Anti-HBsAg antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an anti-hepatitis B virus surface antigen (HBsAg) antibody or a functional fragment thereof and application thereof. The anti-HBsAg 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 better specificity and sensitivity to HBsAg, and can be used for detecting HBsAg and diagnosing or assisting in diagnosing related diseases taking HBsAg as a marker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to an anti-HBsAg 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.

[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, that is, the commonly said "two pairs and a half of hepatitis B". When HBV replicates, a large amount of outer membrane (HBsAg) is produced, and only one in ten thousand of the total virus particles can assemble into complete and mature virus particles. The vast majority of the viruses only have their "surface" (HBsAg) but no "interior" (HBeAg). Therefore, the serum test results of many patients are positive for HBsAg and negative for HBeAg; while the core antigen and E antigen promote virus maturation, and their positivity indicates the presence of complete and mature virus particles.

[0004] Common detection methods for HBsAg include enzyme-linked immunosorbent assay, colloidal gold method, chemiluminescence method, etc., and the reaction principle is based on the sandwich method of antigen-antibody reaction. Currently, the sources of commercial antibodies for detecting HBsAg are few, and most of the detection kits use imported kits from abroad. Since the demand for hepatitis B detection in China is large, preparing antibodies against it can effectively improve the domestic detection ability for this marker. Summary of the Invention

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

[0006] Specifically as follows:

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

[0008] CDR-VH1: S-X1-Y-F-X2-N, where X1 is G or A, and X2 is F or W;

[0009] CDR-VH2: Y-X1-T-Y-D-G-Y-N-X2-Y-N-P-S-X3-K-N, where X1 is I or V, X2 is K or E, and X3 is L or I;

[0010] CDR-VH3: D-G-X1-Y-Y-P-Y-X2-Y-T-L-D-Y, where X1 is T or S, and X2 is T or Y;

[0011] CDR-VL1: S-A-S-X1-S-V-N-S-X2-Y, where X1 is T or S, and X2 is M or V;

[0012] CDR-VL2: L-X1-S-N-X2-A-S, where X1 is T or S, and X2 is I or L;

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

[0014] Further, X2 of CDR-VH1 of the complementarity determining region is W, X2 of CDR-VH2 is K, X1 of CDR-VH3 is T, X2 of CDR-VL1 is M, X1 of CDR-VL2 is T, and X1 of CDR-VL3 is Q.

[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 and light chains has 3 CDRs, starting from the N-terminus of the heavy or 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 speaking, CDR residues directly and most substantially participate in affecting antigen binding.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0045]

[0046]

[0047]

[0048] Further, the antibody 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 heavy-chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are sequentially selected from SEQ ID NO: 1-4; the light-chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are sequentially selected from SEQ ID NO: 5-8.

[0049] Further, the antibody further 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 cow, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken 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] Further, the antibody is a coating antibody.

[0053] The "coating antibody" of the present invention is a coating capable of capturing 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 invention are preferably goat, sheep, mouse, rabbit or rat antibodies, chimeric antibodies or further genetically engineered antibodies, provided that the characteristic properties according to the 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 from 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 from antibody fragments.

[0055] Preferably, the carrier coated with the 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-mentioned antibody or its functional fragment.

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

[0061] On the other hand, the present invention discloses a reagent for detecting HBsAg, which includes 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 HBsAg.

[0063] Beneficial effects:

[0064] Currently, there are few antibody raw materials related to HBsAg on the market, and their performance is uneven. The present invention not only provides a group of monoclonal antibodies with high affinity and specificity for binding to HBsAg, which can well meet the clinical detection needs, especially when applied in chemiluminescent detection reagents, it can significantly improve the detection performance of the reagents and provide multiple references for immunoassay reactions. Detailed implementation manners

[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 described clearly and completely 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 are all conventional products that can be obtained through commercial purchase.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one 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. The techniques employed or considered herein are standard methods unless otherwise indicated. The materials, methods, and examples are illustrative only and not limiting.

[0067] As used herein, the terms "comprising," "including," "having," "may," and variations thereof 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 well 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 HBs protein at a concentration of 2 mg / ml in equal volumes 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, and 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 2.0 or more. Screen mice with a serum titer of 10 6 or more, and take lymph to isolate lymphocytes for cell fusion.

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

[0074] Isolate lymphocytes from immunized mice and perform PEG1500-mediated fusion or electrofusion with cultured SP2 / 0 cells. The fused cells are cultured and screened in HAT-1640 medium containing 20% FBS serum. After one week, change the medium, and after culturing for another 4 days, take the culture supernatant for positive clone screening. Use HBs protein for positive well screening. Select wells with a higher ratio of ELISA positive value to cell number for multiple subcloning. Coat the ELISA plate with HBs protein. Take the culture supernatant of subcloning and screen for monoclonal antibodies that can show affinity under the condition of antigen coating, and select the monoclonal hybridoma cell with the highest affinity from them. Finally, obtain a hybridoma cell line with a high antibody titer that can secrete HBs monoclonal antibody, named 15#, and it 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 mice. One week after the injection, inject 0.5 ml of cell 15# 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 15#.

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

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

[0079] 1) Dilute the HBs antigen with coating solution (0.05 M carbonate and bicarbonate buffer, pH 9.5) to 1 μg / mL, 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 solution).

[0080] 2) Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with dilution solution (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 the dilution solution.

[0081] 3) After washing the plate 3 times with the washing solution, 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 15# is IgG1, 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 in good growth state, obtain the total RNA of hybridoma cells using the 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 purifying 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 buffer (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 diluent (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 HBs protein to 500 ng / mL with diluent. Take out the ELISA plate incubated at 37°C, wash the plate 3 times, add the prepared biotin-labeled HBs protein diluent at 150 μL per well to row A of the 96-well ELISA plate, and add diluent at 100 μL per well to rows B - H of the 96-well ELISA plate. Take 50 μL of the antibody diluent from 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 diluent. 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 15# antibody in Example 1 is SEQ ID NO:11, and the light chain sequence is SEQ ID NO:12. Among them, the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0089] CDR-VH1: S-A(X1)-Y-F-F(X2)-N;

[0090] CDR-VH2: Y-V(X1)-T-Y-D-G-Y-N-E(X2)-Y-N-P-S-I(X3)-K-N;

[0091] CDR-VH3: D-G-S(X1)-Y-Y-P-Y-T(X2)-Y-T-L-D-Y;

[0092] Its light chain variable region is as shown in SEQ ID NO:10. Among them, the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0093] CDR-VL1: S-A-S-T(X1)-S-V-N-S-M(X2)-Y;

[0094] CDR-VL2: L-S(X1)-S-N-I(X2)-A-S;

[0095] CDR-VL3: Q-N(X1)-W-S-S-Y(X2)-P-Y-

[0096] On the basis of antibody 15#, mutations were made at the sites related to antibody activity in the complementarity-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] As can be seen from the results in the above table, the activity effect of the mutant 4 antibody is the best. Therefore, using the mutant 4 antibody as the backbone sequence, other mutation sites with better affinity were screened, and some of the results are as follows:

[0102] Table 3 Mutation sites related to antibody affinity

[0103]

[0104]

[0105] For HBs protein at 500 ng / ml, the absorbance values of OD450 for each mutation were measured, and the results are shown in the following table. It can be seen that the affinities of all mutant antibodies are relatively high.

[0106] Table 4 Affinity test of antibody mutations

[0107]

[0108]

[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 values at 4°C and the accelerated values. The measurement results are as follows. The concentration of the antibody in the present invention is 1 μg / mL, and the concentration of HBs protein is 500 ng / mL.

[0111] Table 5 Stability study

[0112]

[0113]

[0114] Example 3 Antibody Application Research

[0115] 1. Application of antibodies in HBsAg luminescent detection kit

[0116] Since the above mutant antibodies all have relatively high affinity, it can be estimated that the effects of mutant antibodies 4-1 to 4-30 applied to immunoassays should be roughly similar. In view of the fact that the examples are not exhaustive, this example selects mutant 4-13 for testing, couples it with biotin, selects another paired antibody to label alkaline phosphatase, and evaluates the performance of the antibody.

[0117] (1) Reagent configuration

[0118] R1 reagent: dilute the biotin-labeled mutant 4-13 to 1ug / ml with HBsAg-specific anti-reagent 1 buffer, prepare 7mL, mark it and place it on a vortex mixer for at least 60s for later use.

[0119] R2 reagent: dilute the antibody labeled with alkaline phosphatase to 1ug / ml with HBsAg-specific anti-reagent 2 buffer, prepare 7mL, mark it and place it on a vortex mixer, mix it for no less than 60s, and set aside.

[0120] Magnetic bead reagent: dilute the magnetic beads labeled with avidin to 1ug / ml with magnetic bead reagent buffer, prepare 7mL, mark it and place it on a vortex mixer, mix it for no less than 60s, and set aside.

[0121] 1 ml of standard sample for each point, one reference sample from the enterprise, one set of gradient serum, 9 genotype samples, and 20 mutant samples;

[0122] (2) Computer test

[0123] Set up the instrument according to the "EXI1800 Fully Automatic Chemiluminescence Immunoassay Instrument Operation and Maintenance Instructions" and HBsAb project parameters, and load R1, R2, and magnetic bead reagents.

[0124] Testing company reference products and clinical samples: Place HBsAb company reference products and clinical samples into the EXI1800 instrument sample tray, and apply for testing according to the "EXI1800 Fully Automatic Chemiluminescence Immunoassay Instrument Use and Maintenance Operating Instructions".

[0125] (3) Data results

[0126] a. Calibration experiment

[0127] Table 6 HBsAg calibration test data

[0128]

[0129] As shown in the above table, 0.05 IU / ml is the critical point for positive / negative judgment;

[0130] b. Clinical tests

[0131] Table 7 Detection data of genotype samples

[0132]

[0133]

[0134] Table 8 Detection data of mutant strain samples

[0135] Sample Control Positive and negative judgment 2-15 Positive and negative judgment Mutant strain - 01 1437264 ﹢ 860472 ﹢ Mutant strain - 02 2341091 ﹢ 1340004 ﹢ Mutant strain - 03 879163 ﹢ 631046 ﹢ Mutant strain - 04 517773 ﹢ 245493 ﹢ Mutant strain - 05 2828084 ﹢ 1989678 ﹢ Mutant strain - 06 2066743 ﹢ 1134179 ﹢ Mutant strain - 07 1699695 ﹢ 321520 ﹢ Mutant strain - 08 2890161 ﹢ 1047291 ﹢ Mutant strain - 09 1087825 ﹢ 381881 ﹢ Mutant strain - 10 4466 ﹣ 440559 ﹢ Mutant strain - 11 2598489 ﹢ 1189943 ﹢ Mutant strain - 12 1295649 ﹢ 457670 ﹢ Mutant strain - 13 5293 ﹣ 150206 ﹢ Mutant strain - 14 3861 ﹢ 368241 ﹢ Mutant strain - 15 4257 ﹢ 80899 ﹢ Mutant strain - 16 4598566 ﹢ 2373439 ﹢ Mutant strain - 17 436399 ﹢ 213917 ﹢ Mutant strain - 18 351888 ﹢ 2433006 ﹢ Mutant strain - 19 628747 ﹢ 771354 ﹢ Mutant strain - 20 1247107 ﹢ 689295 ﹢

[0136] It can be seen from the test results that this strain of antibody can be effectively applied to the quantitative detection of the HBsAg item, and can effectively distinguish the positive and negative of hepatitis B surface antigen. While quantitatively determining positive samples, compared with some antibodies of the same type on the market, it can more effectively detect mutant samples.

[0137] 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 examples here.

Claims

1. An anti-HBsAg 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-Y-F-X2-N, wherein X1 is G or A, and X2 is F or W; CDR-VH2: Y-X1-T-Y-D-G-Y-N-X2-Y-N-P-S-X3-K-N, wherein X1 is I or V, X2 is K or E, and X3 is L or I; CDR-VH3: D-G-X1-Y-Y-P-Y-X2-Y-T-L-D-Y, wherein X1 is T or S, and X2 is T or Y; CDR-VL1: S-A-S-X1-S-V-N-S-X2-Y, wherein X1 is T or S, and X2 is M or V; CDR-VL2: L-X1-S-N-X2-A-S, wherein X1 is T or S, and X2 is I or L; CDR-VL3: Q-X1-W-S-S-X2-P-Y, wherein X1 is Q or N, and X2 is T or Y.

2. The anti-HBsAg antibody or its functional fragment according to claim 1, characterized in that, In the complementarity-determining regions, X2 of CDR-VH1 is W, X2 of CDR-VH2 is K, X1 of CDR-VH3 is T, X2 of CDR-VL1 is M, X1 of CDR-VL2 is T, and X1 of CDR-VL3 is Q.

3. The anti-HBsAg antibody or its functional fragment according to claim 1, characterized in that, In the CDR-VH1, X1 is G; Preferably, in the CDR-VH1, X1 is A; Preferably, in the CDR-VH1, X2 is F; Preferably, in the CDR-VH1, X2 is W; Preferably, in the CDR-VH2, X1 is I; Preferably, in the CDR-VH2, X1 is V; Preferably, in the CDR-VH2, X2 is K; Preferably, in the CDR-VH2, X2 is E; Preferably, in the CDR-VH2, X3 is L; Preferably, in the CDR-VH2, X3 is I; Preferably, in the CDR-VH3, X1 is T; Preferably, in the CDR-VH3, X1 is S; Preferably, in the CDR-VH3, X2 is T; Preferably, in the CDR-VH3, X2 is Y; Preferably, in the CDR-VL1, X1 is T; Preferably, in the CDR-VL1, X1 is S; Preferably, in the CDR-VL1, X2 is M; Preferably, in the CDR-VL1, X2 is V; Preferably, in the CDR-VL2, X1 is T; Preferably, in the CDR-VL2, X1 is S; Preferably, in the CDR-VL2, X2 is I; Preferably, in the CDR-VL2, X2 is L; Preferably, in the CDR-VL3, X1 is Q; Preferably, in the CDR-VL3, X1 is N; Preferably, in the CDR-VL3, X2 is T; Preferably, in the CDR-VL3, X2 is Y.

4. The anti-HBsAg 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-HBsAg antibody or its functional fragment according to any one of claims 1-4, characterized in that, The antibody or its functional fragment further comprises heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H with sequences successively shown as SEQ ID NO: 1-4, and / or, light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L with sequences successively shown as SEQ ID NO: 5-8.

6. The anti-HBsAg antibody or its functional fragment according to claim 5, characterized in that, The antibody further comprises 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 bovine, equine, dairy cow, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

7. The anti-HBsAg antibody or its functional fragment 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 beads, and latex microspheres; Preferably, the magnetic beads can be selected from at least one of carboxyl magnetic beads, methylacrylamide magnetic beads, and streptavidin magnetic beads; Preferably, the latex microspheres 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 microspheres 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 as described in claim 8.

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

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