus core antibodies to obtain the first coated magnetic particles;

[0016] Step 3: The magnetic particles coated for the first time are mixed with the hepatitis B virus core antigen, reacted, and sealed to obtain the magnetic particles for virus detection.

[0017] In some embodiments, in step 1, the activated reagent includes EDC and NHS, the activated buffer includes at least one of PBS buffer, MES buffer and Hepes buffer, the mass ratio of the EDC to the magnetic particles is 1:(0.2~10), the concentration of the activated buffer is 0.005~0.02M, and the pH of the activated buffer is 6.0~7.35.

[0018] In some specific embodiments, the mass ratio of the EDC to the magnetic particles is 1:0.2, and the activated buffer is a PBS buffer with a concentration of 0.01 M and a pH of 7.35.

[0019] In some embodiments, in step 2 and step 3, the blocking solution includes a blocking buffer containing 1 vol% to 2 vol% bovine serum albumin or casein, the blocking buffer includes at least one of PBS buffer, Tris buffer, BSA solution and casein solution, the concentration of the blocking buffer is 0.005 to 0.02 M, and the pH of the blocking buffer is 6.0 to 7.35.

[0020] In some specific embodiments, the blocking solution is a PBS buffer containing 1 vol% bovine serum albumin, the concentration of the PBS buffer is 0.01 M, and the pH of the PBS buffer is 7.35.

[0021] The present invention provides the use of the magnetic particles for virus detection or the magnetic particles for virus detection prepared by the preparation method in the preparation of a hepatitis B virus detection kit.

[0022] The invention provides a hepatitis B virus detection kit, comprising the magnetic particles for virus detection or the magnetic particles for virus detection prepared by the preparation method.

[0023] In some embodiments, the method further comprises an enzyme conjugate solution and a sample diluent, wherein:

[0024] The enzyme conjugate solution comprises a horseradish peroxidase-labeled hepatitis B virus core antibody and an enzyme conjugate buffer in a volume ratio of 1:(300-800), wherein the enzyme conjugate buffer is a 0.01M-0.05M PBS buffer and / or a Tris buffer at a pH of 6.0-7.35 containing 1vol%-2vol% of bovine serum albumin;

[0025] The sample diluent includes at least one of PBS buffer, Tris buffer and sodium citrate buffer with a pH of 4.0 to 7.35 and a concentration of 0.01M to 0.05M.

[0026] In some specific embodiments, the enzyme conjugate buffer is a 0.05M Tris buffer at pH 7.35 containing 1 vol% bovine serum albumin, and the volume ratio of the horseradish peroxidase-labeled hepatitis B virus core antibody to the enzyme conjugate buffer is 1:500;

[0027] The sample diluent is 0.01 M sodium citrate buffer at pH 4.0.

[0028] Compared with the prior art, the magnetic particles for virus detection provided by the present invention are coated with hepatitis B virus core antibodies first and then with hepatitis B virus core antigens, thereby reducing the influence of various factors such as mechanical shear forces and ionic strength changes in the reaction process on the dynamic equilibrium of viral particle assembly of hepatitis B virus core antigens, thereby improving the precision of detection. The hepatitis B virus core antibody detection kit provided by the present invention is used to perform detection and analysis, and the detection precision of hepatitis B virus core antibodies is significantly improved, so that the CV value of the detection luminescence value is reduced from about 7.79%-33.39% to 1.74%-3.10%. DETAILED DESCRIPTION

[0029] The present invention provides magnetic particles for virus detection and their preparation methods and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0030] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0031] Example 1

[0032] A method for improving the precision of hepatitis B virus core antibody detection (magnetic particle chemiluminescence method), characterized in that: the magnetic particles are first coated with hepatitis B virus core antibodies immunized with hepatitis B virus core antigen, and then coated with hepatitis B virus core antigen.

[0033] Experimental group 1 specifically includes:

[0034] The preparation process of magnetic particle suspension is as follows: add magnetic particles and cross-linking agent EDC / NHS to a coating buffer with non-magnetic stirring for activation (the ratio of EDC to magnetic particles is 1 mg / mg), add hepatitis B virus core antibody immunized with hepatitis B virus core antigen for one coating after activation, add blocking solution for blocking after coating, add HBV CoreAntigen for a second coating after blocking, and add magnetic particle preservation solution for storage at 2-8°C after coating.

[0035] The reaction system of the magnetic particle suspension in the experimental group was: coating buffer, magnetic particle stock solution, core antibody to be coated, HBV CoreAntigen, EDC / NHS and magnetic particle blocking solution, magnetic particle sealing solution;

[0036] Coating buffer: 0.01M PBS buffer, pH 7.35;

[0037] Magnetic particles: MS300carboxyl magnetic particle suspension (100 mg / mL) provided by Merck;

[0038] Hepatitis B virus core antibody to be coated: commercially available HBV CoreAntigen monoclonal antibody; purchased from Zhengzhou Imino Biotechnology Co., Ltd., catalog number: MB32

[0039] HBV CoreAntigen to be coated: Commercially available yeast expressing HBV CoreAntigen; purchased from Zhengzhou Imino Biotechnology Co., Ltd., catalog number: 21HBC. The preparation process of HBV CoreAntigen is as follows: First, the gene encoding HBcAg is introduced into yeast cells by recombinant DNA technology, for example, yeast strains such as Pichia pastoris or Hansenula polymorpha are used. During the culture process, yeast cells are grown in a specific culture medium and expression is initiated by adding inducers such as methanol. After induction, the core antigen accumulates in the nucleus and cytoplasm and assembles into core particles. Finally, high-purity HBcAg is obtained through centrifugation and purification steps.

[0040] EDC / NHS: Commercially available;

[0041] Activation buffer: 0.01M PBS buffer, pH 7.35;

[0042] Magnetic particle blocking solution: 0.01M pH7.35 PBS buffer + 1vol% bovine serum albumin;

[0043] Control group 1: The preparation process of magnetic microparticle suspension is as follows: magnetic microparticles and cross-linking agent EDC / NHS are added to the coating buffer with non-magnetic stirring for activation (the ratio of EDC to magnetic microparticles is 1 mg / mg), HBV CoreAntigen is added for coating after activation, blocking solution is added for blocking after coating, and magnetic microparticle preservation solution is added for storage at 2-8°C after blocking.

[0044] The difference between the control group 1 and the above experimental group is that the control group 1 only coated the HBVCoreAntigen on the magnetic particles, and the other steps were the same as those of the above experimental group.

[0045] Control group 2: The difference from the above experimental group is that the activation buffer used in control group 2 is 0.01 M pH 6.0 MES buffer only in the preparation process of magnetic particle suspension, and the other steps are the same as those of the above experimental group.

[0046] Control group 3: The difference from the above experimental group is that the activation buffer used in control group 3 is 0.01 M pH 6.8 Hepe buffer only in the preparation process of magnetic particle suspension, and the other steps are the same as those of the above experimental group.

[0047] Control group 4: The difference from the above experimental group is that the blocking buffer used in control group 4 is 0.01M pH 7.35 PBS buffer + 2 vol% bovine serum albumin only in the preparation process of the magnetic particle suspension, and the other steps are the same as the above experimental group.

[0048] Control group 5: The difference from the above experimental group is that the blocking buffer used in control group 5 is 0.01M pH 7.35 PBS buffer + 2 vol% casein only in the preparation process of magnetic particle suspension, and the other steps are the same as the above experimental group.

[0049] Control group 6: The difference from the above experimental group is that the blocking buffer used in control group 6 is 0.01M MES buffer with pH 6.0 + 1 vol% bovine serum albumin only in the preparation process of magnetic particle suspension, and the other steps are the same as the above experimental group.

[0050] Control group 7: The difference from the above experimental group is that the blocking buffer used in control group 7 is 0.01M pH 6.8 Hepe buffer + 1 vol% bovine serum albumin only in the preparation process of the magnetic particle suspension, and the other steps are the same as the above experimental group.

[0051] The effects of magnetic particle suspensions prepared by different magnetic particle coating processes, different activation buffers and different blocking buffers on the precision of core antibody immunoassay were compared.

[0052] The experimental group and control group reagents were used to test 3 known Anti-HBc (+) human serum samples and 3 known Anti-HBc (-) human serum samples, including 20uL of magnetic particle suspension, 50uL of enzyme conjugate, 30uL of sample diluent, and 50uL of sample, and the test was performed by a fully automatic chemiluminescence immunoassay analyzer. Each sample was tested 10 times, and the CV value of the luminescence value was calculated. The test results are shown in Table 1-8:

[0053] Table 1

[0054]

[0055] Table 2

[0056]

[0057] Table 3

[0058]

[0059] Table 4

[0060]

[0061] Table 5

[0062]

[0063] Table 6

[0064]

[0065] Table 7

[0066]

[0067] Table 8

[0068]

[0069] The results show that:

[0070] Different activation buffers and blocking buffers have different effects on precision. The preferred activation buffer is: 0.01M pH7.35 PBS buffer; magnetic particle blocking solution is: 0.01M pH7.35 PBS buffer + 1 vol% bovine serum albumin.

[0071] Compared with magnetic particles coated with hepatitis B virus core antigen alone, the coating method of the magnetic particles provided by the present invention, while optimizing the formulation, significantly improves the detection precision, and the CV value of the detection luminescence value is reduced from about 14.73%-20.32% to 1.75%-3.14%.

[0072] Example 2

[0073] A hepatitis B virus core antibody detection (magnetic particle chemiluminescence method) kit, comprising: a magnetic particle suspension, an enzyme conjugate and a sample diluent;

[0074] The preparation process of the magnetic particle suspension of experimental group 2 was the same as that of Example 1.

[0075] Experimental Group 2 Enzyme Conjugate:

[0076] Includes horseradish peroxidase-labeled hepatitis B virus core antibody; the volume ratio to buffer is 1:500.

[0077] Wherein, the buffer solution is a 0.05M Tris buffer solution with a pH of 7.35, containing a final concentration of 1% bovine serum albumin.

[0078] Sample dilution of experimental group 2:

[0079] Wherein, buffer: 0.01M sodium citrate buffer with pH 4.0.

[0080] Control group 8 is different from the above experimental groups in that control group 8 is only in the enzyme conjugate:

[0081] The volume ratio of the hepatitis B virus core antibody labeled with horseradish peroxidase to the buffer solution was 1:300. The remaining steps were consistent with those of the above experimental group 2.

[0082] Control group 9 is different from the above experimental groups in that control group 9 is only in the enzyme conjugate:

[0083] The volume ratio of the hepatitis B virus core antibody labeled with horseradish peroxidase to the buffer solution was 1:800. The remaining steps were consistent with those of the above experimental group 2.

[0084] The difference between the control group 10 and the above experimental group is that the control group 10 contains only 2% final concentration of bovine serum albumin in the enzyme conjugate buffer: 0.05M Tris buffer with pH 7.35. The rest of the steps are the same as those of the above experimental group 2.

[0085] The difference between the control group 11 and the above experimental group is that the control group 11 is only in the enzyme conjugate buffer: 0.05M pH 7.35 PBS buffer, containing 1% final concentration of bovine serum albumin. The rest of the steps are the same as those of the above experimental group 2.

[0086] The difference between the control group 12 and the above experimental groups is that the control group 12 is a sample dilution:

[0087] Buffer: 0.01M PBS buffer, pH 7.35. The remaining steps were the same as those of Experimental Group 2 above.

[0088] The difference between the control group 13 and the above experimental groups is that the control group 13 is a sample dilution:

[0089] Buffer: 0.01M Tris buffer, pH 7.35. The remaining steps were the same as those of Experimental Group 2 above.

[0090] Compare the effects of the experimental group 2 and control group 8-control group 13 kits on the precision of hepatitis B virus core antibody immunoassay.

[0091] The experimental group and control group reagents were used to test 3 known Anti-HBc (+) human serum samples and 3 known Anti-HBc (-) human serum samples, including 20uL of magnetic particle suspension, 50uL of enzyme conjugate, 30uL of sample diluent, and 50uL of sample. The test was performed by a fully automatic chemiluminescence immunoassay analyzer. Each sample was tested 10 times and the CV value of the luminescence value was calculated. The test results are shown in Table 9-15:

[0092] Table 9

[0093]

[0094] Table 10

[0095]

[0096] Table 11

[0097]

[0098] Table 12

[0099]

[0100] Table 13

[0101]

[0102] Table 14

[0103]

[0104] Table 15

[0105]

[0106] The results show that:

[0107] Different enzyme conjugate formulas and sample diluent formulas have different effects on precision. Horseradish peroxidase-labeled hepatitis B virus core antibody is preferred; the volume ratio to the buffer is 1:500. Buffer: 0.05M Tris buffer at pH 7.35, containing 1% final concentration of bovine serum albumin.

[0108] Diluent: 0.01 M sodium citrate buffer, pH 4.0.

[0109] Compared with magnetic particles coated with hepatitis B virus core antigen alone, the coating method of the magnetic particles provided by the present invention and the optimization of the formula, the hepatitis B virus core antibody detection kit prepared by this method has significantly improved detection precision, and the detection luminescence value CV value is reduced from about 7.79%-33.39% to 1.74%-3.10%.

[0110] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Magnetic particles for virus detection, characterized in that The core is a magnetic particle, and the surface of the magnetic particle is coated with hepatitis B virus core antibody and hepatitis B virus core antigen in sequence.

2. The method for preparing magnetic particles for virus detection according to claim 1, characterized in that: include: Step 1, taking magnetic particles for activation; Step 2, mixing, reacting and blocking the activated magnetic particles with hepatitis B virus core antibodies to obtain the first coated magnetic particles; Step 3: The magnetic particles coated for the first time are mixed with the hepatitis B virus core antigen, reacted, and sealed to obtain the magnetic particles for virus detection.

3. The preparation method according to claim 2, characterized in that: In step 1, the activated reagent includes EDC and NHS, the activated buffer includes at least one of PBS buffer, MES buffer and Hepes buffer, the mass ratio of EDC to the magnetic particles is 1: (0.2~10), the concentration of the activated buffer is 0.005~0.02M, and the pH of the activated buffer is 6.0~7.

35.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the EDC to the magnetic particles is 1:0.2, and the activated buffer is a PBS buffer with a concentration of 0.01 M and a pH of 7.

35.

5. The preparation method according to any one of claims 2 to 4, characterized in that In step 2 and step 3, the blocking solution includes a blocking buffer containing 1 vol% to 2 vol% bovine serum albumin or casein, the blocking buffer includes at least one of PBS buffer, Tris buffer, BSA solution and casein solution, the concentration of the blocking buffer is 0.005 to 0.02 M, and the pH of the blocking buffer is 6.0 to 7.

35.

6. The preparation method according to claim 5, characterized in that: The blocking solution is a PBS buffer containing 1 vol% bovine serum albumin, the concentration of the PBS buffer is 0.01 M, and the pH of the PBS buffer is 7.

35.

7. Use of the magnetic particles for virus detection according to claim 1 or the magnetic particles for virus detection prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a hepatitis B virus detection kit.

8. A hepatitis B virus detection kit, characterized in that: Including the magnetic particles for virus detection according to claim 1 or the magnetic particles for virus detection prepared by the preparation method according to any one of claims 2 to 6.

9. The hepatitis B virus detection kit according to claim 8, characterized in that: Also included is an enzyme conjugate solution and a sample diluent, wherein: The enzyme conjugate solution comprises a horseradish peroxidase-labeled hepatitis B virus core antibody and an enzyme conjugate buffer in a volume ratio of 1:(300-800), wherein the enzyme conjugate buffer is a 0.01M-0.05M PBS buffer and / or a Tris buffer at a pH of 6.0-7.35 containing 1vol%-2vol% of bovine serum albumin; The sample diluent includes at least one of PBS buffer, Tris buffer and sodium citrate buffer with a pH of 6.0 to 7.35 and a concentration of 0.01 to 0.05 M.

10. The hepatitis B virus detection kit according to claim 9, characterized in that: The enzyme conjugate buffer is a 0.05M Tris buffer with a pH of 7.35 and containing 1 vol% bovine serum albumin, and the volume ratio of the horseradish peroxidase-labeled hepatitis B virus core antibody to the enzyme conjugate buffer is 1:500; The sample diluent is 0.01 M sodium citrate buffer at pH 4.0.

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