Monoclonal antibody for resisting human hepatitis B e antigen and application thereof

Abstract

The invention discloses an anti-human hepatitis B e antigen monoclonal antibody and application thereof, and relates to the technical field of hepatitis B detection. In a heavy chain variable region of the monoclonal antibody, an amino acid sequence of a complementarity determining region CDR1-3 is as shown in SEQ ID NO.1-3 or has at least 95% of homology with the sequence as shown in SEQ ID NO.1-3; in the light chain variable region, amino acid sequences of complementary determining regions CDR1 and CDR3 are shown as SEQ ID NO.4 and SEQ ID NO.5 or have at least 95% of homology with the sequences shown as SEQ ID NO.4 and SEQ ID NO.5, and an amino acid sequence of a complementary determining region CDR2 is FAS. The monoclonal antibody provided by the invention can well recognize a natural hepatitis B e antigen, and the titer of a supernatant cultured by a B cell can reach 1: 10000, so that the monoclonal antibody can be completely applied to detection research of the human hepatitis B e antigen. The chemiluminescence reagent prepared by using the monoclonal antibody has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, almost has no leak detection, and can replace an imported reagent.

Description

Technical Field

[0001] The present invention relates to the technical field of hepatitis B detection, and in particular to a monoclonal antibody against human hepatitis B e antigen and an application thereof. Background Art

[0002] Hepatitis B (HBsAg) is an infectious disease caused by the hepatitis B virus (HBV) that can lead to liver inflammation and liver damage. Hepatitis B e antigen (HBeAg) is a soluble protein that is generally only found 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 high infection and low response period. After HBeAg disappears, it is replaced by anti-HBe.

[0003] Hepatitis B e antigen is the secretory form of the nucleocapsid protein encoded by the C gene and is the product of post-translational processing of the preC protein. The precursor of HBeAg is a protein (P25e protein) with a complete preC sequence and a molecular weight of 25KD. The full-length PreC / C protein transcribed and translated from the C region (second) ATG needs to be processed to remove the 20 amino acids at the carboxyl terminal and the 19 amino acids at the amino terminal before it can be secreted outside the cell as mature HBeAg. HBeAg is about 1014 molecules / ml serum and is the main form of HBcrAg in HBeAg-positive patients. Serum HBeAg and PreC have similar buoyant density and size distribution, and both show heterogeneity in density and size. Neither HBeAg nor PreC proteins can form capsids in cells or in vitro under physiological conditions.

[0004] At present, the serological examination items of hepatitis B virus generally include HBsAg, Anti-HBs, HBeAg, Anti-HBe, and Anti-HBc, which are commonly known as "two pairs and a half of hepatitis B". The current mainstream HBeAg detection methods include enzyme-linked immunosorbent assay (ELISA), colloidal gold method, chemiluminescence (CLIA), electrochemiluminescence (ECL), time-resolved fluorescence immunoassay (TRFIA), etc. The biggest problem with the detection of HBeAg (hepatitis B e antigen) on the market is missed detection. After analysis, the reason for missed detection may be that the genome of hepatitis B virus is prone to mutation. Although most of them are silent mutations and have no biological significance, mutations in certain key sites (pre-C region, PreC) will cause the e antigen to be detected as negative; however, the subject is indeed a hepatitis B virus carrier. It should be pointed out that the e antigen detection mentioned here is negative, not that the e antigen is not expressed, but that it is missed due to the inherent disadvantages of the detection reagent.

[0005] Therefore, it is very necessary to develop an anti-human hepatitis B e antigen monoclonal antibody with high sensitivity, high activity and low missed detection rate that can be used by downstream reagent manufacturers. Summary of the invention

[0006] In view of the above deficiencies in the prior art, the present invention provides a monoclonal antibody against human hepatitis B e antigen, which is specifically achieved through the following technology.

[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 complementary 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 complementary 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 complementary 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 the framework 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 the framework 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 the framework 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 the framework 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 the framework 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 the framework 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 the framework 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 the framework 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] Furthermore, 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, comprising any one of the following:

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

[0015] (2) an expression vector / vector set containing the nucleic acid molecule;

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

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

[0018] An application of the above monoclonal antibody or composition for preparing a product of human hepatitis B e antigen, or for detecting human hepatitis B e antigen for purposes other than disease diagnosis and treatment.

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

[0020] Furthermore, 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 kit; the detection test strip is a colloidal gold test strip or an ELISA test strip.

[0021] Compared with the prior art, the present invention is beneficial in that the monoclonal antibody provided by the present invention can well identify natural hepatitis B e antigen, and the titer of the supernatant of B cell culture can reach 1:10000, which can be fully applied to the detection research of human hepatitis B e antigen. The chemiluminescent reagent prepared by using the monoclonal antibody of the present invention has the advantages of high specificity, strong anti-interference ability, high detection sensitivity and good stability, and almost no missed detection, and can replace imported reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the peak value result of hepatitis B e antigen elution in Example 1;

[0023] Figure 2 This is the gel electrophoresis diagram of the antibody molecular mass identification during the monoclonal antibody property identification in Example 2. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

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

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

[0028] In this specification, the term "monoclonal antibody" means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present 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 all or part of transgenic animals comprising human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0029] In the specification of the present 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" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, and different types of antibodies can be formed through different combinations. For example, a conventional IgG antibody is a tetramer composed of two light chains and two heavy chains. Among them, VL and VH are both composed of 3 complementary determining regions (CDR) and 4 framework regions (FR). Among them, CDR contains residues that contact the antigen and determine the antigen specificity of VL and VH, and FR is used to maintain the variable region structure and determine the position of the CDR loop. Usually, the heavy chain variable region and the heavy chain constant region constitute a complete heavy chain, while the light chain variable region and the light chain constant region constitute a complete light chain.

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

[0031] In the specification of the present 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 (micro RNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0032] In the specification of the present application, the term "vector" (expression vector or vector group) refers to a vector through which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to transform the host and promote the expression (such as transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, a "viral vector" is a vector transformed from a viral genome, which introduces foreign genes into a host cell through viral infection. "Plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can also maintain a constant copy number in daughter cells and express the genetic information it carries.

[0033] In the specification of the present application, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free impurities with the target protein bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis thereof. The principle of ELISA is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens. The principle of the double antibody sandwich method is to adsorb the antiserum containing known antibodies into the small holes of the microtiter plate and wash it once; add the antigen to be tested, if the two are specific, they will bind, and then wash away the excess antibody; add an enzyme-linked antibody that specifically reacts with the antigen to be tested to form a "sandwich" structure; add the substrate of the enzyme, and if colored enzymatic hydrolysis products are seen, it indicates the presence of the corresponding antigen.

[0034] Experimental Example 1: Naturally extracted hepatitis B e antigen, preparation of anti-human hepatitis B e antigen mouse monoclonal antibody

[0035] 1. Naturally extracted hepatitis B e antigen

[0036] (1) Sample pretreatment

[0037] The positive serum of hepatitis B patients was collected, centrifuged at 10000 rpm, 4°C for 30 min, and filtered through 0.45 μm.

[0038] (2) Ammonium sulfate precipitation

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

[0040] (3) Gel filtration chromatography

[0041] A Chromdex 75PG column (16 mm × 600 mm) was fully equilibrated with a mobile phase (20 mM Tris, pH = 8.5), and 3 ml of the pre-treated protein was added. The system flow rate was 0.2 ml / min, and a portion of 1.5 ml was collected in each tube. The target protein was the portion outlined by the red line, and peak 4 was collected. The chromatogram is shown in FIG. Figure 1 As shown, Figure 1 Among them, peak 1 is macroglobulin, α-globulin, etc., peak 2 is thrombin-like proteins and β-globulin, etc., peak 3 is human serum albumin, peak 4 is the protein component containing HBe-Ag, and peaks 5 and 6 are 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% with 20mM PBS; filter them with 0.22μm filter membranes respectively, and then spread them in ultracentrifuge tubes from high to low density. Spread 1ml of the above-treated protein concentrate on the density gradient sucrose solution, and centrifuge at 30000rpm for 4h at 20℃. After centrifugation, draw out the protein bands and dialyze them in PBS at 4℃; collect the proteins and store them at -20 degrees.

[0044] 2. Animal immunization

[0045] Five female Balb / c mice aged 6-8 weeks were selected for immunization. The purified hepatitis B e antigen was mixed with Freund's complete adjuvant at a dose of 50ug / mouse. After complete emulsification, 200μL was administered to each mouse at multiple points on the back, ventral groove and armpit for the first immunization of mice. The second immunization was performed two weeks later, with the same dosage route as the first, and the adjuvant was changed to incomplete adjuvant; the third immunization was performed 15 days later, with the same dosage as above, and Freund's incomplete adjuvant was added for intraperitoneal injection. One week later, all immunized mice were tail-cut and blood was collected, and the serum antibody titer was detected by ELISA. The test results are shown in Table 1 below.

[0046] Table 1 Results of hepatitis B e antigen immune titer in mice

[0047]

[0048]

[0049] As can be seen from Table 1 above, of the five immunized mice, two (Hbe-left and Hbe right) had tail blood titers greater than 1 / 10W after three immunizations, indicating successful immunization. Immune mice with serum titers of more than 100,000 were selected for fusion. Three days before fusion, mice were immunized by intraperitoneal injection without adjuvant, and the immunization dose was 100 μg / mouse.

[0050] 3. Construction of B cells

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

[0052] The present embodiment adopts SP / 20 myeloma cell strain, which has good growth and fusion efficiency, and a doubling time of 10-12h. When fusion, select myeloma cells with good logarithmic growth phase, cell morphology and activity. Myeloma cells should be cultured adaptively before fusion to make cell growth reach the optimal state (i.e. logarithmic growth phase).

[0053] On the day of fusion, use an elbow pipette to gently blow the myeloma cells off the bottle wall, collect them in a 50ml centrifuge tube or fusion tube, and centrifuge them at 1000r / min for 5-10min; discard the supernatant, add 30ml culture medium to the precipitate, wash it once by centrifugation, and centrifuge it for 5-10min; discard the supernatant, mix the precipitate with 20ml culture medium for later use.

[0054] (2) Preparation of splenocytes

[0055] Take BALB / c mice that have completed animal immunization, remove the eyeballs to collect blood, and use a centrifuge to separate the serum as the positive control serum for antibody detection.

[0056] Mice were killed by cervical dislocation and immersed in 75% alcohol for 5 min. The spleen was removed aseptically in a clean bench and placed in a plate containing 10 ml of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was pierced with a sterile syringe until the spleen turned white. The spleen cells in the plate were collected in a 50 ml centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, 10 ml of culture medium was added, and a small amount of 10 was taken for dilution counting.

[0057] (3) Cell fusion

[0058] Three days before fusion, positive mice were boosted with immunization, mouse spleen cells were mixed with myeloma cells sp2 / 0 at a ratio of 10:1, PEG fused, and then cultured in HAT selection medium. Ten days later, hybridoma cell supernatants were screened by ELISA, and the screened positive hybridoma cells were cloned by limiting dilution. After five screenings, 10 positive hybridoma cell lines were finally determined.

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

[0060] Hepatitis B e antigen was coated on a 96-well plate at 10 μg / mL, 50 μL / well, and adsorbed overnight at 4 degrees. The next morning, the liquid in the well was discarded, and the well was washed 3 times with washing solution, patted dry, and 100 μL of blocking solution was added to each well to block for 2 hours at 37°C, washed twice, and patted dry. 100 μL of the culture supernatant of the 6 hybridoma cells to be tested was added to each well, and positive, negative and blank control standards were set up at the same time. Incubate at 37°C for 0.5h, wash 4 times, and pat dry. Take horseradish enzyme-labeled sheep anti-mouse IgG 1:10000, 100 μL per well was added to the ELISA plate, incubated at 37°C for 30min, washed 4 times, and patted dry.

[0061] Finally, 100 μL / well of TMB substrate colorimetric solution was added, and after 15 min of color development, 50 μL / well of 2 mol / L dilute hydrochloric acid was used to terminate the reaction. The OD value of each well was measured at dual wavelengths of 450 nm and 630 nm, as shown in Table 2 below.

[0062] Table 2 ELISA titer determination results of monoclonal antibodies

[0063] plate HBe natural antigen Unrelated antigen Sample Cell supernatant Cell supernatant Enzymes Sheep anti-mouse Sheep anti-mouse 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 can be seen from Table 2, the blank well value is below 0.02, the negative well value is below 0.1, and the yin and yang are clearly distinguished. It can be determined that the hybridoma cells against human hepatitis B e antigen secrete antibodies that can specifically recognize human hepatitis B e antigen, so the monoclonal cell line HBe-3C3 was screened for subsequent monoclonal antibody tests. The same method was used to measure the mouse eyeball blood titer, which reached 1:100000 and can be used for cell fusion.

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

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

[0067] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs. 1-3; the amino acid sequences of the complementary determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NOs. 4 and 5; the amino acid sequence of the complementary determining region CDR2 of the light chain variable region is FAS;

[0068] The amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs.6-9; the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NOs.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 chain and light chain genes of the monoclonal antibody obtained in step 4 were loaded onto the pCDN3.4 expression vector, respectively, and transfected into 293F cells; the culture supernatant containing the recombinant monoclonal antibody recognizing human hepatitis B e antigen was obtained 72-96 hours after transfection. The recombinant mouse monoclonal antibody recognizing human hepatitis B e antigen was purified from the culture supernatant after transfection using protein A affinity gel resin, and the antibody was packaged after identification and stored at -20°C for future use.

[0073] 6. Monoclonal antibody characterization

[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 UV spectrophotometry. The protein content was calculated according to the following formula:

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

[0077] (2) Antibody molecular mass identification

[0078] Monoclonal antibodies were determined by SDS-PAGE. Figure 2 As shown, the heavy chain of the monoclonal antibody is about 46 KD, and the light chain is about 25 KD.

[0079] (3) ELISA titer determination

[0080] The purified ascites monoclonal antibodies were determined by indirect ELISA, as shown in Table 3. As can be seen from Table 3, the results showed that the titers after purification were all 1:100,000.

[0081] Table 3 Anti-human hepatitis B e antigen monoclonal antibody ascites titer results

[0082] plate HBe natural antigen HBe natural antigen Sample 3C3 Ascites Negative ascites Enzymes Sheep anti-mouse Sheep anti-mouse 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 Dilution 0.018 0.004

[0083] Experimental Example 2: Application of 3C3 antibody in hepatitis B virus e antigen latex chromatography test strip

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

[0085] The magnetic microparticles with a particle size of 1.5-3 μm, EDC and NHS are added to a 50 mM MES solution with a pH of 5.0 in a mass ratio of 5:1:2 for reaction. During the reaction, the concentration of the magnetic microparticles is 10 mg / mL, the reaction environment temperature is 26°C, and the reaction time is 30 min. The magnetic microparticles after the reaction are coupled with 3C3 monoclonal antibodies. During the coupling, the ratio of magnetic microparticles to antibodies is 10 ug of 3C3 antibodies per milligram of magnetic microparticles. The coupling process should be carried out at 25°C for 5 hours. The magnetic microparticles after coupling are washed 3 times with a washing solution, and a phosphate buffer containing 2% glycine, 1% BSA, 0.05% Tween20, 0.05% ProClin300 and pH 7.4 is added to make the concentration of the magnetic microparticles 10 mg / mL. After standing at 25°C for 2 hours, the magnetic microparticles are stored at 2-8°C for later use.

[0086] 2. Preparation of Alkaline Phosphatase-labeled Monoclonal Antibody

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

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

[0089] (3) The alkaline phosphatase and 3A3 antibody reacted in steps 2.1 and 2.2 were mixed in a mass ratio of 1:1 and reacted at 25°C for 1 h. After the reaction was completed, 0.1 mL of phosphate buffer containing sodium chloride, 2% cysteine, and pH 7.4 was added. After standing for 1 h, 1 mL of glycerol was added and stored at -20°C for later use.

[0090] 3. Reagent performance test

[0091] After the prepared coating membrane and latex pad were combined, the sample pad was attached and cut into 3 mm test strips to test the national reference product of hepatitis B virus e antigen rapid diagnostic reagent purchased from the China Food and Drug Inspection Institute and 500 clinical samples collected from hospitals for testing by Shanghai Kehua HbeAg reagent. The sample pad was a glass cellulose membrane with a specification of 300 mm × 17 mm.

[0092] (1) Testing of the national reference product of the hepatitis B virus e antigen rapid diagnostic reagent, the results are as follows:

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

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

[0095] Precision: The precision reference product was tested 10 times repeatedly, all of which were positive and uniform in color, meeting the requirements;

[0096] Minimum detection amount: For the detection sensitivity reference, the minimum detection amount can reach 0.1IU / mL, which is much higher than the standard requirement of 5IU / mL.

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

[0098] Table 4 Comparison results of clinical samples

[0099]

[0100]

[0101] It can be seen from Table 4 above that the negative coincidence rate and the positive coincidence rate of the test strip of the present invention and the control reagent are both 100%, and the clinical Kappa consistency is 100%.

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

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

[0104] The magnetic microparticles with a particle size of 1.5-3 μm, EDC and NHS are added to a 50 mM MES solution with a pH of 5.0 in a mass ratio of 5:1:2 for reaction. The concentration of the magnetic microparticles during the reaction is 10 mg / mL, the reaction environment temperature is 26°C, and the reaction time is 30 min. The magnetic microparticles after the reaction are coupled with 3C3 monoclonal antibodies. During the coupling, the ratio of magnetic microparticles to antibodies is 10 ug of 3C3 antibodies per milligram of magnetic microparticles. The coupling process should be carried out at 25°C for 5 hours. The magnetic microparticles after coupling are washed 3 times with a washing solution, and a phosphate buffer containing 2% glycine, 1% BSA, 0.05% Tween20, 0.05% ProClin300 and pH=7.4 is added to make the concentration of the magnetic microparticles 10 mg / mL. After standing at 25°C for 2 hours, the magnetic microparticles are stored at 2-8°C for standby use.

[0105] 2. Preparation of Alkaline Phosphatase-labeled Monoclonal Antibody

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

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

[0108] (3) The alkaline phosphatase and 3A3 antibody that have completed the reaction in steps 2.1 and 2.2 were mixed in a mass ratio of 1:1 and reacted at 25°C for 1 h. After the reaction was completed, 0.1 mL of phosphate buffer containing sodium chloride, 2% cysteine, and pH = 7.4 was added. After standing for 1 h, 1 mL of glycerol was added and stored at -20°C for later use.

[0109] 3. Reagent performance test

[0110] The magnetic microparticles coated with 3C3 antibody were diluted 20 times with phosphate buffer containing sodium chloride, and the 3A3 antibody labeled with alkaline phosphatase was diluted 1000 times with phosphate buffer containing sodium chloride for testing. The test samples were 1093 clinical samples tested by Abbott Germany GmbH's 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] It can be seen from Table 5 above that the negative coincidence rate and the positive coincidence rate of the test strip of the present invention and the Abbott control reagent are both 100%, the clinical Kappa consistency is 100%, and there is no missed detection and false positive.

[0115] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A monoclonal antibody against human hepatitis B e antigen, characterized in that: In the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO.1 or has at least 95% homology to the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO.2 or has at least 95% homology to the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO.3 or has at least 95% homology to the sequence shown in SEQ ID NO.3; 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.

2. The monoclonal antibody against human hepatitis B e antigen according to claim 1, characterized in that: In the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the framework 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 the framework 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 the framework 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 the framework 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; In the light chain variable region, the amino acid sequence of the framework 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 the framework 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 the framework 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 the framework 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.

3. The monoclonal antibody against human hepatitis B e antigen according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14, or has at least 95% homology to the sequence shown in SEQ ID NO.14; the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.15, or has at least 95% homology to the sequence shown in SEQ ID NO.

15.

4. The monoclonal antibody against human hepatitis B e antigen according to claim 3, characterized in that: The gene sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID NO.16 or has at least 95% homology with the sequence shown as SEQ ID NO.16; the gene sequence encoding the light chain of the monoclonal antibody is shown as SEQ ID NO.17 or has at least 95% homology with the sequence shown as SEQ ID NO.

17.

5. A biomaterial, characterized in that: The biological material includes any one of the following: (1) A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4; (2) an expression vector / vector set containing the nucleic acid molecule; (3) A host cell containing the nucleic acid molecule or containing an expression vector / vector set.

6. A composition for resisting human hepatitis B e antigen, characterized in that: The invention comprises the monoclonal antibody according to any one of claims 1 to 4.

7. A use of the monoclonal antibody according to any one of claims 1 to 4, or the composition according to claim 6, characterized in that: Products used to prepare human hepatitis B e antigen, or for detecting human hepatitis B e antigen for purposes other than disease diagnosis and treatment.

8. The use according to claim 7, characterized in that: The product is a detection kit, a detection test strip or a detection chip.

9. The use according to claim 8, 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 kit; The test strip is a colloidal gold test strip or an ELISA test strip.

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