Hepatitis B virus HBcrAg detection method and kit

Abstract

The invention discloses a hepatitis B virus HBcrAg detection kit, which comprises an immunomagnetic bead working solution, a sample treatment solution and an HBcrAg labeling working solution, the immunomagnetic bead working solution comprises immunomagnetic beads coated with a coating antibody and a magnetic bead diluent; the sample treatment liquid is used for removing outer membranes of hepatitis B virus particles under mild conditions and dissociating hepatitis B virus core particles to release HBcrAg; the HBcrAg marking working solution comprises a marking antibody coupled with a marker and a marker diluent. The detection kit can effectively split the hepatitis B virus core particles under mild conditions, and can capture the hepatitis B virus core particles so as to avoid interference of endogenous antibodies, so that the content of HBcrAg in a sample is more accurately detected, the HBcAg index is measured, the traditional defect of two-in-half hepatitis B is made up, and the detection kit can be used for detecting the HBcrAg content of the sample and detecting the HBcrAg index. And iteration from'two pairs of hepatitis B 'to'three pairs of hepatitis B' is realized. The invention further discloses an HBcrAg detection method adopting the HBcrAg detection kit for the hepatitis B virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a hepatitis B virus HBcrAg detection method and a kit. Background Art

[0002] Hepatitis B virus (HBV) infection is a global public health issue. Viral biomarker testing plays an important role in the diagnosis, prognosis, and monitoring of patients with chronic HBV infection before and after treatment. However, currently used HBV DNA or serological two-pair half assays for viral activity and antiviral efficacy evaluation have limitations.

[0003] The fundamental reason chronic hepatitis B is difficult to cure is the presence of highly stable covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. Currently, the most commonly used serological marker for cccDNA activity in clinical practice is the hepatitis B "two-and-a-half" test, which specifically measures five HBV antigens and antibodies: HBsAg, HBeAg, HBsAb, HBeAb, and HBcAb. HBsAg and HBeAg, as antigenic markers, do not accurately reflect cccDNA activity. First, during prolonged treatment for chronic hepatitis B, many patients develop termination mutations in the e antigen, leading to negative results and thus losing their ability to indicate cccDNA activity in the liver. Second, the presence of integrated HBV DNA within hepatocytes that can continuously produce HBsAg decouples the HBsAg marker from cccDNA activity, rendering it inaccurate. While antibodies have different meanings, they all serve to assess whether a patient has established specific immunity and serve as an auxiliary reference for HBV infection status, rather than directly indicative of cure.

[0004] (1) HBeAg seroconversion (small three positive) cannot indicate the biological activity status of cccDNA.

[0005] Traditionally, it is believed that when a hepatitis B patient changes from big three positive (HBsAg positive, HBeAg positive and HBcAb positive) to small three positive (HBsAg positive, HBeAb positive and HBcAb positive), specific immunity is established in the patient's body and the patient enters the immune clearance period, accompanied by a decrease in HBV replication level, and the disease is effectively controlled.

[0006] However, the reality is that a significant proportion of hepatitis B patients have or develop the G1896A mutation in the precore region (PreC) either in the natural state or during the treatment process. This mutation leads to the premature termination of HBeAg translation. In another case, the A1762T and G1764A mutations in the precore / core promoter (PreC promoter) region inhibit the translation of precore RNA into HBeAg. The results of precore region mutations and BCP mutations both cause the termination of HBeAg expression, and the serological hepatitis B surface antigen test shows the negative conversion of HBeAg. In this case, the result of HBeAg has deviated directionally from the active state of cccDNA, thus losing its indication function for intrahepatic cccDNA. The negative HBeAg does not necessarily mean the cessation of virus replication or the stability of the disease.

[0007] (2) The long-term positive HBsAg does not necessarily mean that cccDNA still has biological activity

[0008] In the current WHO criteria for defining HBV cure, the negative conversion of hepatitis B surface antigen (HBsAg) is taken as the main criterion for functional cure. However, under the current treatment system, the annual negative conversion rate of HBsAg in patients with chronic hepatitis B (CHB) is only 1%. The main reasons are as follows:

[0009] The expression level of HBsAg in serum is affected by multiple factors. For example, factors such as mutations in the pre-S1, pre-S2, or S regions can lead to reduced expression or secretion of HBsAg; amino acid mutations in the "a" determinant or post-translational modification of HBsAg may affect the sensitivity of the detection reagent, resulting in an underestimated HBsAg test value; in addition, HBsAg in CHB patients (especially HBeAg-negative CHB patients) may also be derived from the expression of integrated HBV DNA in hepatocyte chromosomes, rather than the expression of HBV cccDNA.

[0010] Currently, the clinical significance of HBsAg expressed after the integration of HBV DNA into the host chromosome is not clear in the field of hepatology, and current diagnostic reagents cannot distinguish HBsAg from different sources. There is integrated HBV DNA in hepatocytes that can continuously produce HBsAg, resulting in a directional deviation between the HBsAg result and the active state of cccDNA, thus also losing its indication function for the activity of intrahepatic cccDNA.

[0011] (3) HBeAb and HBcAb have nothing to do with the biological activity of cccDNA

[0012] HBeAb and HBcAb are antibody products resulting from the immune response of the human body against HBsAg, HBeAg, and HBcAg of hepatitis B virus. Among them, HBcAb is produced in large quantities and stably expressed for a long time within a relatively short period (1 - 2 weeks) after hepatitis B virus infection; the positivity of HBeAb is accompanied by the decline of HBeAg concentration to negative conversion, indicating that the body can neutralize and clear HBeAg, but it does not reflect whether HBeAg is still in the expression state.

[0013] Similarly, the appearance of HBsAb indicates the natural immune reconstruction of the body against HBV, effectively clearing hepatitis B virus and virus-like particles in the blood, which is a prerequisite for the negative conversion of HBsAg. However, as mentioned above, the annual conversion rate of the negative conversion of HBsAg accompanied by the positivity of HBsAb is only 1%.

[0014] From the above analysis, it can be seen that existing hepatitis B detection indicators all have certain defects more or less, and clinical diagnosis faces a "black box" - chronic hepatitis B (CHB) patients are treated with nucleoside analogs (NAs) for a long time without knowing whether viral replication will rebound after drug withdrawal. Therefore, there is an urgent need to develop new serum markers to make up for this situation and reduce the serious consequences brought by the uncertainty during the treatment of hepatitis B patients.

[0015] Non-invasive serological markers, as surrogate markers for the replication activity of intrahepatic virus, can avoid the harm caused by liver biopsy and may indirectly reflect the level and transcriptional activity of HBVcccDNA, so they have more practical application value. HBV core-related antigen (HBcrAg) is a new type of HBV serum complex marker, which has a high homology with hepatitis B core antigen (HBcAg). Its components include hepatitis B virus core antigen (HBcAg), hepatitis B virus e antigen (HBeAg), and hepatitis B virus precore antigen (p22cr). Therefore, the biological activity of HBVcccDNA can be indicated by detecting HBcrAg.

[0016] However, although HBcrAg detection has obvious advantages in theory, this indicator has not been applied to clinical practice so far. An important reason is that there are two difficulties in the detection of hepatitis B core antigen (HBcAg), making the detection relatively difficult.

[0017] The first difficulty: It is difficult to lyse the viral nucleocapsid. Since HBcAg is encapsulated within the envelope of the virus particle, unlike the surface antigen that is directly exposed on the surface or the E antigen that is free in the serum. To detect HBcAg, it is first necessary to release it from the virus particle. Moreover, when releasing HBcAg, its structure cannot be damaged for subsequent detection. Although there are existing methods that can lyse the virus envelope to release HBcAg, these methods generally use high-concentration protein denaturants, strong acids or bases, or long-time high-temperature heating treatment to inactivate the antibodies corresponding to HBcrAg in the sample, and then detect the content of HBcrAg. Although this method can be used for detection, this approach will not only damage the corresponding antibodies but also the released HBcrAg, affecting the accuracy of subsequent detection.

[0018] The second difficulty: It is difficult to remove endogenous interfering antibodies. Since core antibodies are generally present in the infected individuals, after HBcAg is released, it can be bound by these antibodies, resulting in the blocking of antibody binding sites and preventing subsequent detection.

[0019] Although people have recognized the potential superiority of HBcrAg detection, the above difficulties have always prevented its successful application in clinical practice. Due to the lack of HBcrAg detection indicators, more than 40 years after the invention of hepatitis B serological testing, the "hepatitis B five-item test" has still not evolved into the "hepatitis B six-item test". Therefore, developing an HBcrAg detection kit has important market value and social significance. Summary of the Invention

[0020] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a hepatitis B virus HBcrAg detection kit for detecting the content of HBcrAg, and the second objective of the present invention is to provide an HBcrAg detection method using the hepatitis B virus HBcrAg detection kit.

[0021] The technical solution adopted by the present invention is as follows:

[0022] A hepatitis B virus HBcrAg detection kit includes an immunomagnetic bead working solution, a sample treatment solution, and an HBcrAg labeling working solution. The immunomagnetic bead working solution includes immunomagnetic beads coated with coating antibodies and a magnetic bead diluent. The immunomagnetic beads are one of SA magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads. The immunomagnetic bead working solution is used for specifically capturing hepatitis B virus particles and HBcrAg; the sample treatment solution is used to remove the outer membrane of hepatitis B virus particles under mild conditions and dissociate hepatitis B virus core particles to release HBcrAg; the HBcrAg labeling working solution includes labeled antibodies conjugated with labels and a label diluent, and the HBcrAg labeling working solution is used for labeling the captured HBcrAg.

[0023] As a preferred embodiment of the present invention, the coated antibody conjugated with biotin ester is coated on the immunomagnetic beads in proportion. The coated antibodies include anti-HBcrAg antibody-mAb03, anti-HBcrAg antibody-mAb25, and anti-HBsAg antibody-mAb40. The multiple types of antibodies coated on the immunomagnetic beads of the present invention can compete for more binding sites on the hepatitis B virus, thereby having stronger binding sensitivity to hepatitis B virus core particles.

[0024] As a preferred embodiment of the present invention, the label is one of acridinium ester, alkaline phosphatase, and horseradish peroxidase, and the labeled antibodies include anti-HBcrAg antibody-mAb08 and anti-HBcrAg antibody-mAb29.

[0025] As a preferred embodiment of the present invention, the assembly inhibitor includes one or more of 20-deoxyingenol, canconavirus, sophoreol, and BAY41-4109.

[0026] As a preferred embodiment of the present invention, the assembly inhibitor is mainly prepared from 20-deoxyingenol, canconavirus, sophoreol, and BAY41-4109.

[0027] The present invention also provides another technical solution: a method for detecting hepatitis B virus HBcrAg, including the above-mentioned hepatitis B virus HBcrAg detection kit, and the detection method includes the following steps:

[0028] S1. Calibrate the hepatitis B virus HBcrAg detection kit with a calibrator and establish a standard curve for the detection of the kit;

[0029] S2. Add the immunomagnetic bead working solution to the sample to be tested. After the immunomagnetic bead working solution is mixed with the sample to be tested, perform the first incubation to capture the hepatitis B virus particles and free HBcrAg in the sample;

[0030] S3. After washing and removing the endogenous antibodies in the sample with a washing solution, obtain the immunomagnetic beads adsorbed with hepatitis B virus particles and free HBcrAg;

[0031] S4. Add the sample treatment solution and perform the second incubation under mild conditions to remove the outer membrane of the hepatitis B virus particles while dissociating the hepatitis B virus core particles, and the immunomagnetic beads specifically capture the HBcrAg in the hepatitis B virus core particles;

[0032] S5. Wash and remove the excess added sample treatment solution with a washing solution;

[0033] S6. Add the HBcrAg labeling working solution for the third incubation, and the labeled antibody in the HBcrAg labeling working solution binds to HBcrAg;

[0034] S7. Wash with a cleaning solution to remove impurities, obtaining immunomagnetic beads adsorbed with HBcrAg;

[0035] S8. Add a substrate solution. After the substrate solution is mixed with the immunomagnetic beads, an oxidant is added under an alkaline environment, and the labeled antibody undergoes a chemiluminescence reaction upon oxidation;

[0036] S9. Use a fully automatic chemiluminescent immunoassay analyzer to detect the luminescence intensity, and obtain the content of HBcrAg in the sample by comparing the luminescence intensity with the standard curve graph.

[0037] As a preferred embodiment of the present invention, the calibrator includes C149 protein, and the C149 protein is a recombinant protein recombinantly expressed from the 1-149 amino acid sequence commonly owned by three proteins, namely HBeAg, HBcAg, and P22cr.

[0038] As a preferred embodiment of the present invention, in step S1, the kit calibration steps include:

[0039] S11. Prepare calibrators with different concentrations and add them to the hepatitis B virus HBcrAg detection kit respectively;

[0040] S12. Measure and record the different luminescence intensities corresponding to the calibrators with different concentrations;

[0041] S13. Use the concentration of the calibrator as the abscissa and the luminescence intensity as the ordinate, and obtain the standard curve graph detected by the kit through fitting of the recorded corresponding data.

[0042] As a preferred embodiment of the present invention, in steps S3, S5, and S7, wash repeatedly 4 times with the cleaning solution.

[0043] As a preferred embodiment of the present invention, in steps S2, S4, and S6, the incubation time each time is 1-30 min, and the incubation temperature is 37 °C.

[0044] The working principle of the present invention:

[0045] In the first step of detecting with the kit of the present invention, add the immunomagnetic bead working solution. This is the first key link. In this link, the immunomagnetic bead working solution realizes the first capture of hepatitis B virus particles and free HBcrAg in the sample. Since the antibody binding sites of hepatitis B virus particles and free HBcrAg are blocked after binding with the immunomagnetic beads, the endogenous anti-HBcrAg antibody in the sample will not bind to hepatitis B virus particles and free HBcrAg. Furthermore, the endogenous antibody can be washed away to avoid interference with the capture of subsequent exposed core particles, solving the current problem of endogenous antibody interference.

[0046] In the second step, add the sample treatment solution, which is the second key link. In this link, the sample treatment solution can crack the outer membrane of hepatitis B virus under mild conditions, and at the same time dissociate the hepatitis B virus core particles, so that the HBcrAg in the hepatitis B virus particles is fully exposed. At this time, the immunomagnetic beads can perform secondary capture on the exposed HBcrAg. Since the dissociation environmental conditions are mild and will not damage the corresponding antigen, sufficient capture can be achieved, which can further improve the detection accuracy.

[0047] In the third step, add the HBcrAg labeling working solution. The labeled antibody in the HBcrAg labeling working solution can bind to the target antigen HBcrAg to achieve the labeling of HBcrAg. After labeling HBcrAg, remove the excess impurities to avoid the influence of impurities on the detection of the labeled substance, thereby improving the detection accuracy.

[0048] Finally, add the alkaline substrate solution. Add an oxidant in an alkaline environment, and the oxidant triggers a luminescence reaction of the labeled substance. Since the labeled antibody binds to HBcrAg, the luminescence intensity detected at this time is positively correlated with the content of HBcrAg in the sample. By detecting the luminescence intensity, the content of HBcrAg in the sample can be judged.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] 1. The immunomagnetic bead working solution of the present invention is coated with a coating antibody that specifically captures hepatitis B virus particles and HBcrAg. This antibody has extremely strong specificity and sensitivity for competitively binding hepatitis B virus core particles, and can effectively capture hepatitis B virus particles and HBcrAg, playing a key role in the subsequent separation of endogenous antibodies in serum, and can effectively avoid the interference of endogenous antibodies on the detection.

[0051] 2. The sample treatment solution of the present invention contains a specific hepatitis B virus core particle assembly inhibitor, which can fully dissociate the hepatitis B virus core particles under mild conditions. After the hepatitis B virus core particles are fully dissociated, the HBcrAg in the hepatitis B virus core particles can be specifically captured by immunomagnetic beads. The sample treatment solution adopted by the present invention can avoid the influence on the antigen and antibody to be detected under high-concentration denaturants or high temperatures, which is beneficial to improving the accuracy of subsequent detection.

[0052] 3. As the currently known optimal serological index reflecting the active state of cccDNA, HBcrAg has too much interference in detection, resulting in too low accuracy, so that there is currently no related product applied clinically. By using the kit of the present invention for detection, the content of HBcrAg in the sample can be accurately detected, thereby measuring the HBcAg index, filling the domestic blank of HBcrAg detection, making up for the deficiencies of the traditional "hepatitis B five-item test", realizing the iteration from "hepatitis B five-item test" to "hepatitis B six-item test", and bringing huge social and economic effects to the medical market.

[0053] 4. In the detection method of the present invention, before lysing the outer membrane of hepatitis B virus particles, a step of capturing hepatitis B virus particles with the immunomagnetic beads is added, which can completely remove the endogenous antibodies in the serum, and then avoid the problem that HBcrAg binds to the endogenous antibodies after the subsequent lysis of hepatitis B virus particles, affecting the detection accuracy.

[0054] 5. In the detection method of the present invention, the calibrator of the kit used is the C149 protein. C149 is recombinantly expressed from the 1-149 amino acid sequence shared by the three proteins HBeAg, HBcAg, and P22cr contained in HBcrAg. All the above anti-HBcrAg antibodies are directed against the antigenic epitopes in this region. As long as HBcrAg (any one of the three proteins HBeAg, HBcAg, and P22cr) appears in the sample, it can be captured, and the light emitted by capturing one C149 molecule and capturing one HBcrAg (any one of HBeAg, HBcAg, and P22cr) protein molecule is the same. Therefore, the molar concentration of the protein to be detected in the sample can be traced back to the C149 molar concentration, and the detection system can be traced back to the C149 protein. The standard curve graph obtained by the present invention with this calibrator reflects the concentration of HBcrAg in the sample 1:1 during the sample detection process, and the measured concentration content has high accuracy. Detailed implementation mode

[0055] Typical implementation modes reflecting the features and advantages of the present invention will be specifically described in the following description. It should be understood that the present invention can have various changes in different implementation modes, all of which do not depart from the scope of the present invention, and the descriptions and illustrations therein are essentially for illustrative purposes and not for limiting the present invention.

[0056] This embodiment discloses a kit for detecting hepatitis B virus HBcrAg, which includes an immunomagnetic bead working solution, a sample treatment solution, and an HBcrAg labeling working solution.

[0057] The immunomagnetic bead working solution includes immunomagnetic beads coated with coating antibodies and a magnetic bead diluent. The immunomagnetic beads are one of SA magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads. The coating antibodies include anti-HBcrAg antibody-mAb03, anti-HBcrAg antibody-mAb25, and anti-HBsAg antibody-mAb40.

[0058] Preparation of the undiluted immunomagnetic bead solution:

[0059] Couple biotin ester to the coating antibody to obtain biotinylated antibody. Take 0.1 mg each of anti-HBcrAg antibody-mAb03, anti-HBcrAg antibody-mAb25, and anti-HBsAg antibody-mAb40 and dialyze them separately. The dialysis solution is 20 mol / L PBS. After dialysis is completed, recover the antibodies, add 5 - 100 μl of biotin ester to each of them and mix well. After standing at room temperature for 30 minutes, add 10 mmol / L Tris and mix well. After standing at room temperature for 15 minutes, terminate the reaction. Transfer the coupled antibodies into a dialysis bag and dialyze again to remove the excess biotin ester. Recover the biotinylated antibody. Add an equal volume of glycerol to the biotinylated antibody, label it, and store it at -20 ± 5 °C for later use.

[0060] Coat the biotinylated antibody onto the immunomagnetic beads: Take 1 mg of magnetic beads and place them on a magnetic separator. After the magnetic beads are completely adsorbed and the supernatant is clear, aspirate the excess supernatant; Wash the magnetic beads (resuspend the magnetic beads with 0.5 mL of TBST, place them on the magnetic separator, and aspirate the supernatant after the magnetic beads are completely adsorbed and the supernatant is clear) 3 times; For the 4th wash of the magnetic beads, the difference is that after resuspending the magnetic beads, add 1 - 20 μg of biotinylated antibody, roll and mix well at room temperature for 30 minutes, add 10 μl of biotin and mix well for 30 minutes to block the magnetic beads; For the 5th wash of the magnetic beads, the undiluted immunomagnetic bead solution is obtained.

[0061] Preparation of the magnetic bead diluent:

[0062] The magnetic bead diluent is prepared by mixing a buffer (one or several of common buffers such as PBS, Tris-HCl, Hepes, Mes, etc.), a protective agent (one or several of BSA, Casein, trehalose, sucrose, mannitol, etc.), a surfactant (one or several of Tween 20, Tween 60, Tween 80, Triton X-100, SDS, etc.), and a preservative (proclin-300 or proclin-950).

[0063] Add the magnetic bead diluent to the undiluted immunomagnetic bead solution and dilute it to obtain an immunomagnetic bead working solution with a concentration of 0.01 - 10 mg / ml. The immunomagnetic bead working solution is used for specific capture of hepatitis B virus particles and HBcrAg.

[0064] The sample processing solution includes a buffer (one or more of common buffers such as PBS, Tris-HCl, Hepes, Mes, etc.), a denaturant (one or more of HCl, H2SO4, urea, NaOH, etc.), a detergent (one or more of Tween 20, Tween 60, Tween 80, Triton X-100, SDS, CTAB, etc.), a reducing agent (one or more of DTT, mercaptoethanol, TCEP, etc.) and an assembly inhibitor (one or more of 20-deoxyingenol, canconavir, kushenol, BAY41-4109, etc.). The sample processing solution is used to remove the outer membrane of hepatitis B virus particles under mild conditions and dissociate hepatitis B virus core particles to release HBcrAg.

[0065] The HBcrAg labeling working solution includes a labeled antibody conjugated with a label and a label diluent. The label is one of acridinium ester, alkaline phosphatase, and horseradish peroxidase. The labeled antibody includes anti-HBcrAg antibody-mAb08 and anti-HBcrAg antibody-mAb29.

[0066] Preparation of acridinium ester-labeled antibody:

[0067] Take 0.1 mg each of anti-HBcrAg antibody-mAb08 and anti-HBcrAg antibody-mAb29 and dialyze them with 20 mmol / L PBS respectively. Recover the dialyzed antibodies, add 5 - 100 μL of 10 mmol / L acridinium ester to each of them immediately and mix well. Mix evenly at room temperature in the dark for 1 hour. Add 10 mmol / L Tris to it and mix evenly at room temperature in the dark for 30 minutes to terminate the reaction. Then transfer it to a desalting column with a volume of 0.5 mL and centrifuge at 1500 g * 2 min to collect the filtrate. Add an equal volume of glycerol, label it, and store it at -20 ± 5 °C for later use.

[0068] The label diluent is the same as the magnetic bead diluent. Add the label diluent to the acridinium ester-labeled antibody for dilution to prepare an HBcrAg labeling working solution with a concentration of 0.01 - 10 μg / ml, that is, the acridinium ester-labeled antibody working solution. The acridinium ester-labeled antibody working solution is used to label HBcrAg on immunomagnetic beads.

[0069] This embodiment also discloses an HBcrAg detection method using a hepatitis B virus HBcrAg detection kit. In this detection method, a calibrator, a fully automatic chemiluminescence immunoassay analyzer, a substrate solution, and a cleaning solution are used.

[0070] The specific detection steps include:

[0071] S1. Calibrate the hepatitis B virus HBcrAg detection kit using a calibrator and establish a standard curve for the detection of the kit. The calibrator of the present invention includes the C149 protein, which is a recombinant protein recombinantly expressed from the 1-149 amino acid sequence commonly shared by the three proteins HBeAg, HBcAg, and P22cr. The calibration steps of the kit include:

[0072] S11. Prepare calibrators with different concentrations and add them to the hepatitis B virus HBcrAg detection kit respectively;

[0073] S12. Measure and record the different luminescence intensities corresponding to the calibrators with different concentrations;

[0074] S13. Using the concentration of the calibrator as the abscissa and the luminescence intensity as the ordinate, fit the recorded corresponding data to obtain the standard curve for the detection of the kit.

[0075] S2. Add 10 - 100 μL of the immunomagnetic bead working solution to 50 μL of the sample to be tested. After mixing the immunomagnetic bead working solution and the sample to be tested, incubate at 37 °C for 30 min at room temperature. In this step, the immunomagnetic beads capture the hepatitis B virus particles and free HBcrAg in the sample, preventing the hepatitis B virus particles and HBcrAg from binding to the endogenous antibodies in the sample.

[0076] S3. Wash with a washing solution, separate and remove the supernatant, and retain the magnetically adsorbed immunomagnetic beads. This washing step is repeated 4 times.

[0077] S4. Add 10 - 100 μL of the sample treatment solution to the immunomagnetic beads, mix well, and incubate at 37 °C for 30 min at room temperature. In this step, the sample treatment solution can remove the outer membrane of the hepatitis B virus particles under mild conditions and simultaneously dissociate the hepatitis B virus core particles to fully expose the HBcrAg in the hepatitis B virus core particles, and then the immunomagnetic beads specifically capture the exposed HBcrAg.

[0078] S5. Wash with a washing solution to remove the excess added sample treatment solution. The specific operation is the same as that in S3.

[0079] S6. Add 10 - 100 μL of the acridinium ester-labeled antibody working solution to the immunomagnetic beads, mix well, and incubate at 37 °C for 30 min at room temperature. In this step, the acridinium ester-labeled antibody binds to the HBcrAg.

[0080] S7. Wash with a washing solution to remove impurities to obtain the immunomagnetic beads adsorbed with HBcrAg. The specific operation is the same as that in S3 or S5.

[0081] S8. Add 100-200 μL of substrate solution to the immunomagnetic beads and mix them. Then, add an oxidant in an alkaline environment to oxidize the acridinium ester-labeled antibody to produce a luminescent reaction.

[0082] S9. Use a fully automatic chemiluminescence immunoassay to detect the luminescence intensity, and compare the luminescence intensity with the standard curve to obtain the HBcrAg content in the sample.

[0083] The cleaning solution used in the HBcrAg detection method is generally a conventional cleaning solution. The substrate solution used is the substrate solution of the acridinium ester system.

[0084] Test kit detection effect test

[0085] (1) The above kit was used to test a batch of HBsAg-positive serum samples using the above detection method. The test results are shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089] (2) The above kit was used to test a batch of HBsAg-negative serum samples using the above detection method. The test results are shown in Table 2.

[0090] Table 2

[0091] HBV sample (HBsAg -) Negative / total Positive coincidence rate HBVDNA - / HBeAg - 300 / 300 100％

[0092] In Table 1, HBsAg+ / HBV DNA+ / HBeAg+ means that HBsAg, HBV DNA, and HBeAg are all positive, indicating that there is active hepatitis B virus replication in the patient's body, the viral load is high, and the infectivity is strong.

[0093] HBsAg+ / HBV DNA+ / HBeAg- means that HBsAg and HBV DNA are both positive and HBeAg is negative, indicating that there is still active hepatitis B virus replication in the patient's body, but the viral load is relatively low and the infectiousness is weak.

[0094] HBsAg+ / HBV DNA- / HBeAg- means that HBsAg is positive while HBV DNA and HBeAg are both negative, indicating that although the patient is still infected with hepatitis B virus, the viral replication level is below the detection limit, the viral activity is suppressed, and the infectivity is weak.

[0095] Under the current detection system, the first case (HBsAg+ / HBV DNA+ / HBeAg+) is in the active phase of chronic hepatitis B or the acute infection phase, and it can usually be accurately detected; the second case (HBsAg+ / HBV DNA+ / HBeAg-) may be due to seroconversion brought about by the production of e antibody or termination mutation of e antigen expression. Under the current detection system, the detection accuracy is relatively low; the third case (HBsAg+ / HBV DNA- / HBeAg-) belongs to the non-active carrier state, after HBeAg seroconversion or the quiescent phase of chronic infection. For this kind of sample at the present stage, the detection accuracy is very low.

[0096] However, as can be seen from Table 1, when using the kit of the present invention to detect samples in the above three cases, after detecting a large number of samples, the detection accuracies of the above three cases can reach 99.25%, 90.23% and 84.51% respectively, and the average accuracy can be as high as 91.34%. And as can be seen from Table 2, when detecting HBsAg-negative serum samples, the detection accuracy can reach 100%. Generally speaking, the detection of the kit of the present invention has significantly high accuracy, can effectively reduce misdiagnosis (false positive) and missed diagnosis (false negative), and greatly improves the reliability of diagnosis and the treatment effect.

[0097] The above embodiments are only the preferred embodiments of the present invention, and the scope of protection of the present invention cannot be limited thereby. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention belong to the scope of protection required by the present invention.

Claims

1. A hepatitis B virus HBcrAg detection kit, characterized in that: It includes an immunomagnetic bead working solution, a sample treatment solution, and an HBcrAg labeling working solution. The immunomagnetic bead working solution includes immunomagnetic beads coated with a coating antibody and a magnetic bead diluent. The immunomagnetic beads are one of SA magnetic beads, carboxyl magnetic beads, and tosyl magnetic beads. The immunomagnetic bead working solution is used for specifically capturing hepatitis B virus particles and HBcrAg; the sample treatment solution is used for removing the outer membrane of hepatitis B virus particles under mild conditions and dissociating hepatitis B virus core particles to release HBcrAg; the HBcrAg labeling working solution includes a labeled antibody conjugated with a label and a label diluent, and the HBcrAg labeling working solution is used for labeling the captured HBcrAg.

2. The hepatitis B virus HBcrAg detection kit according to claim 1, wherein: The coating antibody is conjugated with biotin ester and then coated on the magnetic beads in proportion. The coating antibodies include anti-HBcrAg antibody - mAb03, anti-HBcrAg antibody - mAb25, and anti-HBsAg antibody - mAb40.

3. The hepatitis B virus HBcrAg detection kit according to claim 1, characterized in that: The label is one of acridinium ester, alkaline phosphatase, and horseradish peroxidase. The labeled antibodies include anti-HBcrAg antibody - mAb08 and anti-HBcrAg antibody - mAb29.

4. The hepatitis B virus HBcrAg detection kit according to claim 1, characterized in that: The sample treatment solution includes a buffer, a denaturant, a detergent, a reducing agent, and an assembly inhibitor. The assembly inhibitor is used for lysing hepatitis B virus core particles under mild conditions.

5. The hepatitis B virus HBcrAg detection kit according to claim 4, characterized in that: The assembly inhibitor includes one or several of 20-deoxyingenol, canconavirus, kushenol, and BAY41-4109.

6. A method for detecting hepatitis B virus HBcrAg, characterized in that: It includes a hepatitis B virus HBcrAg detection kit according to claims 1-5. The detection method includes the following steps: S1. Calibrate the hepatitis B virus HBcrAg detection kit using a calibrator and establish a standard curve graph for the detection of the kit. S2. Add the immunomagnetic bead working solution to the sample to be tested. After the immunomagnetic bead working solution is mixed with the sample to be tested, perform the first incubation to capture hepatitis B virus particles and free HBcrAg in the sample. S3. After washing with a washing solution to remove endogenous antibodies in the sample, obtain immunomagnetic beads adsorbed with hepatitis B virus particles and free HBcrAg. S4. Add the sample treatment solution and perform the second incubation under mild conditions to remove the outer membrane of hepatitis B virus particles while dissociating hepatitis B virus core particles. The immunomagnetic beads specifically capture HBcrAg in the hepatitis B virus core particles. S5. Wash with a washing solution to remove the excess added sample treatment solution. S6. Add the HBcrAg labeling working solution and perform the third incubation. The labeled antibody in the HBcrAg labeling working solution binds to HBcrAg. S7. Wash with a washing solution to remove impurities and obtain immunomagnetic beads adsorbed with HBcrAg. S8. Add a substrate solution. After the substrate solution is mixed with the immunomagnetic beads, add an oxidant in an alkaline environment, and the labeled antibody undergoes a luminescence reaction upon oxidation. S9. Use an automatic chemiluminescence immunoassay analyzer to detect the luminescence intensity, and obtain the content of HBcrAg in the sample according to the comparison between the luminescence intensity and the standard curve graph.

7. The hepatitis B virus HBcrAg detection method according to claim 6, wherein: The calibrator includes C149 protein, which is a recombinant protein recombinantly expressed from the 1-149 amino acid sequence shared by three proteins, namely HBeAg, HBcAg, and P22cr.

8. The hepatitis B virus HBcrAg detection method according to claim 6, characterized in that: In the step S1, the calibration steps of the kit include: S11. Prepare calibrators with different concentrations and add them to the hepatitis B virus HBcrAg detection kit respectively; S12. Measure and record the different luminescence intensities corresponding to the calibrators with different concentrations; S13. Using the concentration of the calibrator as the abscissa and the luminescence intensity as the ordinate, fit the recorded corresponding data to obtain the standard curve graph detected by the kit.

9. The hepatitis B virus HBcrAg detection method according to claim 6, characterized in that: In the steps S3, S5, and S7, wash repeatedly 4 times with the washing solution.

10. The hepatitis B virus HBcrAg detection method according to claim 6, characterized in that: In the steps S2, S4, and S6, the incubation time each time is 1-30 min, and the incubation temperature is 37 °C.

Images