Anti-HBeAg antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an anti-hepatitis B E antigen (HBeAg) antibody or a functional fragment thereof and application thereof. The anti-HBeAg antibody or the functional fragment thereof disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and the antibody or the functional fragment thereof has relatively good specificity and sensitivity to HBeAg, and can be used for detecting the HBeAg and diagnosing or assisting in diagnosing related diseases taking the HBeAg as a marker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an anti-HBeAg antibody or its functional fragment and its application. Background Art

[0002] Hepatitis B (HBV) is a viral hepatitis caused by the hepatitis B virus, which can be transmitted through unprotected sexual contact, blood transfusion, reuse of contaminated needles and syringes, mother-to-child transmission during childbirth, etc. Due to its extremely strong infectivity, there are approximately 350 million HBV carriers worldwide. In China, there are about 93 million hepatitis B carriers and 30 million hepatitis B patients, with a total infection rate as high as 35.5%-61.1%. Establishing the detection and prevention of hepatitis B virus is one of the primary tasks in China at present.

[0003] The main markers for current hepatitis B diagnosis are the "five hepatitis B items", including hepatitis B virus surface antigen, hepatitis B virus surface antibody, hepatitis B virus e antigen, hepatitis B virus e antibody, and hepatitis B virus core antigen, which is commonly referred to as the "two pairs and a half of hepatitis B". Hepatitis B e antigen (HBeAg) is a soluble protein in the core particles of the hepatitis B virus. The detection rate of HBeAg increases during the active stage of hepatitis B, indicating severe damage to liver cells and strong infectivity of the patient. Hepatitis B e antibody (HBeAb) is one of the serological marker indicators for human infection with the hepatitis B virus. The English abbreviation of hepatitis B e antibody is anti-HBeAb. The positive hepatitis B e antibody mainly appears in asymptomatic hepatitis B surface antigen carriers and chronic hepatitis B patients. This indicates that the virus replication in their bodies is inhibited to varying degrees, indicating a decrease in HBV in the blood and a reduction in infectivity.

[0004] Common detection methods for HBeAg and HBeAb include enzyme-linked immunosorbent assay, colloidal gold method, chemiluminescence method, etc. The reaction principle is based on the sandwich method of antigen-antibody reaction. When detecting HBeAb, one solution is to bind the neutralizing antigen to the HBeAb to be detected, and then use a double-antibody sandwich to detect the amount of the neutralizing antigen to determine the content of the HBeAb to be detected. Currently, the antibody sources for detecting HBeAg and HBeAb are scarce, and most of the detection kits are imported from abroad. Since the demand for hepatitis B detection in China is very large, preparing antibodies for it can effectively improve the domestic detection ability for this marker. Summary of the Invention

[0005] The object of the present invention is to provide an anti-HBeAg antibody or its functional fragment, as well as a reagent for detecting HBeAg. This antibody or its functional fragment has good affinity for hepatitis B e antigen, and using this antibody or its functional fragment to detect HBeAg has good sensitivity and specificity.

[0006] Specifically as follows:

[0007] On the one hand, the present invention provides an anti-HBeAg antibody or a functional fragment thereof, and the anti-Hb antibody or its functional fragment has the following complementarity-determining regions:

[0008] CDR-VH1: S-X1-G-X2-H, where X1 is P or F, and X2 is V or L;

[0009] CDR-VH2: V-X1-W-P-G-G-S-T-X2-Y-N-S-A-X3-M-S, where X1 is I or V, X2 is N or Q, and X3 is L or I;

[0010] CDR-VH3: D-G-A-L-X1-L-L-R-A-M-X2-Y, where X1 is R or A, and X2 is W or D;

[0011] CDR-VL1: K-A-S-Q-S-X1-V-T-T-A-X2-A, where X1 is D or E, and X2 is L or V;

[0012] CDR-VL2: S-X1-S-Y-X2-Y-T, where X1 is G or A, and X2 is R or A;

[0013] CDR-VL3: Q-Q-X1-Y-S-X2-P-Y-T, where X1 is H or R, and X2 is T or S.

[0014] Furthermore, X2 of CDR-VH1 of the complementarity-determining region is V, X2 of CDR-VH2 is N, X1 of CDR-VH3 is R, X1 of CDR-VL1 is D, X2 of CDR-VL2 is R, and X2 of CDR-VL3 is T.

[0015] The CDR segment division of the present invention adopts the Kabat algorithm.

[0016] "CDR" is used herein to refer to the "complementarity-determining region" within the variable sequence of an antibody. Each of the variable regions of the heavy chain and the light chain has 3 CDRs, starting from the N-terminus of the heavy chain or the light chain.

[0017] The antigen-binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in the present invention). A polypeptide containing a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of the antigen-antibody complex has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, and the most extensive antigen contacts are with the heavy-chain CDR3. Therefore, the molecular recognition unit may be mainly responsible for the specificity of the antigen-binding site. Generally, CDR residues directly and most substantially participate in affecting antigen binding.

[0018] In an alternative embodiment, X1 in CDR-VH1 is P;

[0019] In an alternative embodiment, X1 in CDR-VH1 is F;

[0020] In an alternative embodiment, X2 in CDR-VH1 is H;

[0021] In an alternative embodiment, X2 in CDR-VH1 is V;

[0022] In an alternative embodiment, X1 in CDR-VH2 is L;

[0023] In an alternative embodiment, X1 in CDR-VH2 is V;

[0024] In an alternative embodiment, X2 in CDR-VH2 is N;

[0025] In an alternative embodiment, X2 in CDR-VH2 is Q;

[0026] In an alternative embodiment, X3 in CDR-VH2 is L;

[0027] In an alternative embodiment, X3 in CDR-VH2 is I;

[0028] In an alternative embodiment, X1 in CDR-VH3 is R;

[0029] In an alternative embodiment, X1 in CDR-VH3 is A;

[0030] In an alternative embodiment, X2 in CDR-VH3 is W;

[0031] In an alternative embodiment, X2 in CDR-VH3 is D;

[0032] In an alternative embodiment, X1 in the CDR-VL1 is D;

[0033] In an alternative embodiment, X1 in the CDR-VL1 is E;

[0034] In an alternative embodiment, X2 in the CDR-VL1 is L;

[0035] In an alternative embodiment, X2 in the CDR-VL1 is V;

[0036] In an alternative embodiment, X1 in the CDR-VL2 is G;

[0037] In an alternative embodiment, X1 in the CDR-VL2 is A;

[0038] In an alternative embodiment, X2 in the CDR-VL2 is R;

[0039] In an alternative embodiment, X2 in the CDR-VL2 is A;

[0040] In an alternative embodiment, X1 in the CDR-VL3 is H;

[0041] In an alternative embodiment, X1 in the CDR-VL3 is R;

[0042] In an alternative embodiment, X2 in the CDR-VL3 is T;

[0043] In an alternative embodiment, X2 in the CDR-VL3 is S;

[0044] Preferably, each of the complementarity determining regions is selected from any one of the following mutation combinations:

[0045]

[0046]

[0047]

[0048] Furthermore, the antibody or its functional fragment further comprises a light chain framework region FR1-L, FR2-L, FR3-L and FR4-L and a heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H; the FR1-H sequence is SEQ ID NO1 or SEQ ID NO2; the sequences of FR2-H, FR3-H and FR4-H are sequentially selected from SEQ ID NO: 3-5; the sequences of FR1-L, FR2-L, FR3-L and FR4-L are sequentially selected from SEQ ID NO: 6-9.

[0049] Furthermore, the antibody also comprises a constant region.

[0050] Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. Preferably, the species origin of the constant region is bovine, equine, dairy cattle, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, or human.

[0051] Preferably, the functional fragment is selected from any one of VHH, F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

[0052] Furthermore, the antibody is a coating antibody.

[0053] The “coating antibody” in the present invention is a coating that can capture an antigen.

[0054] The term “antibody” includes various forms of antibody structures, including but not limited to intact antibodies and antibody fragments. The antibodies according to the present invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, as long as the characteristic properties according to the present invention are retained. “Antibody fragment” comprises a part of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed by antibody fragments. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed by antibody fragments.

[0055] Preferably, the coating carrier of the coating antibody includes but is not limited to nitrocellulose membranes, ELISA plates, magnetic beads, latex microspheres, etc.

[0056] Preferably, the magnetic beads include but are not limited to carboxyl magnetic beads, methylacrylamide magnetic beads, streptavidin magnetic beads, etc.

[0057] Preferably, the latex microspheres are often polyvinyl alcohol toluene, polystyrene, or microspheres formed by copolymerizing these two substances as the main components and adding other monomers.

[0058] Preferably, the latex microspheres include but are not limited to carboxyl latex microspheres, amino latex microspheres, etc.

[0059] On the other hand, the present invention discloses a vector, which contains a nucleic acid fragment encoding the above antibody or its functional fragment.

[0060] On the other hand, the present invention discloses a recombinant cell, which contains the above vector.

[0061] On the other hand, the present invention discloses a reagent for detecting HBeAg, and the reagent comprises the above-mentioned antibody or its functional fragment.

[0062] On the other hand, the present invention discloses the application of the above-mentioned antibody and its antigen-binding fragment in a chemiluminescent detection kit for HBeAg.

[0063] Beneficial effects: Currently, there are few HBeAg-related antibody raw materials on the market, and their performance varies. The present invention not only provides a monoclonal antibody (wild type) with high affinity for binding to HBeAg, but also makes various mutations with excellent performance on this wild type antibody. It has a wide application range and higher applicability, and can be applied to the use of detecting HBeAg in a kit, providing multiple references for immunoassay reactions. Description of the Drawings

[0064] Figure 1 : The correlation curve between the calibrator and the optical value Detailed Embodiments

[0065] Specifically, to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0067] As used herein, the terms "comprising", "including", "having", "may", and their variants are intended to be transitional phrases, terms, or words that are open-ended and do not exclude the possibility of additional acts or structures.

[0068] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0069] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0070] Example 1 Preparation of Monoclonal Antibodies

[0071] 1. Mouse Immunization and Antibody Detection

[0072] Select 5 SPF-grade female BALB / c mice aged 6 - 8 weeks. Mix Freund's complete adjuvant and HBe protein at a concentration of 2 mg / ml in equal volume and emulsify. Immunize the SPF-grade female BALB / c mice aged 6 - 8 weeks with the emulsified antigen. Inject 50 μg of antigen protein into each mouse by plantar injection or subcutaneous injection in the back. Two weeks after the primary immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify it. Then, inject 50 μg of antigen protein into each mouse again by plantar injection or subcutaneous injection in the back. Two weeks later, collect blood from the tail vein, centrifuge to collect the supernatant, and detect the serum titer by ELISA. Immunize once every two weeks and detect the serum titer. After 2 immunizations, the serum titer after million-fold dilution is as high as over 2.0. Screen the mice with a serum titer above 10^6, and take the lymph to isolate lymphocytes for cell fusion.

[0073] 2. Cell fusion, screening and subcloning of positive hybridoma cells

[0074] Isolate the lymphocytes of the immunized mice and fuse them with the cultured SP2 / 0 cells by PEG1500-mediated fusion or electrofusion. The fused cells are cultured and screened in HAT-1640 medium containing 20% FBS serum. After one week, change the medium. After culturing for another 4 days, take the culture supernatant for positive clone screening. Use HBe protein for positive well screening. Select the wells with a higher ratio of ELISA positive value to cell number for multiple subcloning. Coat the ELISA plate with HBe protein. Take the culture supernatant of the subclones and screen for monoclonal antibodies that can show affinity under the condition of antigen coating. Select the monoclonal hybridoma cells with the highest affinity from them. Finally, obtain a hybridoma cell line with a relatively high antibody titer that can secrete HBe monoclonal antibody, named 57#, which has good stability.

[0075] 3. Production and purification of monoclonal antibody

[0076] Select two groups of BALB / c mice aged 6 - 8 weeks, and intraperitoneally inject 500 μL of paraffin oil to inhibit the immune response of the mice. One week after the injection, inject 0.5 ml of cells 57# into the abdominal cavity of the mice, and the cell number is about 1×10^6. Start collecting ascites two weeks later. The collected ascites is subjected to ammonium sulfate precipitation and affinity purification with protein A to obtain the target antibody 57#.

[0077] 4. Subtype identification and gene sequence cloning of monoclonal antibody

[0078] Use the SBAClonotyping System-HRP kit from Southern Biothech to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions of the manual. The specific operation is as follows:

[0079] a. Dilute the HBe antigen to 1 μg / mL with coating buffer (0.05 M carbonate and bicarbonate buffer, pH 9.5), add 100 μL per well to the ELISA plate, and coat overnight at 4 °C. Wash the plate 3 times with PBS buffer containing 0.05% Tween-20 (washing buffer).

[0080] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with dilution buffer (1% BSA, 0.1% PBST), add 100 μL per well to the ELISA plate, and incubate at 37 °C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) 1:3000 with dilution buffer.

[0081] c. After washing the plate 3 times with the washing buffer, add 100 μL of the diluted enzyme-labeled antibody to each well, and incubate at 37 °C for 30 minutes. Wash the plate 3 times again, then add the chromogenic solution. After about 5 minutes (depending on the strength of the reaction), add 2 M sulfuric acid to terminate the reaction, and read the absorbance at OD450. After identification, the heavy chain subtype of antibody 57# is IgG2a, and the light chain is Kappa.

[0082] According to the antibody subtype results, clone the antibody gene sequence using the method based on the RACE technical route. Collect hybridoma cells with good growth status, obtain the total RNA of hybridoma cells using a total RNA extraction kit, reverse transcribe the mRNA into cDNA according to the operation method of the SMARTer RACE instruction manual of Takara Company, and amplify the full-length sequence of the target antibody.

[0083] 5. In vitro expression of antibody

[0084] According to the full-length antibody sequence, synthesize it into the pcDNA3.1(+) vector separately for the heavy and light chains, transfect it into HEK293 for expression verification, and obtain the antibody after purification of the fermentation supernatant with protein G.

[0085] Example 2 Verification of antibody performance

[0086] 1. ELISA affinity test

[0087] Dilute goat anti-mouse to 1 μg / mL with coating solution (0.05 M carbonate and bicarbonate buffer, pH = 9.5), add 100 μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate 3 times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1 μg / mL with dilution solution (1% BSA, 0.1% PBST), add 100 μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Dilute biotin-labeled HBe protein to 1000 ng / mL with dilution solution. Take out the ELISA plate incubated at 37°C, wash the plate 3 times, add 150 μL of the prepared biotin-labeled HBe protein dilution to row A of the 96-well ELISA plate, and add dilution solution 100 μL per well to rows B - H of the 96-well ELISA plate. Take 50 μL of the antibody dilution in row A wells, perform 3-fold serial dilution successively from row B, and incubate at 37°C for 30 minutes. Dilute avidin labeled with horseradish peroxidase 1:5000 with dilution solution. Take out the ELISA plate, wash the plate 3 times, add 100 μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Take out the ELISA plate, wash the plate 3 times, add chromogenic solution, develop color at room temperature for 3 minutes, add 0.5 M sulfuric acid to terminate the reaction, and read the OD450 value on a microplate reader.

[0088] The heavy chain sequence of antibody 57# in Example 1 is SEQ ID NO: 13&14, and the light chain sequence is SEQ ID NO: 15. The heavy chain variable region is as shown in SEQ ID NO: 10&11. The amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0089] CDR-VH1: S-P(X1)-G-L(X2)-H;

[0090] CDR-VH2: V-V(X1)-W-P-G-G-S-T-Q(X2)-Y-N-S-A-I(X3)-M-S;

[0091] CDR-VH3: D-G-A-L-R(X1)-L-L-R-A-M-W(X2)-Y.

[0092] The light chain variable region is as shown in SEQ ID NO: 12. The amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0093] CDR-VL1: K-A-S-Q-S-E(X1)-V-T-T-A-L(X2)-A;

[0094] CDR-VL2: S-G(X1)-S-Y-A(X2)-Y-T;

[0095] CDR-VL3: Q-Q-R(X1)-Y-S-S(X2)-P-Y-T

[0096] Based on antibody 57#, mutations were made at the sites related to antibody activity in the complementary determining regions, where X1, X2, and X3 are all mutation sites.

[0097] Table 1 Mutation sites related to antibody activity

[0098]

[0099] Table 2 Antibody activity analysis data

[0100]

[0101]

[0102] As can be seen from the above table, the activity effect of mutation 3 is the best. Therefore, mutation 3 was used as the backbone sequence, and other mutation sites with better affinity were screened. Some of the results are as follows:

[0103] Table 3 Mutation sites related to antibody affinity

[0104]

[0105]

[0106] For HBe protein at 1000 ng / ml, the absorbance value of OD450 for each mutation was measured. The results are as follows, and it can be seen that the affinity of each mutation is relatively high.

[0107] Table 4 Affinity test of antibody mutations

[0108] Mutation 3-1 2.2002 Mutation 3-11 2.3909 Mutation 3-21 2.3948 Mutation 3-2 2.3874 Mutation 3-12 2.4711 Mutation 3-22 2.2412 Mutation 3-3 2.289 Mutation 3-13 2.4046 Mutation 3-23 2.4492 Mutation 3-4 2.2973 Mutation 3-14 2.3842 Mutation 3-24 2.4077 Mutation 3-5 2.3053 Mutation 3-15 2.4566 Mutation 3-25 2.2814 Mutation 3-6 2.2206 Mutation 3-16 2.3655 Mutation 3-26 2.3681 Mutation 3-7 2.4717 Mutation 3-17 2.4498 Mutation 3-27 2.2238 Mutation 3-8 2.3183 Mutation 3-18 2.3319 Mutation 3-28 2.4445 Mutation 3-9 2.3926 Mutation 3-19 2.3291 Mutation 3-29 2.4351 Mutation 3-10 2.3161 Mutation 3-20 2.2003 Mutation 3-30 2.4102

[0109] 2. Stability determination

[0110] The antibody was thermally accelerated at 37°C for 7 days in a defined buffer (PBS, 0.05% ProClin TM 300), and the accelerated antibody was evaluated by SDS-PAGE and indirect ELISA, with 4°C as the control to identify the long-term stability of the antibody. In addition, by performing 5 cycles of repeated freezing and thawing at -20°C, the test results were shown as the deviation between the value at 4°C and the value after acceleration. The measurement results are as follows. The concentration of the antibody in the present invention is 1 μg / mL, and the concentration of HBe protein is 1000 ng / mL.

[0111] Table 5 Stability study

[0112]

[0113]

[0114] Example 3 Antibody Application Research

[0115] 1. Application of Antibody in HBeAg Luminescence Detection Kit

[0116] Since the affinities of the above mutant antibodies are relatively high, it can be estimated that the effects of applying mutant antibodies 3-1 to 3-30 in immunoassay should be roughly similar. Given that it is impossible to exhaust all examples, mutant 3-25 is selected for testing in this example.

[0117] (1) Reagent Preparation

[0118] R1 Reagent: Dispense 7 ml of the special reagent 1 buffer for HBeAg.

[0119] R2 Reagent: Dilute the biotin-labeled antibody to 1 μg / ml with the special anti-reagent 2 buffer for HBeAg, prepare 7 mL, mark it, and place it on a vortex mixer for at least 60 s of mixing, then set aside for use.

[0120] Magnetic Bead Reagent: Dilute the 3-25 antibody labeled with magnetic beads to 1 μg / ml with the special anti-reagent buffer for HBeAg, prepare 7 mL, mark it, and place it on a vortex mixer for at least 60 s of mixing, then set aside for use.

[0121] (2) Instrument Testing

[0122] Set the instrument according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescence Immunoassay Analyzer" and the HBeAg project parameters, and load R1, R2, and magnetic bead reagents.

[0123] Test enterprise reference products and clinical samples: Put the HBeAg enterprise reference products and clinical samples into the sample tray of the EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.) instrument, and apply for testing according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescence Immunoassay Analyzer".

[0124] (3) Data Results

[0125] a. Calibration Experiment

[0126] Table 6 HBeAg Calibration Test Data

[0127]

[0128] Complete calibration using the method of serial dilution calibration. The calibration results are shown in the above table, and the calibration results meet the requirements: the linear regression equation of the standard curve R 2 ≥0.99 (as shown in the appendix Figure 1 )

[0129] b. Clinical tests

[0130] Table 7 Detection data of positive and negative samples

[0131] Sample Sample Number Mean RLU Concentration Positive Dilution 2-fold S1 18089973 128.45 Positive Dilution 20-fold S2 2322771 16.2 Positive Dilution 200-fold S3 236653 1.6 All-Negative Blood S4 2758 0

[0132] It can be seen from the test results that this strain of antibody can be effectively applied to the quantitative detection of the HBeAg project, can effectively distinguish the positive and negative of hepatitis B antigen, and quantitatively determine the positive samples.

[0133] 2. Application of antibody in HBeAb chemiluminescent detection kit

[0134] Similarly, mutation 3-25 was selected for the test. The reagents to be measured included R1, R2 and magnetic bead components, as well as standards, negative plasma and positive samples;

[0135] (1) Reagent preparation

[0136] R1 reagent: Dilute the neutralizing antigen labeled with biotin to 1 μg / ml with the special anti-reagent 1 buffer for HBeAg, prepare 7 mL, make marks and place it on the vortex mixer, and mix for no less than 60 s for standby.

[0137] R2 reagent: Dilute the antibody labeled with alkaline phosphatase to 1 μg / ml with the special anti-reagent 2 buffer for HBeAg, prepare 7 mL, make marks and place it on the vortex mixer, and mix for no less than 60 s for standby.

[0138] Magnetic bead reagent: Dilute the 3-25 antibody labeled with magnetic beads to 1 μg / ml with the special anti-reagent buffer for HBeAg, prepare 7 mL, make marks and place it on the vortex mixer, and mix for no less than 60 s for standby.

[0139] (2) Instrument test

[0140] Set the instrument according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescent Immunoassay Analyzer" and the HBeAb project parameters, and load R1, R2 and magnetic bead reagents.

[0141] Test the enterprise reference products and clinical samples: Put the HBeAb enterprise reference products and clinical samples into the sample tray of the EXI1800 instrument, and apply for testing according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescent Immunoassay Analyzer".

[0142] (3) Data results

[0143] Table 8 Calibration data of national standard products

[0144]

[0145] Table 9 National Reference Disk Test Data

[0146]

[0147]

[0148] It can be seen from this that this strain of antibody can be effectively applied to the quantitative detection of the HBeAb project, can effectively distinguish the positive and negative of hepatitis B antigens, and quantitatively determine positive samples.

[0149] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. An anti-HBeAg antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment comprises the following complementarity-determining regions: CDR-VH1: S-X1-G-X2-H, where X1 is P or F, and X2 is V or L; CDR-VH2: V-X1-W-P-G-G-S-T-X2-Y-N-S-A-X3-M-S, where X1 is I or V, X2 is N or Q, and X3 is L or I; CDR-VH3: D-G-A-L-X1-L-L-R-A-M-X2-Y, where X1 is R or A, and X2 is W or D; CDR-VL1: K-A-S-Q-S-X1-V-T-T-A-X2-A, where X1 is D or E, and X2 is L or V; CDR-VL2: S-X1-S-Y-X2-Y-T, where X1 is G or A, and X2 is R or A; CDR-VL3: Q-Q-X1-Y-S-X2-P-Y-T, where X1 is H or R, and X2 is T or S.

2. The anti-HBeAg antibody or its functional fragment according to claim 1, characterized in that, For the complementarity-determining regions, X2 of CDR-VH1 is V, X2 of CDR-VH2 is N, X1 of CDR-VH3 is R, X1 of CDR-VL1 is D, X2 of CDR-VL2 is R, and X2 of CDR-VL3 is T.

3. The anti-HBeAg antibody or a functional fragment thereof according to claim 1, characterized in that, In the CDR-VH1, X1 is P; Preferably, in the CDR-VH1, X1 is F; Preferably, in the CDR-VH1, X2 is H; Preferably, in the CDR-VH1, X2 is V; Preferably, in the CDR-VH2, X1 is L; Preferably, in the CDR-VH2, X1 is V; Preferably, in the CDR-VH2, X2 is N; Preferably, in the CDR-VH2, X2 is Q; Preferably, in the CDR-VH2, X3 is L; Preferably, in the CDR-VH2, X3 is I; Preferably, in the CDR-VH3, X1 is R; Preferably, in the CDR-VH3, X1 is A; Preferably, in the CDR-VH3, X2 is W; Preferably, in the CDR-VH3, X2 is D; Preferably, in the CDR-VL1, X1 is D; Preferably, in the CDR-VL1, X1 is E; Preferably, in the CDR-VL1, X2 is L; Preferably, in the CDR-VL1, X2 is V; Preferably, in the CDR-VL2, X1 is G; Preferably, in the CDR-VL2, X1 is A; Preferably, in the CDR-VL2, X2 is R; Preferably, in the CDR-VL2, X2 is A; Preferably, in the CDR-VL3, X1 is H; Preferably, in the CDR-VL3, X1 is R; Preferably, in the CDR-VL3, X2 is T; Preferably, in the CDR-VL3, X2 is S.

4. The anti-HBeAg antibody or its functional fragment according to any one of claims 1-3, characterized in that, Each of the complementarity-determining regions is selected from any one of the following mutation combinations:

5. The anti-HBeAg antibody or its functional fragment according to any one of claims 1-4, characterized in that, The antibody or its functional fragment also includes a light chain framework region FR1-L, FR2-L, FR3-L and FR4-L and a heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H; the FR1-H sequence is SEQ ID NO1 or SEQ ID NO2; the sequences of FR2-H, FR3-H and FR4-H are sequentially selected from SEQ ID NO: 3-5; the sequences of FR1-L, FR2-L, FR3-L and FR4-L are sequentially selected from SEQ ID NO: 6-9..

6. The anti-HBeAg antibody or its functional fragment according to claim 5, characterized in that, The antibody also includes a constant region; Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. Preferably, the species origin of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

7. The anti-HBeAg antibody or functional fragment thereof according to claim 6, wherein, The antibody is a coating antibody; Preferably, the coating carrier of the coating antibody can be selected from at least one of nitrocellulose membrane, ELISA plate, magnetic bead, and latex microsphere; Preferably, the magnetic bead can be selected from at least one of carboxyl magnetic bead, methylacrylamide magnetic bead, and streptavidin magnetic bead; Preferably, the latex microsphere can be selected from at least one of polyvinyl alcohol toluene microspheres, polystyrene microspheres, or microspheres polymerized mainly from polyvinyl alcohol toluene and polystyrene; Preferably, the latex microsphere can be selected from at least one of carboxyl latex microspheres and amino latex microspheres.

8. A carrier, characterized in that, It contains a nucleic acid fragment encoding the antibody or its functional fragment as described in any one of claims 1-7.

9. A recombinant cell, characterized in that, It contains a vector, and the vector contains the vector described in claim 8.

10. A reagent for detecting HBeAg, characterized in that, It includes the antibody or its functional fragment as described in any one of claims 1-7.

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