Monoclonal antibody against hepatitis b hbca and its application and product

Abstract

The application provides a kind of anti-hepatitis B HBcAg monoclonal antibody and its application and product, it is related to the field of biotechnology.The application uses HBcAg to immunize Balb / c mouse, takes mouse spleen cell and fuses with myeloma cell, and obtains hybridoma cell with high specificity by specific high-throughput screening, obtains a large number of mouse ascites by culture and re-immunization, and then obtains high-purity, high-sensitivity and high-specificity anti-HBcAg monoclonal antibody by multi-step separation and purification, which provides the required raw material for developing HBcAg detection immunostrip.The anti-HBcAg monoclonal antibody of the application can be used for immunological detection such as immunoblotting and immunofluorescence, and the obtained antibody has good specific binding capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a monoclonal antibody against hepatitis B HBcAg and application and product thereof. BACKGROUND

[0002] Hepatitis B virus (HBV) is the pathogen causing hepatitis B (HB). Hepatitis B core antigen (HBcAg) is a single polypeptide, mainly existing in Dane particles, and plays an important role in HBV infection. It can reflect the presence of Dane particles in serum and the replication of HBV in liver, and can be used as a marker of active disease of HB. It has more important significance for monitoring, treatment evaluation and prognosis of HB patients. Dynamic monitoring can be used as a basis for clinicians to develop treatment plans. Antibodies against HBcAg can be used as quality control and standard products for the detection of five items of HB, however, the antibodies against HBcAg are currently dependent on imports, with high cost, and the sensitivity and detection rate of existing HBcAg rapid diagnostic products need to be improved.

[0003] Therefore, the present application is proposed. SUMMARY

[0004] The first object of the present application is to provide a monoclonal antibody against HBcAg, which aims to provide a stable and reliable source of raw materials for HBcAg detection methods based on immunological principles, such as immunofluorescence, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, etc. to reduce the dependence of domestic products on imported raw materials and reduce product costs. At the same time, it improves the detection rate and sensitivity of HBcAg rapid diagnostic products such as gold standard test strips and ELISA kits, reduces the false positive rate, and improves the competitiveness of self-produced products.

[0005] The second object of the present application is to provide a biological material.

[0006] The third object of the present application is to provide the use of the above-mentioned monoclonal antibody against HBcAg in the preparation of hepatitis B virus detection products.

[0007] The fourth object of the present application is to provide a HBcAg marker.

[0008] The fifth object of the present application is to provide a kit for hepatitis B virus detection.

[0009] In order to achieve the above objects, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides an anti-HBcAg monoclonal antibody, wherein a variable region of the anti-HBcAg monoclonal antibody comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL having an amino acid sequence of WAS, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 5.

[0011] As a further technical solution, the variable region comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 6.

[0012] As a further technical solution, the variable region comprises a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 7.

[0013] As a further technical solution, the anti-HBcAg monoclonal antibody is an IgG antibody.

[0014] In a second aspect, the present application provides a biological material selected from any one of a-c:

[0015] a. a nucleic acid comprising a nucleic acid sequence encoding the anti-HBcAg monoclonal antibody;

[0016] b. a vector carrying the nucleic acid in a;

[0017] c. a cell carrying the nucleic acid in a, or containing the vector in b, or expressing the anti-HBcAg monoclonal antibody.

[0018] In a third aspect, the present application provides use of the above-mentioned anti-HBcAg monoclonal antibody in the preparation of a hepatitis B virus detection product.

[0019] In a fourth aspect, the present application provides an HBcAg marker comprising the anti-HBcAg monoclonal antibody and a marker.

[0020] The anti-HBcAg monoclonal antibody and the marker are coupled.

[0021] As a further technical solution, the marker comprises an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin.

[0022] In a fifth aspect, the present application provides a kit for detecting hepatitis B virus, which comprises the monoclonal antibody against HBcAg or the HBcAg marker.

[0023] As a further technical solution, the kit comprises an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic micro-particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The present application uses HBcAg to immunize Balb / c mice, fuses the mouse spleen cells with myeloma cells, and obtains hybridoma cells with high specificity through specific high-throughput screening. A large amount of mouse ascites is obtained through culture and re-immunization, and high-purity, high-sensitivity and high-specificity anti-HBcAg monoclonal antibody anti-HBcAg-mab1 is obtained through multi-step separation and purification, which provides the required raw material for developing an immunological test strip for detecting HBcAg. The anti-HBcAg monoclonal antibody anti-HBcAg-mab1 of the present application can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has been verified to have good specific binding capacity. DETAILED DESCRIPTION

[0026] The embodiments of the present application will be described in detail below with reference to the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0027] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods in the art, unless otherwise indicated, and such methods and techniques are explained fully in the literature in the field of chemistry, biochemistry, molecular biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0028] The "variable region" or "variable domain" of an antibody refers to the domain of the antibody's heavy or light chain that recognizes and binds an antigen, the segment's amino acid composition and arrangement determining the antibody's specificity for the antigen. A heavy chain variable region can be referred to as "VH." A light chain variable region can be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen binding site. Each of the variable regions of the heavy chain and light chain is composed of three complementarity determining regions (CDRs) connected by four framework regions (FRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and with respect to each other to form the variable region, typically, the variable regions of the heavy and light chains, VL / VH, can be arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0029] The term "vector" refers to a nucleic acid vehicle into which a nucleotide can be inserted. When a vector is capable of directing the expression of a polynucleotide inserted into it, the vector is said to be an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell assumes the genetic material carried by the vector and expresses it.

[0030] The vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses. In some embodiments, the vectors of the present application comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and polyU sequences).

[0031] In a first aspect, the present application provides an anti-HBcAg monoclonal antibody, wherein the variable region of the anti-HBcAg monoclonal antibody comprises: a complementarity determining region CDR1-VH having an amino acid sequence as set forth in SEQ ID NO. 1, a complementarity determining region CDR2-VH having an amino acid sequence as set forth in SEQ ID NO. 2, a complementarity determining region CDR3-VH having an amino acid sequence as set forth in SEQ ID NO. 3, a complementarity determining region CDR1-VL having an amino acid sequence as set forth in SEQ ID NO. 4, a complementarity determining region CDR2-VL having an amino acid sequence of WAS, and a complementarity determining region CDR3-VL having an amino acid sequence as set forth in SEQ ID NO. 5.

[0032] The amino acid sequences of SEQ ID NO. 1-5 are shown in Table 1.

[0033] Table 1

[0034]

[0035] In some alternative embodiments, the variable region comprises a heavy chain variable region VH having an amino acid sequence as set forth in SEQ ID NO. 6.

[0036] EVQLQESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEITLRSDNFATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRPGDGYWGQGTTLTVSSA (SEQ ID NO. 6).

[0037] In some alternative embodiments, the variable region comprises a light chain variable region VL having an amino acid sequence as set forth in SEQ ID NO. 7.

[0038] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYRQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKL (SEQ ID NO. 7).

[0039] In some alternative embodiments, the anti-HBcAg monoclonal antibody is an IgG antibody.

[0040] In a second aspect, the present application provides a biomaterial selected from any one of a-c:

[0041] a. a nucleic acid comprising a nucleic acid sequence encoding the anti-HBcAg monoclonal antibody;

[0042] b. a vector carrying the nucleic acid in a.

[0043] c. a cell carrying the nucleic acid in a, or containing the vector in b, or expressing the anti-HBcAg monoclonal antibody.

[0044] In a third aspect, the present application provides use of the anti-HBcAg monoclonal antibody in the manufacture of a hepatitis B virus detection product.

[0045] The anti-HBcAg monoclonal antibody provided by the present application can specifically recognize HBcAg, and thus can be used for hepatitis B virus detection.

[0046] In a fourth aspect, the present application provides an HBcAg marker comprising the anti-HBcAg monoclonal antibody and a marker.

[0047] The anti-HBcAg monoclonal antibody and the marker are coupled.

[0048] The marker can be used for specific labeling of HBcAg.

[0049] In some optional embodiments, the marker includes but is not limited to an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

[0050] In a fifth aspect, the present application provides a kit for hepatitis B virus detection, the kit comprising the anti-HBcAg monoclonal antibody or the HBcAg marker.

[0051] In some optional embodiments, the kit includes but is not limited to an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microsphere detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

[0052] The present application is further illustrated by specific examples and comparative examples below, but it should be understood that these examples are only for more detailed illustration and should not be understood as limiting the present application in any form.

[0053] Example 1, Preparation of the anti-HBcAg monoclonal antibody anti-HBcAg-mab1

[0054] I. Preparation of HBcAg antigen

[0055] In order to obtain a mouse monoclonal antibody for specifically detecting hepatitis B core antigen (HBcAg), an HBcAg gene (GenBank: MZ043097.1) without TAA was synthesized by Shanghai Shengong Company, and a BamH I enzyme cutting site was added to the upstream and an EcoR I enzyme cutting site was added to the downstream. After obtaining the gene, the gene was linked to a pET32a (with HIS tag) vector through double enzyme cutting of BamH I and EcoR I, to construct a pET32a-HBcAg vector. The constructed pET32a-HBcAg plasmid was transformed into a BL21 (DE3) expression strain, and a small amount of expression was induced to explore the expression conditions. Then, a large amount of HIS-HBcAg recombinant protein was induced and expressed, and then the HIS protein was purified through a nickel column. After concentration and desalination, the HIS-HBcAg antigen was separated through a gel filtration pre-packed column / Superdex 200 Increase 10 / 300 GL through the action of a molecular sieve, and then 5 mg of the HIS-HBcAg protein was concentrated to be used for immunizing mice.

[0056] II. Preparation of the monoclonal antibody anti-HBCAG-mab1

[0057] Balb / c healthy female mice aged 6-8 weeks were selected, and immunization injection was performed according to a pre-specified immunization scheme. As an immunogen, BALB / c mice were immunized, and the spleen lymphocytes of the successfully immunized mice were extracted. The lymphocytes were fused with mouse myeloma cells SP2 / 0 through cell fusion technology, and after two rounds of subcloning screening, a hybridoma cell strain stably secreting the monoclonal antibody against HBcAg was obtained, so as to obtain the monoclonal antibody against HBcAg.

[0058] The prokaryotic expression and purified HIS-HBcAg antigen was used for periodic immunization of experimental mice.

[0059] The specific steps of the animal immunization experiment include:

[0060] 1. Balb / c mice with consistent average body weight and age were randomly divided into two groups, an aluminum adjuvant (aluminum hydroxide adjuvant) group and a non-aluminum adjuvant group.

[0061] 2. Before the experiment, the pre-immune serum of each mouse (the pre-immune serum was collected on the fifth day, and the blood was taken through the eyeball, and an appropriate amount of blood was taken to ensure the normal state of the mouse), and the collected serum was stored at -80℃.

[0062] 3、Alum adjuvant (aluminum hydroxide adjuvant) group preparation method: before immunization, each antigen was diluted to the corresponding dose (75 μg per mouse) in 75 μL PBS, and mixed with alum adjuvant (1 mg per mouse), according to the volume antigen: adjuvant = 3: 1 (that is, 75 μl of immunogen diluent was added with 25 μl of adjuvant); before use, the adjuvant was shaken well, and the injection adjuvant (25 μl) was slowly added to the immunogen solution; after the adjuvant and the immunogen diluent were mixed well, they were mixed well for 30 minutes. The adjuvant effectively adsorbed the antigen; the subsequent operation was carried out according to the experimental operation of immunized animals.

[0063] 4、No aluminum adjuvant group: the antigen was diluted to the corresponding dose (75 μg per mouse) in 100 μL PBS in the above table (100 μL of immunogen), and the subsequent operation was carried out according to the experimental operation of immunized animals.

[0064] 5、Subcutaneous injection at 2-week intervals: the experimental design was 3 times of immunization, but the supernatant of some mice was obtained by centrifugation after eye blood was taken 7 days after each immunization, and the serum titer was detected first, and the maximum blood volume was taken by heart blood after 7 days of the last immunization, and the supernatant was obtained by centrifugation and stored at-80℃.

[0065] 6、Detection of serum titer.

[0066] (1), 3 mice were immunized, and the mouse numbers were A0, A1, and A2 in turn. After 3 times of immunization, the serum titer was detected. The detection data are shown in Table 2.

[0067] Table 2: Serum titer detection data

[0068]

[0069] The immune mouse serum titer was detected by indirect ELISA and competitive ELISA using HBcAg standard as antigen competition detection and HBcAg hapten Dig-BSA as coating antigen (in the following experiments, indirect ELISA and competitive ELISA detection refer to this place), the enzyme-labeled plate was coated with 1 μg / ml of coating antigen diluted with coating solution 50 μl per well, and the plate was washed 3 times with washing solution (PBST) (the same below), 200 μl of blocking solution (5% skim milk powder) was added to each well, and the plate was placed in a 37°C incubator for 2 hours, then washed, 50 μl of diluted serum was added to each well, and the plate was incubated in a 37°C incubator for 30 minutes, then washed, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated in a 37°C incubator for 30 minutes. After washing, 100 μl of substrate solution was added, and the plate was incubated in a 37°C incubator for 10 minutes in the dark, then 50 μl of 2 mol / L H2SO4 was added to stop the reaction, and the A450 value was read on an enzyme-labeled instrument. The orbital blood titer of three mice after three immunizations was >62500.

[0070] The process of competitive ELISA is mostly the same as that of indirect ELISA, except that after the plate is blocked and washed, 50 μl of diluted 50 ng / ml and 300 ng / ml small molecule HBcAg standard solution is added, then 50 μl of diluted serum antibody is added, and the remaining steps are the same. The 50 ng / ml and 300 ng / ml small molecule HBcAg competition detection can reach more than 50% at 1:12500, and fusion can be arranged.

[0071] The immune spleen cells were fused with myeloma cell line SP2 / 0 cells, and the fused cells were screened by HAT selection medium (HAT selection medium containing hypoxanthine, aminopterin and thymine), and the fused cells were subjected to ELISA positive screening and subcloning; the positive monoclonal selected was taken from ascites, and the antibody was purified by Protein A / G antibody purification column, and the ELISA titer of the purified antibody was >1:128,000, and the purity was >90%.

[0072] III. Enzyme-linked reaction ELISA detection of HBcAg binding activity

[0073] 1. IgG antibody titer detection method

[0074] (1) Bottom plate coating: the used antigen was diluted to 3 μg / ml with coating diluent, 100 μl of the prepared coating solution was added to each well, and the plate was placed in a 4°C refrigerator for 24 hours.

[0075] (2) After 24 h, take out from the refrigerator and equilibrate at 37°C for 30 min, then discard the liquid in the well; wash the well with the washing solution for 3 times, 3 min each time.

[0076] (3) Seal the enzyme label reaction well: add 200 μl of 5% calf serum to each well, and equilibrate at 37°C for 90 min. After the end of the sealing, wash the well with the washing solution for 3 times, 3 min each time.

[0077] (4) Add the sample to be detected: dilute the sample according to the required proportion, and add the diluted sample to the enzyme label reaction well, 100 μl per well, and equilibrate at 37°C for 90 min; wash the well with the washing solution for 3 times, 3 min each time.

[0078] (5) Add the enzyme label antibody: add the secondary antibody with the appropriate concentration according to the instruction; equilibrate at 37°C for 90 min, and wash the well with 100 μl of the washing solution as before.

[0079] (6) Add the substrate solution: add 100 μl of the substrate to each well, and equilibrate at 37°C for 15-30 min in the dark.

[0080] (7) Terminate the reaction: add 50 μl of the termination solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0081] Four, detect the binding activity of the monoclonal antibody to recognize HBcAg.

[0082] (1) Cell fusion and cloning screening data

[0083] The mouse numbers are A0, A1, A2 in turn, and four rounds of fusion are completed.

[0084] A0 mouse fusion screening selected 22 positive wells in total, subcloned, and finally completed 3 cell strains. Through fusion screening, 43 positive clones with OD450 values > 2.2 were selected for detection of titer by doubling dilution, and second and third subcloning screening was performed. Three cell strains were obtained, which were named A0-1 to A0-3 respectively.

[0085] A1 mouse fusion screening selected 18 positive wells in total, subcloned, and finally completed 3 cell strains. Through fusion screening, 30 positive wells with OD450 values > 2.1 were selected for detection of titer by doubling dilution, and second and third subcloning screening was performed. Three cell strains were obtained, which were named A1-1 to A1-3 respectively.

[0086] A2 mouse fusion screening selected 25 positive wells in total, subcloned, and finally completed 4 cell strains. Through second and third subcloning screening, four cell strains were obtained, which were named A2-1 to A2-4 respectively.

[0087] After four cell fusion, we got 10 cell strains in total.

[0088] (2) Ascites preparation and detection data

[0089] Each complete cell strain was injected into 3 F1 mice, and 10 ascites were prepared. The detection titer data of all ascites are shown in Table 3.

[0090] Table 3

[0091]

[0092] (3) Antibody purification condition exploration and detection data

[0093] The above ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and 10 antibodies were obtained. The titer detection data of all antibodies are shown in Table 4.

[0094] Table 4

[0095]

[0096] The above data show that the monoclonal antibodies of the 10 cell strains have good specific binding ability to HBcAg antigen. The HBcAg colloidal gold product detects urine samples, so the standard HBcAg is used for competition experiment. The results show that the A0-3, A1-1, A1-2, A1-3, A2-1, A2-2 and A2-3 antibodies have competition effect on small molecule HBcAg. Therefore, the A0-3 and A1-1 antibodies are selected for testing of the HBcAg colloidal gold product.

[0097] Five, application of monoclonal antibody anti-HBCAG-mab1 in product.

[0098] Verify the HBcAg antibodies A0-3 and A1-1 by immunocolloidal gold platform.

[0099] 5.1 Purpose of the experiment

[0100] Using the samples sent by Dean Medical Test Center as experimental objects, the HBcAg antibodies A0-3 and A1-1 were evaluated by competition method. The control is the HBcAg reagent strip (competition method) of Alibio and Mike Biology.

[0101] 5.2 Experimental materials

[0102] 5.2.1 Reagent strip information

[0103] The A0-3 and A1-1 antibodies are labeled with colloidal gold through an immune colloidal gold platform, and then a colloidal gold chromatography test paper is prepared, which includes a sample pad, a conjugate pad and a detection pad, and the detection pad is provided with a detection line and a quality control line. The conjugate pad is coated with the screened A0-3 and A1-1 antibodies labeled with colloidal gold, the detection line is coated with an HBcAg antigen, and the quality control line is coated with a goat anti-mouse IgG antibody. The negative buffer solution sample and the HBcAg standard of different concentrations are detected in parallel. When the negative sample is detected, there is no HBcAg to compete with the antibody labeled with colloidal gold for the HBcAg antigen, so the antibody labeled with colloidal gold is combined with the coated HBcAg antigen on the detection line, and the T line is colored. The reagents of the test paper strip are shown in Table 5.

[0104] Table 5: Reagent strip information

[0105]

[0106] 5.2.2 Control information

[0107] Table 6

[0108]

[0109] 5.3 Experimental method

[0110] The HBcAg antibody A0-3 and the A1-1 antibody are evaluated by using the competitive method with the sample sent by Dean as a standard.

[0111] 5.4 Experimental results

[0112] Table 7

[0113]

[0114] Note: 1-10 represents the level of the color depth of the strip of the test paper strip, and the higher the value, the darker the color, + / - represents slightly darker or lighter than the color of the level. The addition of the HBcAg standard value is lower, indicating that the small molecules have a competitive effect, and the antibody can be combined with the HBcAg standard.

[0115] 5.5 Experimental conclusion

[0116] (1) The preliminary evaluation of the performance of the HBcAg antibody A0-3 and the A1-1 antibody found no obvious abnormalities, and the follow-up experiments can be continued.

[0117] (2) The results of the existing combination are compared with the control reagent and the chemiluminescence data, and it is found that the results of the combination are closer to the chemiluminescence data.

[0118] 5.6 Antibody stability

[0119] The stability of A0-3 antibody was evaluated using the test strip of 5.2.1, three batches of antibody samples were prepared, and the evaluation results are shown in Table 8 below.

[0120] Table 8

[0121]

[0122] Note: G3.5-G9 represents the level of strip color of the test strip, the higher the value, the darker the color, + / - indicates slightly darker or lighter than the color of the level. The addition of HBcAg standard value decreases indicates that the small molecule has a competitive effect, and the antibody can bind to the HBcAg standard.

[0123] The results show that the preliminary evaluation of the performance of the three batches of raw materials found no obvious abnormalities and can be used for products.

[0124] 5.7 Determination of antibody affinity:

[0125] Using the Octet® R8 protein analysis system of Germany Sartorius, using BLI biological film interference technology, using HBcAG antigen fixation, detecting HBcAg antibody A0-3 and A1-1 antibody and HBcAb antibody purchased from Abnova and MyBioSource as a control, detecting the affinity of the antibody, the data are shown in Table 9 below.

[0126] Table 9

[0127]

[0128] The results show that the affinity of A0-3 antibody is the highest, and A0-3 antibody is named anti-HBCAG-mab1 according to the evaluation results of the product, which can be used for the detection of HBcAg antigen detection kit.

[0129] Six, monoclonal antibody anti-HBCAG-mab1 heavy chain V region (VH) and light chain V region (VL) sequence analysis.

[0130] 1. Design primers for amplifying heavy chain V region (VH) and light chain V region (VL) genes.

[0131] The primers are as follows:

[0132] Heavy chain variable region forward primer (VH-FOR):

[0133] GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO. 10);

[0134] Heavy chain variable region reverse primer (VH-BACK):

[0135] GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO. 11);

[0136] Light chain variable region forward primer (VL-FOR):

[0137] CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC (SEQ ID NO. 12);

[0138] Light chain variable region reverse primer (VL-BACK):

[0139] GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG (SEQ ID NO. 13).

[0140] 2. Take anti-HBCAG-mab1 hybridoma cell strain (about 10 7 cells) in logarithmic growth phase, extract total RNA of cells according to the instructions of Trizol RNA extraction kit, synthesize cDNA first strand with total RNA as template, and PCR amplify VH / VL gene of antibody with the above amplification product as template.

[0141] 3. Recover heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-HBCAG-mab1, and send to company for sequencing.

[0142] 4. Then analyze VH / VL gene sequence:

[0143] The obtained sequence is as follows:

[0144] Variable region sequence of heavy chain:

[0145] Anti-HBcAg-VH: 351 bp.

[0146] GAGGTCCAACTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTACATTGAGATCTGATAATTTTGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAGGACACTGGCATTTATTACTGTACCAGGCCGGGGGACGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCC (SEQ ID NO. 8).

[0147] anti-HBcAg-mab1 protein: 117 aa.

[0148] EVQLQESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEITLRSDNFATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRPGDGYWGQGTTLTVSSA (SEQ ID NO. 6).

[0149] Variable region sequence of the light chain:

[0150] anti-HBcAg-mab1 LVK: 330 bp VK3.

[0151] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCGGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTG (SEQ ID NO. 9).

[0152] anti-HBcAg -mab1 LV kappa protein: 110 aa.

[0153] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYRQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKL (SEQ ID NO. 7).

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A monoclonal antibody against HBcAg, characterized in that, The variable region of the anti-HBcAg monoclonal antibody comprises a complementarity determining region CDR1-VH of an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH of an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH of an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL of an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL of an amino acid sequence of WAS, and a complementarity determining region CDR3-VL of an amino acid sequence as shown in SEQ ID NO.

5.

2. The monoclonal antibody against HBcAg according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO.

6.

3. The monoclonal antibody against HBcAg according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO.

7.

4. The monoclonal antibody against HBcAg according to claim 1, characterized in that, The anti-HBcAg monoclonal antibody is an IgG antibody.

5. Biomaterial, characterized in that, The biological material is selected from any one of a-c: a. a nucleic acid comprising a nucleic acid sequence encoding the anti-HBcAg monoclonal antibody according to any one of claims 1-4; b. a vector carrying the nucleic acid of a; c. a cell carrying the nucleic acid of a, or containing the vector of b, or expressing the anti-HBcAg monoclonal antibody according to any one of claims 1-4.

6. Use of the anti-HBcAg monoclonal antibody according to any one of claims 1-4 in the preparation of a hepatitis B virus detection product.

7. A kit for detection of hepatitis B virus, characterized by, The kit comprises the anti-HBcAg monoclonal antibody according to any one of claims 1-4.

8. The kit of claim 7, wherein The kit comprises an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit, or an immunoblotting detection kit.

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