An antibody against HPV16 and / or HPV18 type E2 protein and its application

Abstract

The present invention discloses an antibody against HPV16 and / or HPV18 type E2 protein and its application, and relates to the field of biomedical technology. The antibody includes a heavy chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementary determining region in the light chain variable region as shown in SEQ ID NO: 2. The antibody can specifically bind to HPV16 and / or HPV18 type E2 protein, and based on the antibody provided by the present invention, reagents, kits, test strips, antibody chips, antibody probes or detectors for detecting HPV16 and / or HPV18 type E2 antigens can be developed. After testing, the kit developed based on the monoclonal antibody has the advantages of high detection sensitivity, good specificity / specificity, high precision, high accuracy, fast detection, and strong tolerance, and can be used for quality control in the production process of recombinant human HPV16 and / or HPV18 type E2 antigens.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antibody against HPV16 and / or HPV18 type E2 protein and an application thereof. Background Art

[0002] Human papillomavirus (HPV) is a pathogen that mainly infects the squamous epithelium of the human epidermis and mucosa, causing proliferative lesions. It is mainly transmitted through sexual intercourse or close contact. More than 200 HPV subtypes have been identified, which are divided into low-risk and high-risk types according to the severity of the disease they cause. 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, HPV16 and 18 infections are the most important pathogenic factors for the development of cervical cancer. Cervical cancer is one of the most common malignant tumors and one of the major public health issues affecting women's health worldwide. Its incidence and mortality rate rank fourth among female cancers in the world, second only to lung cancer, breast cancer and colorectal cancer. According to statistics from the World Health Organization, there are nearly 600,000 new cases of cervical cancer and approximately 300,000 deaths worldwide each year. However, in socioeconomically developed countries, the incidence and mortality rates of cervical cancer are lower.

[0003] Human papillomavirus (HPV) is a non-enveloped, double-stranded, circular small DNA virus consisting of a viral protein coat and core DNA. The early transcription region of the HPV genome contains multiple proteins such as E1~E8, among which 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 destroy the keratin intermediate filament network, and E6 and E7 are the main oncogenic proteins that are continuously expressed throughout the entire process of carcinogenesis and participate in the malignant transformation of cells. E6 and E7 are important causes of cervical cancer, so they are also recognized as ideal target antigens for therapeutic HPV vaccines.

[0004] The currently available preventive HPV vaccines are all based on virus-like particles (VLPs). VLPs are formed by a single viral protein and are not infectious or carcinogenic. However, no studies have shown that VLP vaccines have therapeutic effects. Therefore, for subjects who have been infected with HPV before vaccination, it cannot change the infection and development of the virus. Patients infected with HPV still lack treatment options. In recent years, in order to fill this gap in the market, many foreign companies have begun to develop therapeutic HPV vaccines for patients who have already developed cervical cancer or precancerous lesions. However, most of the HPV vaccine companies in my country are still in the stage of preventive vaccine development, and there is little research and development on therapeutic HPV vaccines. Therefore, the development of therapeutic HPV vaccines is an ideal strategy to control existing HPV infections and treat related cancers and precancerous lesions caused by HPV infections. It is also closely linked to clinical needs and in line with market trends.

[0005] During the development and production of HPV vaccines, it is very important to monitor the quality of the main active ingredients of the vaccine. Enzyme-linked immunosorbent assay (ELISA) has the advantages of being sensitive, rapid, and highly tolerant, and can be used for quality control during the production of recombinant HPV16 / 18 E2 antigens.

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

[0007] The object of the present invention is to provide an antibody against HPV16 and / or HPV18 type E2 protein and its application to solve the above technical problems.

[0008] The present invention is achieved in that:

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

[0010] In a second aspect, the present invention further provides the use of an antibody or an antigen-binding fragment thereof against HPV16 and / or HPV18 type E2 protein in any of the following:

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

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

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

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

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

[0016] In a third aspect, the present invention also provides an antibody conjugate, which is formed by coupling the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment and a marker, and the marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent reagent and a nanoparticle marker.

[0017] In a fourth aspect, the present invention also provides an HPV16 and / or HPV18 type E2 protein detection product, which includes the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment, or includes the above-mentioned antibody conjugate, and the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0018] In a fifth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment.

[0019] The present invention has the following beneficial effects:

[0020] The antibodies provided by the present invention can specifically bind to HPV16 and / or HPV18 E2 proteins. Based on the antibodies provided by the present invention, detection products for detecting HPV16 and / or HPV18 E2 antigens, such as reagents, test kits, test strips, antibody chips, antibody probes or detectors, can be developed. After testing, the kit developed based on the monoclonal antibody has the advantages of high sensitivity, good specificity / specificity, high intermediate precision, high accuracy, consistent linear range of the standard curve, fast detection, and strong tolerance for HPV16 and / or HPV18 E2 antigen detection. It can be used for quality control in the production process of recombinant human HPV16 and / or HPV18 E2 antigens, and can efficiently and accurately quantify the antigen content in the vaccine and monitor the quality of the vaccine. It is conducive to the development of therapeutic HPV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 It is the flow chart of sandwich ELISA experiment;

[0023] Figure 2 This is the standard curve for 1F4 and 3B3 monoclonal antibodies to detect HPV16 / 18 E2 antigen. DETAILED DESCRIPTION

[0024] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0025] Definition

[0026] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum 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, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0027] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to HPV16 and / or HPV18 E2 antigens or fragments thereof.

[0028] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0029] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. 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 to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0030] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0031] The variable region VL of the light chain of an antibody can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0032] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against HPV16 and / or HPV18 type E2 protein, which comprises a heavy chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementarity determining region in the light chain variable region as shown in SEQ ID NO: 2.

[0033] SEQ ID NO: 1:

[0034] EVQLQQSGAELVKPGASVKLSCTASGFNIGDTYLHWVKQRPDQGLEWIGWIDPANGHTEYDPTFQGKATMTADTSSNTAYLLLSSLTSEDTALYHCSRGGKYYGLDFWGQGASVTVSS.

[0035] SEQ ID NO: 2:

[0036] DIQMTQSPASLSSASVGETVTITTCRTSGNIHNYLAWYQQKQGKSPQLLVYYAKTLADGVPSRFSGSGSGTQYSLKINSLHPEDFGSYYCQHFWSTPYTFGGGTKLKIK.

[0037] By inputting the above SEQ ID NO: 1 and SEQ ID NO: 2 sequences into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0038] The amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining region are discovered and disclosed for the first time in the present invention. They are novel sequences that can endow the antibody or its antigen-binding fragment with the ability to specifically recognize and bind to HPV16 and / or HPV18 type E2 proteins.

[0039] The antibody screened by the present invention having 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 1F4.

[0040] In a preferred embodiment of the present invention, the heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, whose 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 YAK.

[0041] The sequence of CDR-H1, SEQ ID NO: 3 is: GNFIGDTY;

[0042] The sequence of CDR-H2, SEQ ID NO: 4 is: IDPANGHT;

[0043] The sequence of CDR-H3, SEQ ID NO: 5 is: SRGGKYYGLDF;

[0044] The sequence of CDR-L1, SEQ ID NO: 6 is: GNIHNY;

[0045] The sequence of CDR-L3, SEQ ID NO: 8 is: QHFWSTPYT.

[0046] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 9-12, for example, the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are 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 in SEQ ID NOs: 9-12, for example.

[0047] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 13 to 16, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are 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 in SEQ ID NOs: 13 to 16, for example.

[0048] The sequence of the heavy chain framework region, HFR1, SEQ ID NO: 9 is: EVQLQQSGAELVKPGASVKLSCTAS;

[0049] The sequence of HFR2, SEQ ID NO: 10 is: LHWVKQRPDQGLEWIGW;

[0050] The sequence of HFR3, SEQ ID NO: 11 is: EYDPTFQGKATMTADTSSNTAYLLLSSLTSEDTALYHC;

[0051] The sequence of HFR4, SEQ ID NO: 12 is: WGQGASVTVSS.

[0052] The sequence of the light chain framework region, LFR1, SEQ ID NO: 13 is: DIQMTQSPASLSASVGETVTITCRTS;

[0053] The sequence of LFR2, SEQ ID NO: 14 is: LAWYQQKQGKSPQLLVY;

[0054] The sequence of LFR3, SEQ ID NO: 15, is: TLADGVPSRFSGSGSGTQYSLKINSLHPEDFGSYYC;

[0055] The sequence of LFR4, SEQ ID NO: 16 is: FGGGTKLKIK.

[0056] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof 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 region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0057] The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds.

[0058] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0059] The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an antibody.

[0060] In a preferred embodiment of the present invention, the constant region is derived from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans. Cattle are, for example, dairy cows and cattle.

[0061] In a preferred embodiment of the present invention, the species of origin of the constant region of the antibody or antigen-binding fragment thereof is mouse.

[0062] In a second aspect, the present invention further provides the use of an antibody or an antigen-binding fragment thereof against HPV16 and / or HPV18 type E2 protein in any of the following:

[0063] (1) Detection of HPV16 and / or HPV18 E2 protein, and the application is not for the purpose of disease diagnosis;

[0064] (2) Conduct quality control on HPV16 and / or HPV18 vaccines;

[0065] (3) Preparation of HPV16 and / or HPV18 E2 protein detection products;

[0066] (4) Preparation of HPV16 vaccine quality control products or HPV16 vaccine immune effect evaluation products;

[0067] (5) Prepare HPV18 vaccine quality control products or HPV18 vaccine immune effect evaluation products.

[0068] The antibodies or antigen-binding fragments thereof provided by the present invention can specifically bind to HPV16 and / or HPV18 E2 proteins, and thus detection products for HPV16 and / or HPV18 E2 proteins can be developed. They can also be used for quality control during the production of recombinant human HPV16 and / or HPV18 E2 antigens, and can efficiently and accurately quantify the antigen content in the vaccine and monitor the vaccine quality.

[0069] In one embodiment, antibodies or antigen-binding fragments thereof against HPV16 and / or HPV18 E2 proteins can be coated on a separation column, and separation and enrichment of HPV16 and / or HPV18 E2 proteins can be achieved through the specificity and high affinity of the antibodies to HPV16 and / or HPV18 E2 proteins. For example, the antibodies are coated on a filler and filled in a separation column; used for affinity separation and enrichment of HPV16 and / or HPV18 E2 proteins. Therefore, antibodies or antigen-binding fragments thereof against HPV16 and / or HPV18 E2 proteins have good application prospects in the preparation of HPV16 and / or HPV18 E2 protein enrichment products.

[0070] The detection products are reagents, test kits, test strips, antibody chips, antibody probes or detectors.

[0071] In order to improve the stability of the reagent and extend the shelf life, those skilled in the art can add functional components such as stabilizers and protective agents to the reagent as needed. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protective agent 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 semisolid.

[0072] The antibody chip refers to a chip formed by fixing the above-mentioned anti-HPV16 type E6 protein and E7 protein antibodies or antigen-binding fragments thereof on a carrier.

[0073] In a preferred embodiment of the present invention, the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment is used as a coating antibody or a detection antibody.

[0074] The antibody or its antigen-binding fragment is labeled with a detectable marker; the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers. A detectable marker refers to a class of substances that have characteristics that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which the corresponding target can be qualitatively or quantitatively detected. In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter what marker is used, it belongs to the protection scope of the present invention.

[0075] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0076] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0077] In an alternative embodiment, the radioactive isotopes 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 Such as 62 Cu, 64 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0078] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0079] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0080] In a preferred embodiment of the present invention, the kit includes a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; for example, the antibody or antigen-binding fragment thereof is connected to the solid phase by chemical coupling.

[0081] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0082] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0083] When an antibody or an antigen-binding fragment thereof against HPV16 and / or HPV18 type E2 protein is used as the detection antibody, an antibody or an antigen-binding fragment thereof 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;

[0084] Alternatively, when an antibody or an antigen-binding fragment thereof against HPV16 and / or HPV18 type E2 protein is used as the coating antibody, an antibody or an antigen-binding fragment thereof 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.

[0085] The amino acid sequence of the heavy chain variable region (VH) of another antibody is SEQ ID NO: 17:

[0086] QVHLQQSGAELVRPGTSVRVSCKASGYAFTDHLIEWLRQRPGQGLEWIGVINPRTGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARHYDYQYYFDSWGQGTTLTVSS

[0087] The amino acid sequence of the light chain variable region (VL) of another antibody is SEQ ID NO: 18:

[0088] DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQVLVYAATNLADGVSSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTQLEIK.

[0089] This antibody was also obtained by the inventors through screening for the first time and was named 3B3.

[0090] In particular, when the 1F4 monoclonal antibody is used as the coating antibody, and the antibody or antigen-binding fragment of the heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO: 18 is used as the detection antibody, the prepared kit has the advantages of high detection sensitivity, good specificity / specificity, high intermediate precision, high accuracy, consistent linear range of the standard curve, fast detection, and strong tolerance.

[0091] In other embodiments, another antibody against HPV16 and / or HPV18 E2 antigen may be selected as needed and used in combination with the antibody provided by the present invention.

[0092] In a third aspect, the present invention also provides an antibody conjugate, which is formed by coupling the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment and a marker, and the marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent reagent and a nanoparticle marker.

[0093] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0094] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0095] In an alternative embodiment, the radioactive isotopes 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 Such as 62 Cu, 64 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and18 F.

[0096] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0097] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0098] In a fourth aspect, the present invention also provides an HPV16 and / or HPV18 type E2 protein detection product, which includes the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment, or includes the above-mentioned antibody conjugate, and the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0099] The chip can also be called a suspension array or a liquid array, which includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0100] The aforementioned carrier can be made of a variety of materials and forms, for example, it can be preferably selected from containers with a flat bottom. A more typical preferred example is a multi-well plate widely used in biochemical detection, a microplate, a microfluidic-based device (such as a microfluidic chip), a container similar to a watch glass, etc., and is not limited thereto.

[0101] The microfluidic chip is a PDMS chip or a metal droplet generator selected from a T-type chip, a flow focusing chip or a coaxial flow chip, or a PMMA microfluidic chip.

[0102] Furthermore, the kit may also include at least one of a buffer solution, a detection reagent, a diluent, and a washing solution, but is not limited thereto.

[0103] In a fifth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-HPV16 and / or HPV18 type E2 protein antibody or its antigen-binding fragment.

[0104] In a sixth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned antibody against HPV16 and / or HPV18 type E2 protein or its antigen-binding fragment.

[0105] The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and intersugar (backbone) bonds. 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 may also be contained. Examples of these modified bases include nitrogenous and denitrogenous adenine, guanine, cytosine, thymine and uracil; as well as xanthine and hypoxanthine.

[0106] In a seventh aspect, the present invention also provides a vector comprising the above-mentioned nucleic acid molecule.

[0107] The term "vector" is used herein in its most general sense and includes any intermediate medium for nucleic acid, which enables the nucleic acid to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, 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 relates to a construct of extrachromosomal genetic material, typically a circular DNA double strand, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as they can replicate and be stable in the host.

[0108] In an optional embodiment, the above-mentioned vector is an expression vector. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0109] In an eighth aspect, the present invention also provides a recombinant cell comprising the above-mentioned vector.

[0110] 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 comprises prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats or primates. Cells can be derived from multiple tissue types and include primary cells and cell lines. Nucleic acids can be present in host cells in single copy form or in two or more copy forms, and in one embodiment, are expressed in recombinant cells.

[0111] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0112] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0113] In an alternative embodiment, the recombinant cell is HEK293.

[0114] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0115] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0116] Example 1

[0117] This embodiment provides a method for preparing a monoclonal antibody, which specifically comprises the following steps:

[0118] (1) Mouse immunization: Female BALB / c mice were subcutaneously immunized with HPV16 / 18 type E2 antigen (the amino acid sequence of which is shown in SEQ ID NO:7) emulsified in complete Freund's adjuvant (CFA). Two weeks later, the same antigen was emulsified in incomplete Freund's adjuvant (IFA) for immunization. The immunization was continued for multiple times after an interval of two weeks until the serum titer was qualified.

[0119] The amino acid sequence of HPV16 / 18 E2 antigen is: SEQ ID NO:7.

[0120] MSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSIGGSGG SGGSGGSGGSTSDDTVSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRKLCSGNTTPIIHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVAIPDSVQILVGYMTM.

[0121] (2) Cell fusion: Take mouse spleen cells with qualified immune titer and mouse myeloma SP2 / 0 cells, fuse them with 50% PEG at a ratio of 5:1, suspend them in 1% HAT selection medium, and observe the growth of cloned cells for 7-10 days;

[0122] (3) Screening of hybridoma cell lines: Cell lines that react to HPV16 / 18 E2 antigens are screened by indirect ELISA (using HPV16 / 18 E2 antigen coating), and then cloned repeatedly to obtain stable hybridoma cells;

[0123] (4) Monoclonal antibody preparation: Stable secretory hybridoma cells were injected into the peritoneal cavity of BALB / c mice for in vivo ascites production. The ascites was then collected for purification to obtain monoclonal antibodies (1F4) and (3B3).

[0124] (5) Hybridoma cell sequencing / variable region gene sequencing

[0125] When hybridoma cells grow to the logarithmic growth phase, the cells are collected (clone numbers are 1F4 and 3B3), and the total RNA of the hybridoma cells is extracted. The first-strand cDNA complementary to the full-length mRNA is obtained by RT-PCR using the 3'RACE technology and the 5'RACE technology. The synthesized cDNA is used as a template for PCR amplification to obtain the antibody heavy chain and light chain variable region genes, and the antibody variable region genes are connected to the T vector. The positive clones are transformed and selected for sequencing. The sequencing results are analyzed by bioinformatics to obtain the antibody variable region gene sequence.

[0126] The amino acid sequence of the heavy chain variable region of the 1F4 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. The amino acid sequence of the heavy chain variable region of the 3B3 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.

[0127] Example 2

[0128] This embodiment provides a sandwich ELISA detection method.

[0129] This embodiment provides a sandwich ELISA kit and a detection method. The kit includes a polystyrene microplate, a TMB colorimetric solution, a PBST elution solution, a coating antibody (1F4), and an antibody for detection (3B3).

[0130] The principle of the sandwich ELISA detection method is: the specific antibody is diluted to a certain concentration and then fixed on the surface of the polystyrene microplate by physical adsorption. The sample to be tested is added to specifically bind to the antibody coated on the solid phase carrier, and then the enzyme-labeled secondary antibody and colorimetric solution are added. Finally, the stop solution is added 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. The concentration of the antigen in the test sample is calculated based on the standard curve. See the sandwich ELISA experimental flow chart for details. Figure 1 .

[0131] Determination of linear range and antibody working concentration

[0132] (1) According to conventional coating conditions, dilute the mouse monoclonal antibody (1F4) to 1 μg / ml for coating (coating at 2-8°C for 16-24 hours, coating solution formula: NaHCO3 3.068g, Na2CO3 1.435g, add purified water to 1L);

[0133] (2) The next day, wash off the unbound coated antibody with 1*PBST, then add blocking solution for blocking (blocking at 25°C for 1 hour), thus obtaining the antibody-coated ELISA plate;

[0134] (3) When using, add serially diluted standard, test sample and diluted horseradish peroxidase-labeled 3B3 monoclonal antibody in sequence, react at 25°C, add TMB colorimetric solution of horseradish peroxidase colorimetric system, and finally terminate with 1M phosphoric acid and read the specific absorbance value. The labeling method of horseradish peroxidase-labeled 3B3 monoclonal antibody is as follows:

[0135] (a) Weigh 4.2 mg HRP and dissolve it in 420 μl ultrapure water;

[0136] (b) Weigh 9.7 mg NaIO4 and dissolve it in 755 μl ultrapure water;

[0137] (c) Add 420 μl of solution (b) to solution (a) and react at 4°C in the dark for 30 min.

[0138] (d) After the reaction, add 3.78 μl of ethylene glycol and react at room temperature in the dark for 30 min;

[0139] (e) The solution obtained in (d) and the antibody to be labeled were added into a dialysis bag at a mass ratio of 1:1 and dialyzed against 1× CBS buffer;

[0140] (f) After dialysis, transfer the liquid in the dialysis bag into a beaker; then add 45 μl NaBH4 (5 mg / ml) and react at 4°C in the dark for 3 h;

[0141] (g) After the reaction is completed, add saturated ammonium sulfate equal to the total volume of the first six steps and react at 4°C in the dark for 30 min;

[0142] (h) After the reaction, the liquid was taken out and centrifuged at 11000 rpm and 4°C for 15 min;

[0143] (i) Redissolve the precipitate in PBS, then add an equal volume of glycerol, mix well, and store.

[0144] (4) After testing, the results showed that we finally concluded that the optimal working dilution ratio of 3B3 enzyme-labeled antibody was 1:400; in the range of 240ng / ml~1.88ng / ml, the absorbance value was highly linearly correlated with the detection concentration (ng / ml) (R 2 >0.98).

[0145] Example 3

[0146] The present invention verifies the specificity of the sandwich ELISA detection method provided in the above-mentioned Example 2.

[0147] The experimental design is shown in the following table:

[0148]

[0149] illustrate:

[0150] Protein 717 is a recombinant HPV16 / 18 type E2 antigen, and its amino acid sequence is shown in SEQ ID NO. 7. The amino acid sequences of proteins 715, 716, 718, and 721 are shown below, respectively.

[0151] The 715 protein is a recombinant HPV16 E6 / E7 antigen, and its amino acid sequence is shown in SEQ ID NO: 19:

[0152] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKVSEYRYYCYSLYGTTLEQQYNKPLCDLLIRGINCQKPLCPDEKQRHLDKKQRFHNI RGRWTGRCMSCCRSSRTRRETQLgsgsgsgsgsgsgMHGDTPTLHEYMLDLQPETTDLYGYGQLHDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP.

[0153] The 716 protein is a recombinant HPV18 E6 / E7 antigen, and its amino acid sequence is shown in SEQ ID NO: 20:

[0154] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRGLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQC HSCCNRARQERLQRRRETQVgsgsgsgsgsgsgMHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQ.

[0155] The 718 protein is a recombinant HPV16 E1 antigen, and its amino acid sequence is shown in SEQ ID NO: 21:

[0156] MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLYLVSPLSDISGCVDNNEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMVVLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTL。

[0157] The 721 protein is a recombinant HPV18 E1 antigen, and its amino acid sequence is shown in SEQ ID NO:22:

[0158] .

[0159] Acceptance criteria: The recovery rate of each sample is 80%~120%; the presence of other antigens does not interfere with the detection of the test antigen.

[0160] Recovery rate (%) = 717 mixed protein measured value / 717 protein measured value × 100%

[0161] The actual measured value of 717 protein is the detection value containing only 717 protein.

[0162] Specificity verification results

[0163]

[0164] Specificity verification conclusion:

[0165] In the presence of 715, 716, 718 and 721 proteins, the content of 717 protein 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.

[0166] Example 4

[0167] This example verifies the precision (repeatability) of the sandwich ELISA detection method provided in Example 2.

[0168] Experimental design: A batch of 717 proteins was selected and diluted with diluent to high, medium and low concentrations of 240 ng / ml, 120 ng / ml and 60 ng / ml respectively, and then 8 gradients of 2-fold dilution were tested in the plate.

[0169] During the test, each concentration was measured three times, and the CV value of the three repeated tests was calculated.

[0170] Acceptance criteria: The test sample must have at least three dilutions within the linear range of the standard curve; the CV of three replicate tests of the same batch of test samples must be ≤20%.

[0171] The precision (repeatability) verification results are shown in the following table:

[0172]

[0173] Precision (repeatability) conclusion: The CV between the three retest values ​​of each concentration is less than 20% (5.0%, 1.0%, and 0.9%, respectively). The repeatability verification is qualified.

[0174] Example 5

[0175] This example verifies the precision (intermediate precision) of the sandwich ELISA detection method provided in Example 2.

[0176] Experimental design: Three experimenters repeated the "repetitive" experiment on another day and calculated the day-to-day and individual differences.

[0177] Acceptance criteria: CV of test values ​​of the same batch of test products for different dates and different personnel is ≤20%.

[0178] The precision (intermediate precision) verification results are as follows:

[0179]

[0180] Precision (intermediate precision) conclusion: The CV of the 717 antigen test results on different dates and by different testers was less than 20% (4.2%, 1.7%, and 2.7%, respectively). The intermediate precision verification was qualified.

[0181] Example 6

[0182] This example verifies the accuracy of the sandwich ELISA detection method provided in Example 2.

[0183] Experimental design: Select a batch of 717 stock solution (i.e., 717 protein), dilute it with diluent according to Table 1-2, and then add spikes. The spike-adding process is shown in Table 3. Take C5, C6, C7, M4, M5, M6, c1, c2, and c3 for determination, and calculate the recovery rate.

[0184] Recovery rate (%) = (measured value of spiked sample - measured value of sample) / measured value of standard product × 100%

[0185] Acceptance criteria: Recovery rate between 80% and 120%.

[0186] Table 1 The dilution process of 717 stock solution is as follows

[0187]

[0188] Table 2. Dilution process of 717 reference product (i.e. standard product)

[0189]

[0190] Table 3 Accuracy standard addition process

[0191]

[0192] The accuracy verification results are shown in the following table:

[0193]

[0194] Note: Spiked measured value = Spiked sample measured value - Sample measured value.

[0195] The sample measured value is the concentration measurement value of the original solution, the spiked sample measured value refers to the sample concentration after an equal volume of reference material is added to the original solution; the standard sample measured value is the measured value of the reference material.

[0196] Accuracy verification conclusion: The recovery rates of spiked concentrations were all between 80% and 120% (93%, 98%, and 103%, respectively), and the accuracy verification was qualified.

[0197] Example 7

[0198] This example verifies the standard curve (linearity and range) of the sandwich ELISA detection method provided in Example 2.

[0199] Experimental design: Summarize the standard curve data in the precision, accuracy, and specificity verification to obtain the linearity and optimal detection range of the standard curve.

[0200] Acceptance criteria: The correlation coefficient of the standard curve (four-parameter fitting curve) is not less than 0.98, and the R 2 The CV was ≤10% and the optimal detection range of the standard curve was consistent.

[0201] The standard curve (linearity and range) verification results are shown in the following table:

[0202]

[0203] Standard curve (linearity and range) verification conclusion: Based on the previous verifications, the standard curve has good linearity and R 2 All greater than 0.99, R 2 The CV% is less than 10%, which meets the verification standard; the linear range of the standard curve is consistent, which is 240~1.88ng / ml. Figure 2 .

[0204] Example 8

[0205] This embodiment provides a kit, which includes a polystyrene microplate, on the surface of which a 3B3 monoclonal antibody (coating antibody) is immobilized. In addition, the kit also includes: an enzyme-labeled detection antibody (1F4), TMB and a PBST washing solution.

[0206] (1) According to conventional coating conditions, the 8E6 antibody was diluted in a certain ratio for coating (coating at 2-8°C for 16-24 hours, coating solution formula: NaHCO3 3.068g, Na2CO3 1.435g, add purified water to make up to 1L).

[0207] (2) The next day, wash off the unbound coated antibody with 1*PBST, then add blocking solution for blocking (blocking at 25°C for 1 hour), thus obtaining the antibody-coated ELISA plate;

[0208] (3) When using, add serially diluted standard, test sample and diluted horseradish peroxidase-labeled (1F4) monoclonal antibody in sequence, react at 25°C, add TMB colorimetric solution of horseradish peroxidase colorimetric system, and finally terminate with 1M phosphoric acid and read the specific absorbance value.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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. The amino acid sequence of CDR-L2 is YAK.

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 heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 1, and the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to claim 2 or 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. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The species origin of the constant region of the antibody or antigen-binding fragment thereof is mouse.

6. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 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 / or HPV18 vaccines, wherein the HPV16 and / or 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.

7. The use according to claim 6, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 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.

8. 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 5 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.

9. A detection product, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the antibody conjugate according to claim 8, and the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

10. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

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