An antibody against HPV16 and / or HPV18 E2 protein, an antibody conjugate and its application

Abstract

The present invention discloses an antibody against HPV16 and / or HPV18 type E2 protein, an antibody conjugate and their applications, which relate to the field of medical biotechnology. The antibody comprises the heavy chain complementarity determining regions in the heavy chain variable region shown in SEQ ID NO: 1, and the light chain complementarity determining regions in the light chain variable region shown in SEQ ID NO: 2. After testing, the kit developed based on this monoclonal antibody has the advantages of high detection sensitivity for HPV16 and / or HPV18 type E2 antigen, good specificity, high intermediate precision, high accuracy, consistent linear range of the standard curve, rapid detection and strong tolerance. It can be used for quality control in the production process of recombinant human HPV16 and / or HPV18 type E2 antigen, can efficiently and accurately quantify the antigen content in the vaccine, and monitor the vaccine quality.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and more particularly, to an antibody against HPV16 and / or HPV18 E2 protein, an antibody conjugate, and their applications. Background Art

[0002] Human papillomavirus (HPV) is a pathogen that mainly infects human epidermal and mucosal squamous epithelia, causing proliferative lesions, and is mainly transmitted through sexual intercourse or close contact. More than 200 HPV subtypes have been identified so far, and they are classified into low-risk and high-risk types according to the severity of the diseases they cause. The high-risk types mainly include HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68, which can cause genital warts, external genital cancer, cervical cancer, and high-grade cervical intraepithelial neoplasia. In particular, the infections of HPV16 and 18 are the most important pathogenic factors in the development of cervical cancer. Cervical cancer is one of the most common malignant tumors and also one of the major public health problems affecting the health of women globally. Its incidence and mortality rank fourth among female cancers globally, after lung cancer, breast cancer, and colorectal cancer. According to the statistics of the World Health Organization, there are nearly 600,000 new cases of cervical cancer and about 300,000 deaths globally every year. In countries with more developed social and economic conditions, the incidence and mortality of cervical cancer are relatively low.

[0003] Human papillomavirus (HPV) is an enveloped double-stranded closed-loop small DNA virus composed of a viral protein coat and core DNA. The early transcription region of the HPV genome contains multiple proteins such as E1~E8. Among them, E1 plays a key role in the initiation of viral DNA replication, E2 is involved in the trans-activation of viral DNA transcription, E4 can disrupt the keratin intermediate filament network, and E6 and E7 are the main oncoproteins that are continuously expressed throughout the process of carcinogenesis and participate in the process of cellular malignant transformation. E6 and E7 are important causes of cervical cancer. Therefore, both of them are also recognized as ideal target antigens for therapeutic HPV vaccines.

[0004] Currently, all the marketed prophylactic HPV vaccines are virus-like particle (VLP)-based vaccines. VLPs are formed by a single viral protein and are non-infectious and non-carcinogenic. However, there is no study indicating that VLP vaccines have a therapeutic effect. Therefore, for subjects already infected with HPV before vaccination, it cannot change the infection and development of the virus. And patients infected with HPV still lack treatment options. In recent years, to fill this gap in the market, several foreign companies have started the research and development of therapeutic HPV vaccines for patients with cervical cancer or precancerous lesions. Currently, most domestic companies are still in the stage of developing prophylactic vaccines for HPV, and there is less research and development on therapeutic HPV vaccines. Therefore, developing therapeutic HPV vaccines is an ideal strategy to control existing HPV infections and treat related cancers and precancerous lesions caused by HPV infections. It also meets the clinical needs and conforms to the market trend.

[0005] Quality control of the main active ingredients of the vaccine is very important during the R & D and production process of HPV vaccines. Enzyme-linked immunosorbent assay (ELISA) has the advantages of sensitivity, rapidity, and strong tolerance, and can be used for quality control during the production process of recombinant HPV16 / 18 E2 antigen.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide an antibody, antibody conjugate against HPV16 and / or HPV18 E2 protein and their applications to solve the above technical problems.

[0008] The present invention is implemented as follows:

[0009] In the first aspect, the present invention provides an antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein, which comprises the heavy-chain complementarity-determining region in the heavy-chain variable region shown in SEQ ID NO: 1, and the light-chain complementarity-determining region in the light-chain variable region shown in SEQ ID NO: 2.

[0010] In the second aspect, the present invention also provides the application of the antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein in any one of the following:

[0011] (1) Detecting HPV16 and / or HPV18 E2 protein, and the application is not for the purpose of disease diagnosis;

[0012] (2) Conducting quality control on HPV16 and / or HPV18 vaccines;

[0013] (3) Preparing HPV16 and / or HPV18 E2 protein detection products;

[0014] (4) Prepare quality control products for HPV16 vaccine or immune effect evaluation products for HPV16 vaccine;

[0015] (5) Prepare quality control products for HPV18 vaccine or immune effect evaluation products for HPV18 vaccine;

[0016] The detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector.

[0017] In the third aspect, the present invention also provides an antibody conjugate, which is formed by conjugating the above-mentioned antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0018] In the fourth aspect, the present invention also provides a detection product for HPV16 and / or HPV18 E2 protein, which includes the above-mentioned antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment, or includes the above-mentioned antibody conjugate, and the detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector.

[0019] In the fifth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment.

[0020] In the sixth aspect, the present invention also provides a preparation method for the antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment, and culturing cells including the above-mentioned nucleic acid molecule.

[0021] The present invention has the following beneficial effects:

[0022] The antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment provided by the present invention can specifically recognize HPV16 and / or HPV18 E2 protein, and can be used to develop detection products for detecting HPV16 and / or HPV18 E2 antigen, such as reagents, kits, test strips, antibody chips, antibody probes or detectors. After testing, the kit developed based on this monoclonal antibody has the advantages of high detection sensitivity for HPV16 and / or HPV18 E2 antigen, good specificity, high intermediate precision, high accuracy, consistent linear range of the standard curve, fast detection, and strong tolerance. It can be used for quality control in the production process of recombinant human HPV16 and / or HPV18 E2 antigen, can efficiently and accurately quantify the antigen content in the vaccine, and monitor the vaccine quality. It is beneficial to the development of therapeutic HPV vaccines. Description of the Drawings

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a flow chart of the sandwich ELISA experiment.

[0025] Figure 2 It is a standard curve graph for detecting HPV16 / 18 type E2 antigen using 1F4 and 3B3 monoclonal antibodies. Detailed implementation manners

[0026] Reference to the embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0027] 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 of ordinary skill 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 (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0028] Definition of Terms

[0029] The term "antigen-binding fragment" generally refers to any protein / protein fragment that contains CDR regions, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-compound binding fragments of the above-mentioned antibodies, including Fab, F(ab’)2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and minimal antibody recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0030] The term "antibody" in this document is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the required biological activity, such as specifically binding to the HPV16 and / or HPV18 E2 antigen or fragments thereof.

[0031] In the present invention, the term "complementary determining region or complementarity-determining region", "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to regions that contain one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0032] In the present invention, the heavy-chain complementarity-determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light-chain complementarity-determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0033] Under normal circumstances, the variable region VH of the heavy chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0034] The variable region VL of the light chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0035] In a first aspect, the present invention provides an antibody against HPV16 and / or HPV18 E2 protein or an antigen-binding fragment thereof, which comprises the heavy chain complementary determining regions in the heavy chain variable region shown in SEQ ID NO: 1, and the light chain complementary determining regions in the light chain variable region shown in SEQ ID NO: 2.

[0036] SEQ ID NO: 1:

[0037] QVHLQQSGAELVRPGTSVRVSCKASGYAFTDHLIEWLRQRPGQGLEWIGVINPRTGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARHYDYQYYFDSWGQGTTLTVSS;

[0038] SEQ ID NO: 2:

[0039] DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQVLVYAATNLADGVSSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTQLEIK.

[0040] Inputting the above SEQ ID NO: 1 and SEQ ID NO: 2 sequences into the CDR labeling system can obtain the corresponding CDR sequences.

[0041] The amino acid sequences of the above heavy chain complementary determining regions and light chain complementary determining regions are first discovered and revealed by the present invention, and are a new sequence, which can endow the antibody or its antigen-binding fragment with the ability to specifically recognize and bind to HPV16 and / or HPV18 E2 protein.

[0042] The antibody screened out with the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2 is named monoclonal antibody 3B3.

[0043] In a preferred embodiment of the application of the present invention, the heavy chain complementarity determining regions include: CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 3-5 in sequence. The light chain complementarity determining regions include CDR-L1, CDR-L2 and CDR-L3. The amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 6 and SEQ ID NO: 8 respectively, and the amino acid sequence of CDR-L2 is AAT.

[0044] Heavy chain complementarity determining region, CDR-H1, the sequence of SEQ ID NO: 3 is: GYAFTDHL;

[0045] CDR-H2, the sequence of SEQ ID NO: 4 is: INPRTGGT;

[0046] CDR-H3, the sequence of SEQ ID NO: 5 is: ARHYDYQYYFDS;

[0047] Light chain complementarity determining region, CDR-L1, the sequence of SEQ ID NO: 6 is: ENIYNN;

[0048] The sequence of CDR-L2 is: AAT;

[0049] CDR-L3, the sequence of SEQ ID NO: 8 is: QHFWGTPRT.

[0050] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region, and / or, a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 which are respectively at least 80% homologous to the amino acid sequences shown as SEQ ID NO: 9-12 in sequence. For example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 which are respectively at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown as SEQ ID NO: 9-12 in sequence.

[0051] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 which are respectively at least 80% homologous to the amino acid sequences shown as SEQ ID NO: 13-16 in sequence. For example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 which are respectively at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown as SEQ ID NO: 13-16 in sequence.

[0052] Heavy chain framework region, HFR1, the sequence of SEQ ID NO: 9 is: QVHLQQSGAELVRPGTSVRVSCKAS;

[0053] HFR2, the sequence of SEQ ID NO: 10 is: IEWLRQRPGQGLEWIGV;

[0054] HFR3, the sequence of SEQ ID NO: 11 is: NYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC;

[0055] HFR4, the sequence of SEQ ID NO: 12 is: WGQGTTLTVSS.

[0056] Light chain framework region, LFR1, the sequence of SEQ ID NO: 13 is: DIQMTQSPASLSVSVGETVTITCRAS;

[0057] LFR2, the sequence of SEQ ID NO: 14 is: LAWYQQKQGKSPQVLVY;

[0058] LFR3, the sequence of SEQ ID NO: 15 is: NLADGVSSRFSGSGSGTQFSLKINSLQSEDFGSYYC;

[0059] LFR4, the sequence of SEQ ID NO: 16 is: FGGGTQLEIK.

[0060] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further comprises a constant region, the constant region comprises a heavy chain constant region, and / or, a light chain constant region, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the kappa-type or lambda-type light chain constant regions.

[0061] The antigen-binding fragment of the above antibody generally has the same binding specificity as its source antibody. Those skilled in the art can easily understand from the content recorded in the present invention that the functional fragment of the above antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds.

[0062] The antigen-binding fragment of the above antibody can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0063] The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, Fab′-SH, and scFv of an antibody.

[0064] In a preferred embodiment of the application of the present invention, the species source of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human. Bovine, for example, is dairy cow, yellow cattle, etc.

[0065] In a preferred embodiment of the application of the present invention, the species source of the constant region of the antibody or its antigen-binding fragment is murine.

[0066] In a second aspect, the present invention also provides the use of an antibody against HPV16 and / or HPV18 type E2 protein or its antigen-binding fragment in any one of the following:

[0067] (1) Detecting HPV16 and / or HPV18 type E2 protein, and the application is not for the purpose of disease diagnosis;

[0068] (2) Conducting quality control on HPV16 and / or HPV18 type vaccines;

[0069] (3) Preparing a detection product for HPV16 and / or HPV18 type E2 protein;

[0070] (4) Preparing a quality control product for HPV16 type vaccine or an immune effect evaluation product for HPV16 type vaccine;

[0071] (5) Preparing a quality control product for HPV18 type vaccine or an immune effect evaluation product for HPV18 type vaccine;

[0072] The detection product is a reagent, kit, test strip, antibody chip, antibody probe, or detector.

[0073] In order to improve the stability of the reagent and extend its shelf life, those skilled in the art can add functional components such as stabilizers and protectants to the reagent as needed. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100, and Tween-20. The protectant is selected from cryoprotectants, such as polyols and sugars. The polyol is selected from sorbitol, mannitol, or a mixture thereof. The form of the reagent includes, but is not limited to, solid, liquid, and semi-solid.

[0074] The antibody chip refers to: a chip formed by immobilizing the above-mentioned antibody against HPV16 type E6 protein and E7 protein or its antigen-binding fragment on a carrier.

[0075] In a preferred embodiment of the application of the present invention, the kit comprises a solid phase, to which an antibody or its antigen-binding fragment is coated; for example, by means of chemical coupling, the antibody or its antigen-binding fragment is connected to the solid phase.

[0076] In a preferred embodiment of the application of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0077] In a preferred embodiment of the application of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, latex microspheres, microtiter plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0078] Based on the property that the antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein specifically binds to HPV16 and / or HPV18 E2 protein, this antibody is expected to be used for developing detection products against HPV16 and / or HPV18 E2 protein, and can also be used for quality control in the production process of recombinant human HPV16 and / or HPV18 E2 antigen, capable of efficiently and accurately quantifying the antigen content in the vaccine and monitoring the vaccine quality.

[0079] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein serves as a coating antibody or a detection antibody; for example, it can be coated on an enzyme-linked immunosorbent assay (ELISA) plate or used as an enzyme-labeled secondary antibody, or embedded in the conjugate pad of a test strip or in the nitrocellulose membrane of a test strip.

[0080] When the antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein is a detection antibody, an antibody or antigen-binding fragment having a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18 is used as the coating antibody;

[0081] Or, when the antibody or its antigen-binding fragment against HPV16 and / or HPV18 E2 protein is a coating antibody, an antibody or antigen-binding fragment having a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18 is used as the detection antibody.

[0082] SEQ ID NO:17:

[0083] EVQLQQSGAELVKPGASVKLSCTASGFNIGDTYLHWVKQRPDQGLEWIGWIDPANGHTEYDPTFQGKATMTADTSSNTAYLLLSSLTSEDTALYHCSRGGKYYGLDFWGQGASVTVSS;

[0084] SEQ ID NO:18:

[0085] DIQMTQSPASLSASVGETVTITCRTSGNIHNYLAWYQQKQGKSPQLLVYYAKTLADGVPSRFSGSGSGTQYSLKINSLHPEDFGSYYCQHFWSTPYTFGGGTKLKIK。

[0086] This antibody was also first screened and obtained by the inventors and was named 1F4. In particular, when the monoclonal antibody 1F4 is used as the coating antibody and the monoclonal antibody 3B3 is used as the detection antibody, the prepared kit has the advantages of high detection sensitivity, good specificity, high intermediate precision, high accuracy, consistent linear range of the standard curve, rapid detection, and strong tolerance.

[0087] In other embodiments, another antibody against HPV16 and / or HPV18 E2 antigen can also be selected as needed to be paired with the antibody provided by the present invention for use.

[0088] In one embodiment, an antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment can be coated on a separation column, and the separation and enrichment of HPV16 and / or HPV18 E2 protein can be achieved through the specificity and high affinity between the antibody and HPV16 and / or HPV18 E2 protein. For example, the antibody is coated on a filler and filled in a separation column; it is used for the affinity separation and enrichment of HPV16 and / or HPV18 E2 protein. Therefore, the antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment has good application prospects in the preparation of HPV16 and / or HPV18 E2 protein enrichment products.

[0089] In a third aspect, the present invention also provides an antibody conjugate, which is formed by conjugating the above-mentioned antibody against HPV16 and / or HPV18 E2 protein or its antigen-binding fragment with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0090] The above-mentioned label refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through this characteristic. In the actual use process, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it belongs to the protection scope of the present invention.

[0091] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, including but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, including but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, including but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, including but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, including but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0092] In alternative embodiments, the enzymes that catalyze the substrate to develop color include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0093] In alternative embodiments, the radioisotopes include, but are not limited to 212 Bi 131 I 111 In 90 Y 186 Re 211 At 125 I 188 Re 153 Sm 213 Bi 32 P 94 mTc 99 mTc 203 Pb 67 Ga 68 Ga 43 Sc 47 Sc 110 mIn 97 Ru 62 Cu 64 Cu 86 Y 88 Y 121 Sn 161 Tb 166 Ho 105 Rh 177 Lu 172 Lu and18 F.

[0094] In an alternative embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

[0095] In an alternative embodiment, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids; the nanoparticles include, but are not limited to: organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0096] In a fourth aspect, the present invention further provides an HPV16 and / or HPV18 type E2 protein detection product, which includes the above-mentioned antibody against HPV16 and / or HPV18 type E2 protein or its antigen-binding fragment, or, includes the above-mentioned antibody conjugate, and the detection product is a reagent, kit, test strip, antibody chip, antibody probe, or detector.

[0097] The chip can also be referred to as a suspension array or a liquid array. It includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0098] The aforementioned carrier can be of various materials and forms, for example, it can preferably be selected from containers with a flat bottom. A more typical preferred example is the multi-well plates, microplates, microfluidics-based devices (such as microfluidic chips), petri-dish-like containers, etc., which are widely used in biochemical detection, and is not limited thereto.

[0099] The microfluidic chip is selected from a PDMS chip or a metal droplet generator of a T-type chip, a flow-focusing type chip, or a coaxial flow type chip, or a PMMA microfluidic chip.

[0100] Furthermore, the kit may further include at least one of a buffer, a detection reagent, a diluent, and a washing solution, and is not limited thereto.

[0101] In a fifth aspect, the present invention further provides a nucleic acid molecule that encodes the above-mentioned antibody against HPV16 and / or HPV18 type E2 protein or its antigen-binding fragment.

[0102] The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of such modified bases include nitrogen-containing and deazaanine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0103] In a sixth aspect, the present invention also provides a vector containing the above-mentioned nucleic acid molecule.

[0104] The term "vector" is used herein in its most general sense and includes any intermediate vehicle for nucleic acids that is capable of introducing the nucleic acid, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrating it into the genome. This type of vector preferably replicates and / or expresses in the cell. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

[0105] In an alternative embodiment, the above-mentioned vector is an expression vector, and an important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0106] In a seventh aspect, the present invention also provides a recombinant cell containing the above-mentioned vector.

[0107] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. The term "recombinant cell" according to the present invention includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, especially human cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, or primates. The cells can be derived from multiple tissue types and include primary cells and cell lines. The nucleic acid can be present in the host cell in single copy or in two or more copies, and in one embodiment, is expressed in the recombinant cell.

[0108] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0109] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0110] In an alternative embodiment, the recombinant cell is HEK293.

[0111] In an eighth aspect, the present invention also provides a method for preparing an antibody or an antigen-binding fragment thereof against the E2 protein of HPV16 and / or HPV18, by culturing a cell comprising the nucleic acid molecule described above. In order to obtain a target antibody with higher purity, those skilled in the art can easily select a purification step.

[0112] 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. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0113] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0114] Example 1

[0115] This example provides a method for preparing a monoclonal antibody, which specifically includes the following steps:

[0116] (1) Mouse immunization: Female BALB / c mice were subcutaneously immunized with the HPV16 / 18 E2 antigen (the amino acid sequence of which is shown in SEQ ID NO: 7) emulsified with complete Freund's adjuvant (CFA). After two weeks, the same antigen emulsified with incomplete Freund's adjuvant (IFA) was used for immunization. After an interval of two weeks, immunization was continued multiple times until the serum titer was qualified;

[0117] The amino acid sequence of the HPV16 / 18 E2 antigen is: SEQ ID NO: 7.

[0118] MSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSIGGSGGSGGSGGSGGSTSDDTVSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRKLCSGNTTPIIHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVAIPDSVQILVGYMTM。

[0119] (2)Cell fusion: Mouse spleen cells and mouse myeloma cells SP2 / 0 cells with qualified immune titer were taken and fused with 50% PEG at a ratio of 5:1, suspended in 1% HAT selection medium for culture, and the growth of cloned cells was observed after 7 - 10 days;

[0120] (3)Screening of hybridoma cell lines: Cell lines reactive with HPV16 / 18 type E2 antigen were screened by indirect ELISA (coated with HPV16 / 18 type E2 antigen), and then cloned repeatedly to obtain stable hybridoma cells;

[0121] (4)Monoclonal antibody preparation: Hybridoma cells that could secrete stably were injected into the abdominal cavity of BALB / c mice for in vivo ascites production, and then the ascites was taken for purification to obtain monoclonal antibody (1F4) and monoclonal antibody (3B3).

[0122] (5)Sequencing of hybridoma cells / Sequencing of variable region genes

[0123] When the hybridoma cells grew to the logarithmic growth phase, cells (clone numbers 1F4, 3B3) were collected, total RNA of hybridoma cells was extracted, and the first-strand cDNA complementary to the full-length mRNA was obtained by RT-PCR using 3’RACE technology and 5’RACE technology. Using the synthesized cDNA as a template, the variable region genes of the antibody heavy chain and light chain were amplified by PCR. The antibody variable region genes were ligated to the T vector, transformed, and positive clones were selected for sequencing. By bioinformatics analysis of the sequencing results, the antibody variable region gene sequences were obtained.

[0124] The amino acid sequence of the heavy chain variable region of the 1F4 monoclonal antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18. The amino acid sequence of the heavy chain variable region of the 3B3 monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0125] Example 2

[0126] This example provides a sandwich ELISA detection method.

[0127] This example provides a sandwich ELISA kit and a detection method. The kit includes a polystyrene microplate, a TMB chromogenic solution, a PBST elution solution, a coating antibody (1F4), and an antibody for detection (3B3).

[0128] Principle of the sandwich ELISA detection method: Dilute the specific antibody to a certain concentration and fix it on the surface of the polystyrene microplate by physical adsorption. Add the specimen to be tested, which specifically binds to the antibody coated on the solid-phase carrier. Then add the enzyme-labeled secondary antibody and the chromogenic solution. Finally, add the termination solution to terminate the reaction. Quantitative analysis is performed by measuring the absorbance value at a specific wavelength. The absorbance value of the test sample is positively correlated with its antigen concentration, and the concentration of the antigen in the test sample is calculated according to the standard curve. The flow chart of the sandwich ELISA experiment is shown in Figure 1 .

[0129] Determination of the linear range and the working concentration of the antibody

[0130] (1) According to the conventional coating conditions, dilute the mouse monoclonal antibody (1F4) to 1 μg / ml for coating (coat at 2 - 8°C for 16 - 24 hours, coating solution formula: 3.068 g of NaHCO3, 1.435 g of Na2CO3, add purified water to 1 L);

[0131] (2) The next day, wash off the unbound coating antibody with 1*PBST, and then add the blocking solution for blocking (block at 25°C for 1 hour) to obtain the enzyme-labeled plate coated with the antibody;

[0132] (3) When in use, sequentially add serial dilutions of the standard product, the test sample, and the diluted horseradish peroxidase-labeled 3B3 monoclonal antibody. After reacting at 25°C, add the TMB chromogenic solution of the horseradish peroxidase chromogenic system. Finally, terminate with 1 M phosphoric acid and read the specific absorbance value. The labeling method of the horseradish peroxidase-labeled 3B3 monoclonal antibody is as follows:

[0133] (a) Weigh 4.2 mg of HRP and dissolve it in 420 μl of ultrapure water;

[0134] (b) Weigh 9.7 mg of NaIO4 and dissolve it in 755 μl of ultrapure water;

[0135] (c) Add 420 μl of the solution in (b) to (a), and react at 4°C in the dark for 30 min;

[0136] (d) After the reaction, add 3.78 μl of ethylene glycol, and react at room temperature in the dark for 30 min;

[0137] (e) Take the solution obtained from the reaction in (d) and the antibody to be labeled, and add them to a dialysis bag at a mass ratio of 1:1, and dialyze with 1×CBS buffer;

[0138] (f) After dialysis, transfer the liquid in the dialysis bag to a beaker; then add 45 μl of NaBH4 (5 mg / ml), and react at 4°C in the dark for 3 h;

[0139] (g) After the reaction, add saturated ammonium sulfate with the total volume of the first 6 steps of the reaction, and react at 4°C in the dark for 30 min;

[0140] (h) After the reaction, take out the liquid, centrifuge at 11000 rpm and 4°C for 15 min;

[0141] (i) Resuspend the precipitate with PBS, then add an equal volume of glycerol, mix well, and store.

[0142] (4) Through experiments, the results show that we finally obtained the optimal working dilution ratio of the 3B3 enzyme-labeled antibody as 1:400; in the range of 240 ng / ml to 1.88 ng / ml, the absorbance value and the detection concentration (ng / ml) showed a highly linear correlation (R 2 > 0.98).

[0143] Example 3

[0144] The present invention verifies the specificity of the sandwich ELISA detection method provided in Example 2 above.

[0145] The experimental design is shown in the following table:

[0146]

[0147] Note:

[0148] The 717 protein is a recombinant HPV16 / 18 type E2 antigen, and its amino acid sequence is as shown in SEQ ID NO.7. The amino acid sequences of the 715, 716, 718, and 721 proteins are shown as follows.

[0149] The 715 protein is a recombinant HPV16 E6 / E7 antigen, and its amino acid sequence is as shown in SEQ ID NO:19:

[0150] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKVSEYRYYCYSLYGTTLEQQYNKPLCDLLIRGINCQKPLCPDEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLgsgsgsgsgsgsgMHGDTPTLHEYMLDLQPETTDLYGYGQLHDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP。

[0151] The 716 protein is a recombinant HPV18 E6 / E7 antigen, and its amino acid sequence is shown in SEQ ID NO:20 as follows:

[0152] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRGLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVgsgsgsgsgsgsgMHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQ。

[0153] The 718 protein is a recombinant HPV16 E1 antigen, and its amino acid sequence is shown in SEQ ID NO:21 as follows:

[0154] MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLYLVSPLSDISGCVDNNEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMVVLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTL

[0155] The 721 protein is a recombinant HPV18 E1 antigen, and its amino acid sequence is shown in SEQ ID NO:22:

[0156] MADPEGTDGEGTGCNGWFYVQAIVDKKTGDVISDDEDENATDTGSDMVDFIDTQGTFCEQAELETAQALFHAQEVHNDAQVLHVLKRKKRRLFTIADSGYGCSEVEATQIQVTTNGEHGGNVCSGGSTEAIDNGGTEGNNSSVDGTSDNSNIENVNPQCTIAQLKDLLKVNNKQGAMLAVFKDTYGLSFTDLVRNFKSDKTTCTDWVTAIFGVNPTIAEGFKTLIQPFILYAHIQCLDCKWGVLILALLRYKCGKSRLTVAKGLSTLLHVPETCMLIQPPKLRSSVAALYWYRTGISNISEVMGDTPEWIQRLTIIQHGIDDSNFDLSEMVQWAFDNELTDESDMAFEYALLADSNSNAAAFLKSNCQAKYLKDCATMCKHYRRAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRYQHIEFITFLGALKSFLKGTPKKNCLVFCGPANTGKSYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCWTYFDTYMRNALDGNPISIDRKHKPLIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFPFDKNGNPVYEINDKNWKCFFERTWSRLDLHEEEEDADTEGNPFGTFKCVAGQNHRPL。

[0157] Acceptance criteria: The recovery rate of each sample is 80% - 120%; in the presence of other antigens, it does not interfere with the detection of the antigen in the test sample.

[0158] Recovery rate (%) = Measured value of the mixed protein 717 / Measured value of protein 717 × 100%

[0159] The measured value of protein 717 is the detection value containing only protein 717.

[0160] Results of specificity verification

[0161]

[0162] Conclusion of specificity verification:

[0163] In the presence of proteins 715, 716, 718, and 721 respectively, the content of protein 717 can still be accurately detected (the recovery rate is within the acceptable range of 80% - 120%), that is, the specificity of this detection method is good. The specificity verification is qualified.

[0164] Example 4

[0165] In this example, the precision (repeatability) of the sandwich ELISA detection method provided in Example 2 was verified.

[0166] Experimental design: Select one batch of protein 717, dilute it with a diluent to three concentrations of high, medium, and low, which are 240 ng / ml, 120 ng / ml, and 60 ng / ml respectively, and then perform 8 - gradient 2 - fold serial dilutions within the plate for detection.

[0167] During detection, each concentration was measured 3 times. Calculate the CV value of the three repeated measurements.

[0168] Acceptance criteria: There must be at least 3 dilutions of the test sample within the linear range of the standard curve; the CV of the three repeated measurements of the test sample in the same batch ≤ 20%.

[0169] The results of the precision (repeatability) verification are as follows:

[0170]

[0171] Conclusion of precision (repeatability): The CV among the three repeated measurements at each concentration is less than 20% (5.0%, 1.0%, 0.9% respectively). The repeatability verification is qualified.

[0172] Example 5

[0173] In this example, the precision (intermediate precision) of the sandwich ELISA detection method provided in Example 2 was verified.

[0174] Experimental design: Another day, three experimenters repeated the "repeatability" experiment to calculate the between - day and between - person differences.

[0175] Acceptance criteria: For the test samples of the same batch on different days and by different persons, the CV of the detection values ≤ 20%.

[0176] The results of the precision (intermediate precision) verification are as follows:

[0177]

[0178] Conclusion of precision (intermediate precision): The CV of the detection results of the 717 antigen by different experimenters on different days is less than 20% (4.2%, 1.7%, 2.7% respectively). The intermediate precision verification is qualified.

[0179] Example 6

[0180] This example verifies the accuracy of the sandwich ELISA detection method provided in Example 2.

[0181] Experimental design: Select 1 batch of 717 stock solution (i.e., 717 protein), dilute it according to Table 1-2 with the diluent, then add the standard. The process of adding the standard is shown in Table 3. Take C5, C6, C7, M4, M5, M6, c1, c2, c3 for determination, and calculate the recovery rate.

[0182] Recovery rate (%) = (measured value of the spiked sample - measured value of the sample) / measured value of the standard × 100%.

[0183] Acceptance criteria: The recovery rate is between 80% and 120%.

[0184] Table 1 The dilution process of 717 stock solution is as follows

[0185]

[0186] Table 2. Dilution process of 717 reference product (i.e., standard product)

[0187]

[0188] Table 3 Accuracy spiking process

[0189]

[0190] The results of accuracy verification are shown in the following table:

[0191]

[0192] Note: Measured value of the spiked sample = measured value of the spiked sample - measured value of the sample.

[0193] The measured value of the sample is the measured value of the concentration of the stock solution, the measured value of the spiked sample is the sample concentration after adding the same volume of the reference product to the stock solution; the measured value of the standard is the measured value of the reference product.

[0194] Conclusion of accuracy verification: The recovery rates of spiking at each concentration are all between 80% and 120% (93%, 98%, 103% respectively), and the accuracy verification is qualified.

[0195] Example 7

[0196] This example verifies the standard curve (linearity and range) of the sandwich ELISA detection method provided in Example 2.

[0197] Experimental design: Summarize the standard curve data in the verification of precision, accuracy, and specificity to obtain the linearity and the best detection range of the standard curve.

[0198] Acceptance criteria: The correlation coefficients of the standard curves (four-parameter fitting curves) are all not less than 0.98, and the CV of R for each standard curve 2 ≤ 10%; The optimal detection ranges of the standard curves are consistent.

[0199] The verification results of the standard curves (linearity and range) are as follows:

[0200]

[0201] Verification conclusion of the standard curves (linearity and range): From the previous verifications, it can be seen that the standard curves have good linearity, R 2 are all greater than 0.99, and the CV% of R 2 is less than 10%, meeting the verification standards; the linear ranges of the standard curves are consistent, being 240 - 1.88 ng / ml. The standard curves are as Figure 2 .

[0202] Example 8

[0203] This example provides a kit, which includes a polystyrene microplate with the 3B3 monoclonal antibody (coating antibody) fixed on its surface. In addition, the kit also includes: an enzyme-labeled detection antibody (1F4), TMB, and PBST washing solution.

[0204] (1) According to the conventional coating conditions, dilute the 8E6 antibody at a certain ratio for coating (coat at 2 - 8°C for 16 - 24 hours, coating solution formula: 3.068 g of NaHCO3, 1.435 g of Na2CO3, and make up to 1 L with purified water).

[0205] (2) The next day, wash off the unbound coating antibody with 1*PBST, and then add the blocking solution for blocking (block at 25°C for 1 hour), thus obtaining the enzyme-labeled plate coated with the antibody;

[0206] (3) When in use, sequentially add the serially diluted standard products, test samples, and diluted horseradish peroxidase-labeled (1F4) monoclonal antibody. After reacting at 25°C, add the TMB chromogenic solution of the horseradish peroxidase chromogenic system, and finally terminate with 1M phosphoric acid and read the specific absorbance value.

[0207] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: It includes a heavy chain complementary determining region and a light chain complementary determining region. The heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown in SEQ ID NO: 3-5 respectively. The light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3. The amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 6 and SEQ ID NO: 8 respectively, and the amino acid sequence of CDR-L2 is AAT.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof also includes a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 9-12, respectively; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 13-16, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the light chain constant region of κ type or λ type; The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody.

5. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in any of the following: (1) Detection of HPV16 and HPV18 E2 fusion proteins, the amino acid sequences of which are shown in SEQ ID NO.7, and the application is not for the purpose of disease diagnosis; (2) Performing quality control on HPV16 and HPV18 vaccines, wherein the HPV16 and HPV18 vaccines contain HPV16 and HPV18 E2 fusion proteins, and the amino acid sequence of the HPV16 and HPV18 E2 fusion proteins is shown in SEQ ID NO.7; (3) Preparing a detection product for HPV16 and HPV18 E2 fusion proteins, wherein the amino acid sequence of the HPV16 and HPV18 E2 fusion proteins is shown in SEQ ID NO.7; (4) Preparation of HPV16 vaccine quality control products or HPV16 vaccine immune effect evaluation products, wherein the HPV16 vaccine contains HPV16 and HPV18 E2 fusion proteins, and the amino acid sequence of the HPV16 and HPV18 E2 fusion proteins is shown in SEQ ID NO.7; (5) Preparing a quality control product for HPV18 vaccine or a product for evaluating the immune effect of HPV18 vaccine, wherein the HPV18 vaccine contains a fusion protein of HPV16 and HPV18 E2, and the amino acid sequence of the fusion protein of HPV16 and HPV18 E2 is shown in SEQ ID NO.7; The detection product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector.

6. The use according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 as a coating antibody or a detection antibody; When the antibody or antigen-binding fragment thereof is a detection antibody, the antibody or antigen-binding fragment having a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 18 is used as a coating antibody; Or, when the antibody or antigen-binding fragment thereof is a coating antibody, the antibody or antigen-binding fragment having a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 18 is used as a detection antibody.

7. An antibody conjugate, characterized in that: It is formed by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a marker, wherein the marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes substrate color development, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

8. A detection product, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the antibody conjugate according to claim 7, and the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

9. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: Cultivate cells comprising the nucleic acid molecule of claim 9.

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