A monoclonal antibody against human hepatitis B e antigen and its application

Abstract

This invention discloses a monoclonal antibody against human hepatitis B e antigen and its application, relating to the field of hepatitis B detection technology. In the heavy chain variable region of the monoclonal antibody, the amino acid sequences of the complementarity-determining regions (CDR1-3) are as shown in SEQ ID NO. 1-3 or have at least 95% homology with the sequences shown in SEQ ID NO. 1-3; in the light chain variable region, the amino acid sequences of the complementarity-determining regions (CDR1 and CDR3) are as shown in SEQ ID NO. 4 and 5 or have at least 95% homology with the sequences shown in SEQ ID NO. 4 and 5, and the amino acid sequence of the complementarity-determining region (CDR2) is FAS. The monoclonal antibody provided by this invention can effectively recognize natural hepatitis B e antigen, and the titer of the supernatant from B cell culture can reach 1:10000, making it fully applicable to the detection and research of human hepatitis B e antigen. The chemiluminescent reagent prepared using the monoclonal antibody described in this invention has advantages such as high specificity, strong anti-interference ability, high detection sensitivity, and good stability, with almost no missed detections, and can replace imported reagents.

Description

Technical Field

[0001] This invention relates to the field of hepatitis B detection technology, and in particular to monoclonal antibodies against human hepatitis B e antigen and their applications. Background Technology

[0002] Hepatitis B (HBV) is an infectious disease caused by the hepatitis B virus, leading to liver inflammation and liver function impairment. Hepatitis B e antigen (HBeAg) is a soluble protein, generally found only in HBsAg-positive serum. In acute HBV infection, HBeAg appears slightly later than HBsAg. The presence of HBeAg indicates that the patient is in a period of high infection and low response. The disappearance of HBeAg is replaced by anti-HBe.

[0003] Hepatitis B e antigen (HBeAg) is the secretory form of a nucleocapsid protein encoded by the C gene, and is a product of post-translational processing of the preC protein. The precursor of HBeAg is a protein with a molecular weight of 25 kDa (P25e protein) containing the complete preC sequence. The full-length PreC / C protein, transcribed from the second ATG in the C region, requires processing, removing 20 amino acids from the C-terminus and 19 amino acids from the N-terminus, before it can be secreted extracellularly as mature HBeAg. HBeAg is present at approximately 10¹⁴ molecules / ml of serum and is the predominant form of HBcrAg in HBeAg-positive patients. Serum HBeAg and PreC have similar buoyant densities and size distributions, both exhibiting heterogeneity in density and size. Neither HBeAg nor PreC proteins can form a capsid in cells or in vitro under physiological conditions.

[0004] Currently, serological testing for hepatitis B virus generally includes HBsAg, Anti-HBs, HBeAg, Anti-HBe, and Anti-HBc, commonly known as the "hepatitis B two and a half pairs." The mainstream methods for HBeAg detection include enzyme-linked immunosorbent assay (ELISA), colloidal gold method, chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECL), and time-resolved fluorescence immunoassay (TRFIA). The biggest problem with HBeAg (hepatitis B e antigen) testing currently on the market is false negatives. Analysis suggests that the reason for these false negatives may be that the hepatitis B virus genome is prone to mutation. Although most mutations are silent and have no biological significance, mutations at certain key sites (pre-C region, PreC) can cause a negative e antigen test; however, the tested individual is indeed a hepatitis B virus carrier. It should be noted that a negative e antigen test does not mean that the e antigen is not expressed, but rather that it is missed due to the inherent limitations of the testing reagents.

[0005] Therefore, it is essential to develop a monoclonal antibody against human hepatitis B e antigen that has high sensitivity, high activity, and low false negative rate that can be used by downstream reagent manufacturers. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a monoclonal antibody against human hepatitis B e antigen, specifically achieved through the following techniques.

[0007] A monoclonal antibody against human hepatitis B e antigen, wherein in the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity-determining region CDR1 is as shown in SEQ ID NO.1 or has at least 95% homology with the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementarity-determining region CDR2 is as shown in SEQ ID NO.2 or has at least 95% homology with the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO.3 or has at least 95% homology with the sequence shown in SEQ ID NO.3;

[0008] In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity-determining region CDR1 is as shown in SEQ ID NO.4 or has at least 95% homology with the sequence shown in SEQ ID NO.4, the amino acid sequence of the complementarity-determining region CDR2 is FAS, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO.5 or has at least 95% homology with the sequence shown in SEQ ID NO.5.

[0009] Further, in the heavy chain variable region of the monoclonal antibody, the amino acid sequence of backbone region FR1 is as shown in SEQ ID NO. 6 or has at least 95% identity with the sequence shown in SEQ ID NO. 6; the amino acid sequence of backbone region FR2 is as shown in SEQ ID NO. 7 or has at least 95% identity with the sequence shown in SEQ ID NO. 7; the amino acid sequence of backbone region FR3 is as shown in SEQ ID NO. 8 or has at least 95% identity with the sequence shown in SEQ ID NO. 8; and the amino acid sequence of backbone region FR4 is as shown in SEQ ID NO. 9 or has at least 95% identity with the sequence shown in SEQ ID NO. 9.

[0010] In the light chain variable region, the amino acid sequence of backbone region FR1 is as shown in SEQ ID NO.10 or has at least 95% identity with the sequence shown in SEQ ID NO.10; the amino acid sequence of backbone region FR2 is as shown in SEQ ID NO.11 or has at least 95% identity with the sequence shown in SEQ ID NO.11; the amino acid sequence of backbone region FR3 is as shown in SEQ ID NO.12 or has at least 95% identity with the sequence shown in SEQ ID NO.12; and the amino acid sequence of backbone region FR4 is as shown in SEQ ID NO.13 or has at least 95% identity with the sequence shown in SEQ ID NO.13.

[0011] Further, the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.14, or has at least 95% homology with the sequence shown in SEQ ID NO.14; the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.15, or has at least 95% homology with the sequence shown in SEQ ID NO.15.

[0012] Furthermore, the gene sequence encoding the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.16 or has at least 95% homology with the sequence shown in SEQ ID NO.16; the gene sequence encoding the light chain of the monoclonal antibody is as shown in SEQ ID NO.17 or has at least 95% homology with the sequence shown in SEQ ID NO.17.

[0013] A biomaterial, said biomaterial comprising any one of the following:

[0014] (1) A nucleic acid molecule, said nucleic acid molecule encoding the above-described monoclonal antibody;

[0015] (2) An expression vector / vector group containing the nucleic acid molecule;

[0016] (3) A host cell containing the nucleic acid molecule or containing an expression vector / vector group.

[0017] A composition against human hepatitis B e antigen, characterized in that it comprises the above-described monoclonal antibody.

[0018] The above-mentioned monoclonal antibody or composition is used to prepare a product containing human hepatitis B e antigen, or to detect human hepatitis B e antigen for purposes other than disease diagnosis and treatment.

[0019] Furthermore, the product is a test kit, test strip, or test chip.

[0020] Furthermore, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip.

[0021] Compared with existing technologies, the advantages of this invention are: the monoclonal antibody provided by this invention can effectively recognize natural hepatitis B e antigen, and the titer of the supernatant from B cell culture can reach 1:10000, making it fully applicable to the detection and research of human hepatitis B e antigen. The chemiluminescent reagent prepared using the monoclonal antibody described in this invention has advantages such as high specificity, strong anti-interference ability, high detection sensitivity, and good stability, with almost no missed detections, and can replace imported reagents. Attached Figure Description

[0022] Figure 1 The results show the peak elution value of hepatitis B e antigen in Example 1;

[0023] Figure 2 This is a gel electrophoresis image of antibody molecular mass identification during the identification of monoclonal antibody characteristics in Example 2. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0026] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0027] Generally, the terms used in this specification have the following meanings:

[0028] In this specification, the term "monoclonal antibody" refers to an antibody derived from a group of substantially homologous antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles), which are typically present in trace amounts. Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0029] In this application, the terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant region," "light chain constant region," "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("FR"), and "complementarity-determining region" ("CDR") refer to the components of an antibody, which can form different types of antibodies through different combinations. For example, a conventional IgG antibody is a tetramer composed of two light chains and two heavy chains. Both VL and VH consist of three complementarity-determining regions (CDRs) and four framework regions (FRs). The CDRs contain residues that contact the antigen and determine the antigen specificity of VL and VH, while the FRs maintain the variable region structure and determine the position of the CDR loops. Typically, the heavy chain variable region and the heavy chain constant region form a complete heavy chain, while the light chain variable region and the light chain constant region form a complete light chain.

[0030] In the specification of this application, a conjugate refers to a molecule that, after binding with an antibody, can indicate the location or amount of the antibody through color, chemical reaction, excitation light, mass spectrometry, etc.; including but not limited to alkaline phosphatase, peroxidase, luciferase, luciferin, fluorescent protein, biotin, streptomycin, or isotopes.

[0031] In the specification of this application, the terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0032] In this application, the term "vector" (expression vector or vector set) refers to a vector that can introduce a polynucleotide sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, bacteriophage vectors, viral vectors, etc. Among them, a "viral vector" is a vector modified from a viral genome, which introduces a foreign gene into a host cell through viral infection. A "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast, and actinomycetes, which exists in the cytoplasm or nucleus, has the ability to replicate autonomously, maintains a constant copy number in daughter cells, and expresses the genetic information it carries.

[0033] In this application, "enzyme-linked immunosorbent assay (EILSA)" refers to a detection method that utilizes the specific binding of antibody molecules to antigen molecules to bind free impurities and the target protein bound to a solid-phase carrier, and then uses special labels for qualitative or quantitative analysis. The principle of EILSA is as follows: antigens or antibodies can be physically adsorbed onto a solid-phase surface while maintaining their immunogenicity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds, while maintaining their respective immunogenic or enzymatic activities; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of an immune reaction can be determined by the color reaction of the added substrate, and the intensity of the color reaction is directly proportional to the amount of the corresponding antigen or antibody in the sample. Depending on the substance to be detected and the available detection conditions, various types of detection methods can be designed; the double-antibody sandwich method is the most commonly used method for detecting antigens. The principle of the double antibody sandwich method is as follows: antiserum containing known antibodies is adsorbed into the wells of a microtiter plate and washed once; the antigen to be tested is added, and if the two are specific, they will bind; then excess antibody is washed away; an enzyme-linked antibody that specifically reacts with the antigen to be tested is added to form a "sandwich" structure; the substrate of the enzyme is added, and if a colored enzymatic digest is seen, it indicates the presence of the corresponding antigen.

[0034] Experimental Example 1: Preparation of mouse monoclonal antibody against human hepatitis B e antigen extracted from natural sources.

[0035] 1. Naturally extracted hepatitis B e antigen

[0036] (1) Sample pretreatment

[0037] Positive serum from hepatitis B patients was collected, centrifuged at 10,000 rpm and 4°C for 30 min, and filtered through a 0.45 μm filter.

[0038] (2) Ammonium sulfate precipitation

[0039] Collect the pretreated serum, add 15% solid ammonium sulfate according to the mass-volume ratio, stir thoroughly to dissolve, stir at 4°C for 15 h, centrifuge at 10000 rpm at 4°C for 30 min, collect the precipitate and redissolve it in Tris solution at pH=8.5, filter at 0.45 μm for the next step.

[0040] (3) Gel filtration chromatography

[0041] The Chromdex 75PG column (16mm × 600mm) was fully equilibrated with mobile phase (20mM Tris, pH = 8.5). 3 ml of the pretreated protein was loaded onto the column at a flow rate of 0.2 ml / min. Partial collection was performed, with 1.5 ml collected per tube. The target protein is outlined in red. Peak 4 was collected, and its chromatogram is shown below. Figure 1 As shown, Figure 1 In the spectrum, peak 1 represents macroglobulin, α-globulin, etc.; peak 2 represents thrombin-like proteins and β-globulin, etc.; peak 3 represents human serum albumin; peak 4 represents HBe-Ag protein components; and peaks 5 and 6 represent small molecules in serum.

[0042] (4) Density gradient centrifugation

[0043] Prepare sucrose density gradient solutions with mass fractions of 20%, 30%, 40%, 50%, and 60% using 20 mM PBS. Filter each solution through a 0.22 μm filter and then spread them evenly in ultracentrifuge tubes according to density from highest to lowest. Spread 1 ml of the prepared protein concentrate on top of the density gradient sucrose solution and centrifuge at 30,000 rpm for 4 h at 20°C. After centrifugation, aspirate the protein bands and dialyze them against PBS at 4°C. Collect the protein and store it at -20°C.

[0044] 2. Animal immunization

[0045] Five 6-8 week old female Balb / c mice were immunized. The purified hepatitis B e antigen was mixed with Freund's complete adjuvant at a dose of 50 μg / mouse, completely emulsified, and administered in 200 μL per mouse per immunization. The mice were immunized for the first time at multiple sites on the back, groin, and axilla. Two weeks later, a second immunization was administered at the same dose and route, but with an incomplete adjuvant. Fifteen days later, a third immunization was administered at the same dose, with Freund's incomplete adjuvant added, via intraperitoneal injection. One week later, blood was collected from the tails of all immunized mice, and serum antibody titers were detected by ELISA. The results are shown in Table 1 below.

[0046] Table 1. Results of Hepatitis B e Antigen Immunotiter in Mice

[0047]

[0048]

[0049] As shown in Table 1 above, two of the five immunized mice (HBe-left and HBe-right) had tail blood titers greater than 1 / 10W after the third immunization, indicating successful immunization. Mice with serum titers above 100,000 were selected for fusion. Three days before fusion, mice were immunized via intraperitoneal injection without adjuvant at a dose of 100 μg / mouse.

[0050] 3. Construction of B cells

[0051] (1) Culture and preparation of myeloma cell lines

[0052] This embodiment uses the SP / 20 myeloma cell line, which exhibits excellent growth and fusion efficiency, with a doubling time of 10-12 hours. Myeloma cells in the logarithmic growth phase, with optimal cell morphology and viability, are selected for fusion. Myeloma cells should undergo adaptive culture before fusion to allow them to grow to their optimal state (i.e., the logarithmic growth phase).

[0053] On the day of fusion, gently blow myeloma cells off the bottle wall with a bent dropper and collect them in a 50ml centrifuge tube or fusion tube. Centrifuge at 1000r / min for 5-10min. Discard the supernatant, add 30ml of culture medium to the precipitate, centrifuge and wash once, centrifuge for 5-10min. Discard the supernatant, mix the precipitate with 20ml of culture medium and set aside.

[0054] (2) Preparation of spleen cells

[0055] BALB / c mice that have completed animal immunization were enucleated to collect blood, and the serum was separated by centrifugation to serve as a positive control serum for antibody detection.

[0056] Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. The spleen was aseptically removed in a laminar flow hood and placed in a petri dish containing 10 ml of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was punctured with a sterile syringe until it turned white. The spleen cells in the petri dish were collected into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, 10 ml of culture medium was added, and a small amount of 10-fold dilution was taken for counting.

[0057] (3) Cell fusion

[0058] Three days prior to fusion, positive mice were boosted with immunization. Mouse spleen cells and myeloma cells sp2 / 0 were mixed at a ratio of 10:1, fused with PEG, and then cultured in HAT selective medium. Ten days later, the hybridoma cell supernatant was screened using ELISA. The selected positive hybridoma cells were cloned using limiting dilution. After five rounds of screening, ten positive hybridoma cell lines were finally identified.

[0059] (4) Screening of hybridoma cells (ELISA method)

[0060] Hepatitis B e antigen (HBeAg) at 10 μg / mL, 50 μL / well, was coated onto a 96-well plate and incubated overnight at 4°C. The liquid in the wells was discarded the following morning, and the plates were washed three times with washing buffer, patted dry, and 100 μL of blocking buffer was added to each well for blocking at 37°C for 2 h. The plates were then washed twice and patted dry. 100 μL of the supernatant from the culture of six hybridoma cell lines to be tested was added to each well, along with positive, negative, and blank control standards. The plates were incubated at 37°C for 0.5 h, washed four times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG at a 1:10000 concentration was added to each well of the ELISA plate, and the plates were incubated at 37°C for 30 min. The plates were washed four times and patted dry.

[0061] Finally, add 100 μL of TMB substrate chromogenic solution per well, and after 15 min of development, terminate the reaction with 50 μL of 2 mol / L dilute hydrochloric acid per well. The OD values ​​of each well were measured at dual wavelengths of 450 nm and 630 nm, as shown in Table 2 below.

[0062] Table 2 Results of ELISA titer assay for monoclonal antibodies

[0063] plate HBe natural antigen irrelevant antigens Sample Cell supernatant Cell supernatant enzymes Sheep Anti-Rat Sheep Anti-Rat enzyme concentration [1 / 1W] [1 / 1W] HBe-1C5 0.366 0.004 HBe-2C9 0.006 0.004 HBe-3C3 2.664 0.006 HBe-3D1 0.089 0.006 HBe-4D8 0.251 0.005 HBe-5B1 0.010 0.005 HBe-5F1 0.008 0.012 HBe-6C7 0.109 0.008 HBe-7B5 0.010 0.000 HBe-8G5 0.008 0.012 Imported antibodies 2.778 0.009 negative control 0.013 0.005 blank 0.008 0.010

[0064] As shown in Table 2, the blank well values ​​were below 0.02 and the negative well values ​​were below 0.1, indicating a clear distinction between positive and negative. This confirms that hybridoma cells resistant to human hepatitis B e antigen secrete antibodies that specifically recognize human hepatitis B e antigen. Therefore, the monoclonal cell line HBe-3C3 was selected for subsequent monoclonal antibody experiments. Using the same method, the titer in mouse ocular blood reached 1:100,000, indicating its suitability for cell fusion.

[0065] 4. Cloning of the gene encoding mouse monoclonal antibodies

[0066] Positive clones were identified by antigen-coated ELISA using the supernatant of cultured hybridoma cells. Cells from positive clones were collected, lysed, and RNA was extracted. The RNA was then reverse transcribed into cDNA using a total RNA extraction kit (65°C, 5 min). The light and heavy chain variable region genes (VH and VL) of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones using PCR, and their sequences were determined by sequencing.

[0067] The amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO. 1-3; the amino acid sequences of the complementarity-determining regions (CDR1 and CDR3) of the light chain variable region are shown in SEQ ID NO. 4 and SEQ ID NO. 5; the amino acid sequence of the complementarity-determining region (CDR2) of the light chain variable region is FAS.

[0068] The amino acid sequences of the backbone regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO. 6-9; the amino acid sequences of the backbone regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NO. 10-13.

[0069] The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14, and the amino acid sequence of the light chain is shown in SEQ ID NO.15.

[0070] The gene sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.16, and the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.17.

[0071] 5. Production and purification of monoclonal antibodies

[0072] The heavy and light chain genes of the monoclonal antibody obtained in step 4 were loaded into the pCDN3.4 expression vector and transfected into 293F cells. After 72-96 hours of transfection, the culture supernatant contained recombinant monoclonal antibodies recognizing human hepatitis B e antigen. The recombinant mouse monoclonal antibody recognizing human hepatitis B e antigen was purified from the transfected culture supernatant using protein A affinity gel resin. After antibody identification, it was aliquoted and stored at -20°C for later use.

[0073] 6. Characterization of monoclonal antibodies

[0074] (1) Determination of antibody concentration

[0075] The absorbance values ​​(A280) and A260 of the monoclonal antibody at 280 nm and 260 nm were determined by ultraviolet spectrophotometry. Protein content was calculated using the following formula:

[0076] Protein content (mg / ml) = (A280 × dilution factor) / 1.35.

[0077] (2) Identification of antibody molecular weight

[0078] Monoclonal antibodies were measured using SDS-PAGE. Figure 2 As shown, the heavy chain of the monoclonal antibody is approximately 46 kDa, and the light chain is approximately 25 kDa.

[0079] (3) Determination of ELISA titer

[0080] The purified ascites fluid monoclonal antibodies were determined using indirect ELISA. The results are shown in Table 3 below. Table 3 shows that the purified titers were all 1:100,000.

[0081] Table 3. Results of ascites titer of anti-human hepatitis B e antigen monoclonal antibody.

[0082] plate HBe natural antigen HBe natural antigen Sample 3C3 ascites negative ascites enzymes Sheep Anti-Rat Sheep Anti-Rat enzyme concentration [1 / 1W] [1 / 1W] [1 / 500] 3.837 0.004 [1 / 1K] 4.317 0.005 [1 / 2K] 3.553 0.007 [1 / 4K] 3.328 0.005 [1 / 8K] 3.068 0.001 [1 / 1.6W] 2.108 0.002 [1 / 3.2W] 1.362 0.003 [1 / 6.4W] 0.774 0.005 [1 / 12.8W] 0.399 0.005 [1 / 25.6W] 0.213 0.004 [1 / 51.2W] 0.129 0.005 diluent 0.018 0.004

[0083] Experimental Example 2: Application of 3C3 Antibody in Hepatitis B Virus e Antigen Latex Chromatography Test Strips

[0084] 1. Preparation of monoclonal antibodies coated with magnetic microparticles

[0085] Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 26 °C, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 3C3 monoclonal antibody at a ratio of 10 μg of 3C3 antibody per milligram of magnetic microparticle. The conjugation process was carried out at 25 °C for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25 °C for 2 h and then stored at 2-8 °C for later use.

[0086] 2. Preparation of alkaline phosphatase-labeled monoclonal antibodies

[0087] (1) Take 1.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing sodium chloride, and add 0.04 mg TR to it. React at 37°C for 1 h.

[0088] (2) Take 1 mg of 3A3 antibody (purchased from Wuhan Aokebotai Biotechnology Co., Ltd., catalog number A0022), adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC, and react at 37℃ for 1 h.

[0089] (3) Mix the alkaline phosphatase and 3A3 antibody that have been reacted in steps 2.1 and 2.2 at a mass ratio of 1:1 and react at 25°C for 1 hour. After the reaction is complete, add 0.1 mL of phosphate buffer containing sodium chloride, 2% cysteine ​​and pH 7.4, let stand for 1 hour, add 1 mL of glycerol, and store at -20°C for later use.

[0090] 3. Reagent performance testing

[0091] After assembling the prepared coating membrane and latex pad, the sample pad was attached and cut into 3mm test strips. These strips were then used to test 500 clinical samples collected from hospitals using the National Reference Material for Rapid Diagnostic Reagent of Hepatitis B Virus e Antigen (HBeAg) purchased from the China National Institutes for Food and Drug Control and the Shanghai Kehua HBeAg reagent. The sample pad was a 300mm × 17mm glass cellulose membrane.

[0092] (1) The results of testing the national reference material for the rapid diagnostic reagent for hepatitis B virus e antigen are as follows:

[0093] Negative compliance rate: Of the 15 negative national reference samples N1-N15, the compliance rate (- / -) is 15 / 15, which meets the requirements;

[0094] Positive compliance rate: For 10 positive national reference samples P1 to P10, the compliance rate (+ / +) is 10 / 10, which is higher than the standard requirement of 9 / 10.

[0095] Precision: The precision reference sample was tested repeatedly 10 times, and all results were positive with uniform color development, which meets the requirements;

[0096] Minimum detection limit: The minimum detection limit for the detection sensitivity reference material is 0.1 IU / mL, which is far higher than the standard requirement of 5 IU / mL.

[0097] (2) The results of the clinical sample comparison test are shown in Table 4 below.

[0098] Table 4 Comparison Results of Clinical Samples

[0099]

[0100]

[0101] As can be seen from Table 4 above, the negative and positive concordance rates of the test strip described in this invention with the control reagent are both 100%, and the clinical Kappa consistency is 100%.

[0102] Experimental Example 2: Application of 3C3 antibody in chemiluminescent reagent for hepatitis B virus e antigen

[0103] 1. Preparation of monoclonal antibodies coated with magnetic microparticles

[0104] Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 26 °C, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 3C3 monoclonal antibody at a ratio of 10 μg of 3C3 antibody per milligram of magnetic microparticle. The conjugation process was carried out at 25 °C for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25 °C for 2 h and then stored at 2-8 °C for later use.

[0105] 2. Preparation of alkaline phosphatase-labeled monoclonal antibodies

[0106] (1) Take 1.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing sodium chloride, and add 0.04 mg TR to it. React at 37°C for 1 h.

[0107] (2) Take 1 mg of 3A3 antibody (purchased from Wuhan Aokebotai Biotechnology Co., Ltd., catalog number A0022), adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC, and react at 37℃ for 1 h.

[0108] (3) Mix the alkaline phosphatase and 3A3 antibody that have been reacted in steps 2.1 and 2.2 at a mass ratio of 1:1 and react at 25°C for 1 hour. After the reaction is complete, add 0.1 mL of phosphate buffer containing sodium chloride, 2% cysteine, and pH=7.4. Let stand for 1 hour, then add 1 mL of glycerol and store at -20°C for later use.

[0109] 3. Reagent performance testing

[0110] The magnetic microparticles coated with 3C3 antibody were diluted 20 times with phosphate buffer containing sodium chloride, and the alkaline phosphatase-labeled 3A3 antibody was diluted 1000 times with phosphate buffer containing sodium chloride before testing. The test samples were 1093 clinical samples tested by Abbott Laboratories' Hepatitis B virus e antigen assay kit (chemiluminescent microparticle immunoassay). The comparison test results are shown in Table 5 below.

[0111] Table 5

[0112]

[0113]

[0114] As can be seen from Table 5 above, the negative and positive concordance rates of the test strip described in this invention with Abbott's control reagent are both 100%, the clinical Kappa consistency is 100%, and there are no missed detections or false positives.

[0115] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against human hepatitis B e antigen, characterized in that, The amino acid sequence of the complementarity determining region CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, the amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO. 3; The amino acid sequence of the complementarity determining region CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 4, the amino acid sequence of the complementarity determining region CDR2 is FAS, and the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO.

5.

2. The monoclonal antibody against human hepatitis B e antigen according to claim 1, characterized by, The amino acid sequence of the framework region FR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 6, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO. 7, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO. 8, and the amino acid sequence of the framework region FR4 is shown in SEQ ID NO. 9; The amino acid sequence of the framework region FR1 in the light chain variable region is shown in SEQ ID NO. 10, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO. 11, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO. 12, and the amino acid sequence of the framework region FR4 is shown in SEQ ID NO.

13.

3. A biomaterial, characterized by, The biological material comprises any one of the following: (1) a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1-2; (2) an expression vector / vector set containing the nucleic acid molecule; (3) a host cell containing the nucleic acid molecule or containing the expression vector / vector set.

4. The biomaterial of claim 3, wherein, In the nucleic acid molecule, the gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 16, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

17.

5. A composition against human hepatitis B e antigen, characterized by, The monoclonal antibody according to any one of claims 1-2.

6. Use of a monoclonal antibody according to any one of claims 1 to 2, or a composition according to claim 5, characterized in that, A product for preparing an anti-human hepatitis B e antigen, or for detecting a human hepatitis B e antigen without the purpose of disease diagnosis and treatment; the product is a detection kit, a detection test strip or a detection chip.

7. Use according to claim 6, characterized in that, The detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatography kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit or a radioimmunoassay detection kit; The detection test strip is a colloidal gold test strip or an ELISA test strip.

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