CN119529069A

CN119529069A - An antibody against HPV16 and/or HPV18 type E2 protein, an antibody conjugate and its application

Info

Publication number: CN119529069A
Application number: CN202510097271.1A
Authority: CN
Inventors: 方习静; 陈静; 胡敏; 杨增云
Original assignee: Shanghai Maikekang Biotechnology Co ltd; Chengdu Maikekang Biotechnology Co ltd
Current assignee: Shanghai Maikekang Biotechnology Co ltd; Chengdu Maikekang Biotechnology Co ltd
Priority date: 2025-01-22
Filing date: 2025-01-22
Publication date: 2025-02-28

Abstract

The invention discloses an antibody and an antibody conjugate for resisting HPV16 and/or HPV18 type E2 proteins and application thereof, and relates to the technical field of medical biology. The antibody comprises a heavy chain complementarity determining region in a heavy chain variable region as shown in SEQ ID NO. 1, and a light chain complementarity determining region in a light chain variable region as shown in SEQ ID NO. 2. Through testing, the kit developed based on the monoclonal antibody has the advantages of high detection sensitivity, good specificity, high intermediate precision, high accuracy, consistent linear range of standard curve, rapid detection and strong tolerance on HPV16 and/or HPV18 type E2 antigen, 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 vaccine, and monitors the quality of the vaccine.

Description

Antibody for resisting HPV16 and/or HPV18 type E2 protein, antibody conjugate and application thereof

Technical Field

The invention relates to the technical field of medical biology, in particular to an antibody for resisting HPV16 and/or HPV18 type E2 protein, an antibody conjugate and application thereof.

Background

Human papillomavirus (human papillomavirus, HPV) is a pathogen that primarily infects human epidermis and mucosal squamous epithelium, causing proliferative lesions, primarily transmitted by sex life or intimate contact. More than 200 HPV subtypes have been identified, and are classified into low-risk and high-risk types according to the severity of the disease they cause. The high-risk type mainly comprises HPV16/18/31/33/35/39/45/51/52/56/58/59/68, can cause genital wart diseases, external genital cancers, cervical cancers and high cervical intraepithelial neoplasia, and particularly HPV16 and 18 type infections are the most important causative factors for the development of cervical cancers. Cervical cancer is one of the most common malignancies, and one of the major public health problems affecting global female health, with morbidity and mortality in the fourth place of global female cancers, next to lung, breast and colorectal cancers. According to world health organization data statistics, the new incidence of cervical cancer is nearly 60 ten thousand and death is about 30 ten thousand each year worldwide, and in countries with developed socioeconomic performance, the incidence rate and death rate of cervical cancer are lower.

Human papillomaviruses (human papillomavirus, HPV) are non-enveloped, double-stranded closed-loop small DNA viruses, consisting of viral protein coat and core DNA. The early transcribed region of HPV genome contains a plurality of proteins such as E1-E8, wherein E1 plays a key role in viral DNA initial replication, E2 relates to trans-activation of viral DNA transcription, E4 can destroy keratin intermediate fiber network, E6 and E7 are main oncogenic proteins, and are continuously expressed in the whole process of canceration occurrence and participate in the malignant transformation process of cells. E6 and E7 are important causes of cervical cancer, and therefore, both are also recognized as ideal target antigens for therapeutic HPV vaccines.

The prophylactic HPV vaccines currently on the market are all vaccines based on virus-like particles (VLPs), formed from a single viral protein, without infectivity and carcinogenicity. However, no studies have shown that VLP vaccines have therapeutic effects, and thus it is not possible to alter the infection and development of viruses in subjects who have been infected with HPV prior to immunization with the vaccine. While patients infected with HPV currently remain lacking in treatment options. In recent years, in order to make up for the gap in the market, for patients with cervical cancer or precancerous lesions, a plurality of foreign enterprises start to develop therapeutic HPV vaccines, but most of the enterprises in China still are in a preventive vaccine development stage for HPV vaccines, and the development of the therapeutic HPV vaccines is less, so that the development of the therapeutic HPV vaccines is an ideal strategy for controlling the existing HPV infection and treating related cancers and precancerous lesions caused by the HPV infection, is also a tight clinical requirement, and meets the market trend.

Quality monitoring of the main active ingredients of the vaccine is very important in the HPV vaccine research and development production process. The ELISA has the advantages of sensitivity, rapidness and strong tolerance, and can be used for quality control in the production process of recombinant HPV16/18 type E2 antigen.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an antibody for resisting HPV16 and/or HPV18 type E2 protein, an antibody conjugate and application thereof, so as to solve the technical problems.

The invention is realized in the following way:

In a first aspect, the present invention provides an antibody or antigen binding fragment thereof against HPV16 and/or HPV18 type E2 protein comprising a heavy chain complementarity determining region in the heavy chain variable region as set forth in SEQ ID No.1, and a light chain complementarity determining region in the light chain variable region as set forth in SEQ ID No. 2.

In a second aspect, the invention also provides the use of an antibody or antigen binding fragment thereof against HPV16 and/or HPV18 type E2 protein in any one of:

(1) Detecting HPV16 and/or HPV18 type E2 proteins and using for no disease diagnosis purposes;

(2) Quality control of HPV16 and/or HPV18 type vaccines;

(3) Preparing HPV16 and/or HPV18 type E2 protein detection products;

(4) Preparing an HPV16 type vaccine quality control product or an HPV16 type vaccine immune effect evaluation product;

(5) Preparing an HPV18 type vaccine quality control product or an HPV18 type vaccine immune effect evaluation product;

the detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

In a third aspect, the present invention also provides an antibody conjugate formed by coupling an antibody against HPV16 and/or HPV18 type E2 protein or an antigen-binding fragment thereof as described above and a label selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a nanoparticle-based label.

In a fourth aspect, the invention also provides an HPV16 and/or HPV18 type E2 protein detection product comprising an antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein as described above, or comprising an antibody conjugate as described above, the detection product being a reagent, kit, test strip, antibody chip, antibody probe or detector.

In a fifth aspect, the invention also provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof against HPV16 and/or HPV18 type E2 protein as described above.

In a sixth aspect, the invention also provides a method of preparing an antibody or antigen binding fragment thereof against HPV16 and/or HPV18 type E2 protein, culturing a cell comprising a nucleic acid molecule as described above.

The invention has the following beneficial effects:

The antibody or antigen binding fragment thereof for resisting the HPV16 and/or HPV18 type E2 protein provided by the invention can specifically recognize the HPV16 and/or HPV18 type E2 protein, and can be used for developing detection products for detecting HPV16 and/or HPV18 type E2 antigen, such as reagents, kits, test strips, antibody chips, antibody probes or detectors. Through testing, the kit developed based on the monoclonal antibody has the advantages of high detection sensitivity, good specificity, high intermediate precision, high accuracy, consistent linear range of standard curve, rapid detection and strong tolerance on HPV16 and/or HPV18 type E2 antigen, 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 vaccine, and monitors the quality of the vaccine. Is beneficial to developing therapeutic HPV vaccine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a sandwich ELISA assay.

FIG. 2 is a standard graph of HPV16/18 type E2 antigen detection using 1F4 and 3B3 monoclonal antibodies.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, 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 invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, 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 ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

Noun definition

The term "antigen binding fragment" refers broadly to all proteins/protein fragments, particularly antibodies or antibody functional fragments, comprising CDR regions. "antigen binding fragments" include antigen compound binding fragments of these 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 may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD, and the like. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The term "antibody" is used herein in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specifically binding to HPV16 and/or HPV 18E 2 antigen or fragments thereof.

In the present invention, the terms "complementarity determining region or complementarity determining region", "CDR" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions comprising 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 an antigen or epitope recognized by the antibody or antigen binding fragment. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3. CDR labelling methods commonly used in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the novel 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 KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.

Typically, the variable region VH of the heavy chain of an antibody is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with FRs in a combinatorial arrangement. HCDR1 is synonymous with CDR-H1.

The variable region VL of the light chain of an antibody may be obtained by ligating the CDRs numbered LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combination arrangement.

SEQ ID NO:1:

QVHLQQSGAELVRPGTSVRVSCKASGYAFTDHLIEWLRQRPGQGLEWIGVINPRTGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARHYDYQYYFDSWGQGTTLTVSS;

SEQ ID NO:2:

DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQVLVYAATNLADGVSSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTQLEIK.

The corresponding CDR sequences can be obtained by inputting the sequences of SEQ ID NO. 1 and SEQ ID NO. 2 into a CDR marking system.

The amino acid sequences of the heavy chain complementarity determining region and the light chain complementarity determining region are the first discovered and revealed by the present invention as novel sequences that confer upon the antibody or antigen binding fragment thereof the ability to specifically recognize and bind to HPV16 and/or HPV18 type E2 proteins.

The screened antibody having the heavy chain variable region shown in SEQ ID NO.1 and the light chain variable region shown in SEQ ID NO. 2 was designated as monoclonal antibody 3B3.

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

The sequence of the heavy chain complementarity determining region, CDR-H1, SEQ ID NO:3 is GYAFTDHL;

The sequence of CDR-H2 and SEQ ID NO. 4 is INPRTGGT;

CDR-H3, SEQ ID NO. 5 is ARHYDYQYYFDS;

The sequence of the light chain complementarity determining region, CDR-L1, SEQ ID NO. 6 is ENIYNN;

the sequence of CDR-L2 is AAT;

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

In preferred embodiments of the invention, the antibody or antigen binding fragment thereof further comprises a heavy chain framework region comprising HFR1, HFR2, HFR3 and HFR4 which in turn have at least 80% homology to the amino acid sequences shown in SEQ ID NOS 9-12, and/or a light chain framework region comprising HFR1, HFR2, HFR3 and HFR4 which in turn have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequences shown in SEQ ID NOS 9-12.

The light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 that have at least 80% homology with the amino acid sequences shown in SEQ ID NOS 13-16 in sequence. For example, the light chain framework regions include LFR1, LFR2, LFR3 and LFR4 that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequences shown in SEQ ID NOS 13-16.

The sequence of the heavy chain framework region, HFR1 and SEQ ID NO. 9 is QVHLQQSGAELVRPGTSVRVSCKAS;

The sequence of HFR2 and SEQ ID NO. 10 is IEWLRQRPGQGLEWIGV;

the sequence of the HFR3 and SEQ ID NO. 11 is NYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC;

the sequence of HFR4 and SEQ ID NO. 12 is WGQGTTLTVSS.

The sequence of the light chain framework region, LFR1 and SEQ ID NO. 13 is DIQMTQSPASLSVSVGETVTITCRAS;

the sequence of the LFR2 and SEQ ID NO. 14 is LAWYQQKQGKSPQVLVY;

the sequence of the LFR3 and SEQ ID NO. 15 is NLADGVSSRFSGSGSGTQFSLKINSLQSEDFGSYYC;

LFR4, SEQ ID NO. 16 has the sequence FGGGTQLEIK.

In a preferred embodiment of the use of the invention, the antibody or antigen binding fragment thereof further comprises a constant region comprising a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD, and/or a light chain constant region selected from the group consisting of kappa-type or lambda-type light chain constant regions.

The antigen binding fragments of the above antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the antibodies described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds.

The antigen binding fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

The antigen binding fragment is selected from any one of F (ab ') 2, fab ', fab, fv, fab ' -SH and scFv of the antibody.

In a preferred embodiment of the invention, the constant region is derived from bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human. The cattle are, for example, cows, cattle, etc.

In a preferred embodiment of the use of the invention, the constant region of the antibody or antigen binding fragment thereof is of murine species origin.

(2) Quality control of HPV16 and/or HPV18 type vaccines;

(3) Preparing HPV16 and/or HPV18 type E2 protein detection products;

In order to improve the stability of the reagent and prolong the shelf life, the functional components such as a stabilizer, a protective agent and the like can be added into the reagent according to the need by a person skilled in the art, and the protein stabilizer is selected from sucrose, trehalose, BSA, glycerol, mannitol, triton X-100 and Tween-20. The protectant is selected from cryoprotectants such as polyols and sugars. The polyhydric alcohol is selected from sorbitol, mannitol or their mixture. The form of the reagent includes, but is not limited to, solid, liquid, semi-solid.

The antibody chip is formed by fixing the antibodies or antigen binding fragments thereof for resisting the HPV16 type E6 protein and the E7 protein on a carrier.

In a preferred embodiment of the use of the invention, the kit comprises a solid phase, the antibody or antigen binding fragment thereof being coated on the solid phase, and the antibody or antigen binding fragment thereof being attached to the solid phase, e.g. by chemical coupling.

In a preferred embodiment of the use of the invention, the solid phase is selected from the group consisting of microspheres, plates and membranes;

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

Based on the characteristic that the antibody or the antigen binding fragment thereof for resisting the HPV16 and/or HPV18 type E2 protein has the characteristic of specifically binding the HPV16 and/or HPV18 type E2 protein, the antibody is expected to be used for developing detection products aiming at the HPV16 and/or HPV18 type E2 protein, can also be used for quality control in the production process of recombinant human HPV16 and/or HPV18 type E2 antigen, can be used for efficiently and accurately quantifying the antigen content in the vaccine, and can monitor the vaccine quality.

In a preferred embodiment of the invention, the antibodies or antigen binding fragments thereof against HPV16 and/or HPV18 type E2 proteins are used as coating antibodies or detection antibodies, for example coated on an ELISA plate or as a second ELISA antibody, for example embedded on the binding pad of a test strip or on the nitrocellulose membrane of a test strip.

When an antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type 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 a coating antibody;

or, when the antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein is a coated 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 a detection antibody.

SEQ ID NO:17:

EVQLQQSGAELVKPGASVKLSCTASGFNIGDTYLHWVKQRPDQGLEWIGWIDPANGHTEYDPTFQGKATMTADTSSNTAYLLLSSLTSEDTALYHCSRGGKYYGLDFWGQGASVTVSS;

SEQ ID NO:18:

DIQMTQSPASLSASVGETVTITCRTSGNIHNYLAWYQQKQGKSPQLLVYYAKTLADGVPSRFSGSGSGTQYSLKINSLHPEDFGSYYCQHFWSTPYTFGGGTKLKIK.

The antibody was also obtained by the first screening of the inventors and was designated 1F4. Particularly, when the 1F4 monoclonal antibody is used as a coating antibody and the 3B3 monoclonal antibody is used as a detection antibody, the prepared kit has the advantages of high detection sensitivity, good specificity, high intermediate precision, high accuracy, consistent linear range of standard curve, quick detection and strong tolerance.

In other embodiments, another antibody against HPV16 and/or HPV18 type E2 antigen may be selected for use in pairing with the antibodies provided herein, as desired.

In one embodiment, antibodies or antigen binding fragments thereof against HPV16 and/or HPV18 type E2 proteins may be coated on a separation column to effect separation and enrichment of HPV16 and/or HPV18 type E2 proteins by the specificity and high affinity of the antibodies to HPV16 and/or HPV18 type E2 proteins. For example, antibodies are coated on a filler and filled in a separation column, and are used for affinity separation and enrichment of HPV16 and/or HPV18 type E2 proteins. Therefore, the antibody or the antigen binding fragment thereof for resisting the HPV16 and/or HPV18 type E2 protein has good application prospect in preparing HPV16 and/or HPV18 type E2 protein enrichment products.

The above-mentioned markers refer to a substance having characteristics such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, and by which qualitative or quantitative detection of a corresponding target can be achieved. In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polymethylchlorophyll (preCP), and the like).

In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to ²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and ¹⁸ F.

In alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, nanoparticles including, but not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

The chip may also be referred to as a suspension chip (suspension array) or a liquid array (liquid array). Which includes a carrier and nucleic acid molecules (e.g., primers and/or probes) and/or antibodies bound to the surface of the carrier.

The aforementioned carrier may be of various materials and forms, and may preferably be a container having a flat bottom, for example. A more typical preferred example is, but is not limited to, a multi-well plate (multe-WELL PLATES) widely used in biochemical assays, a micro-well plate (microplates), a microfluidic-based device (e.g., a microfluidic chip), a container like a surface dish, and the like.

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

Further, the kit may further include at least one of a buffer solution, a detection reagent, a diluent, and a wash solution, and is not limited thereto.

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

In a sixth aspect, the invention also provides a vector comprising the nucleic acid molecule described above.

The term "vector" is used herein in its most general sense and includes any intermediate vector for a nucleic acid that enables the nucleic acid to be introduced, for example, into a prokaryotic and/or eukaryotic cell, and where appropriate, integrated into the genome. Vectors of this type are preferably replicated and/or expressed in cells. 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, typically circular DNA duplex, that can replicate independently of chromosomal DNA. Any plasmid or vector may be used as long as it is replicable and stable in the host.

In an alternative embodiment, the vector is an expression vector, and an important feature of the expression vector is that it generally contains an origin of replication, a promoter, a marker gene and translational control elements.

In a seventh aspect, the present invention also provides a recombinant cell comprising the vector described above.

The term "recombinant cell" refers to any cell that can be transformed or transfected with an exogenous nucleic acid. The term "recombinant cell" according to the invention comprises prokaryotic (e.g. E.coli) or eukaryotic cells (e.g. mammalian cells, in particular human cells, yeast cells and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats or primates, are particularly preferred. Cells may be derived from a plurality of tissue types and comprise primary cells and cell lines. The nucleic acid may be present in the host cell in a single copy or in two or more copies, and in one embodiment is expressed in a recombinant cell.

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

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

In an alternative embodiment, the recombinant cell is HEK293.

In an eighth aspect, the invention also provides a method of preparing an antibody or antigen binding fragment thereof against HPV16 and/or HPV18 type E2 protein, culturing a cell comprising a nucleic acid molecule as described above. The purification steps are easily selected by those skilled in the art in order to obtain the desired antibody with high purity.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a preparation method of monoclonal antibody, which specifically comprises the following steps:

(1) Selecting female BALB/c mice, performing subcutaneous immunization by using HPV16/18 type E2 antigen (the amino acid sequence of which is shown as SEQ ID NO: 7) emulsified Complete Freund's Adjuvant (CFA), performing immunization by using the same antigen emulsified Incomplete Freund's Adjuvant (IFA) after two weeks, and continuing immunization for multiple times after two weeks interval until the serum titer is qualified;

the amino acid sequence of HPV16/18 E2 antigen is SEQ ID NO. 7.

MSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSIGGSGGSGGSGGSGGSTSDDTVSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRKLCSGNTTPIIHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVAIPDSVQILVGYMTM.

(2) Cell fusion, namely taking mouse spleen cells with qualified immune titers and mouse myeloma cells SP2/0 cells, fusing the spleen cells and the myeloma cells with 50% PEG according to a ratio of 5:1, suspending the fused cells in a 1% HAT selection medium for culture, and observing the growth condition of cloned cells for 7-10 days;

(3) Screening hybridoma cell strains, namely screening cell strains which react with HPV16/18 type E2 antigen by an indirect ELISA method (adopting HPV16/18 type E2 antigen coating), and repeatedly cloning to obtain stable hybridoma cells;

(4) Monoclonal antibody preparation, namely injecting hybridoma cells capable of being stably secreted into the abdominal cavity of a BALB/c mouse, producing ascites in vivo, and purifying the ascites to obtain the monoclonal antibody (1F 4) and the monoclonal antibody (3B 3).

(5) Hybridoma cell sequencing/variable region Gene sequencing

Collecting cells (clone numbers 1F4 and 3B 3) when the hybridoma cells grow to a logarithmic growth phase, extracting total RNA of the hybridoma cells, obtaining first-strand cDNA complementary to the full-length mRNA by RT-PCR (reverse transcription-polymerase chain reaction) through a 3'RACE technology and a 5' RACE technology, obtaining antibody heavy chain and light chain variable region genes by PCR amplification by taking the synthesized cDNA as a template, connecting the antibody variable region genes to a T vector, transforming and selecting positive clones for sequencing, and obtaining an antibody variable region gene sequence through a bioinformatic analysis sequencing result.

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

Example 2

The embodiment provides a sandwich ELISA detection method.

The embodiment provides a sandwich ELISA kit and a detection method, wherein the kit comprises a polystyrene micro-pore plate, TMB color development liquid, PBST eluent, a coated antibody (1F 4) and an antibody (3B 3) for detection.

The sandwich ELISA detection method comprises the following steps of diluting a specific antibody to a certain concentration, fixing the specific antibody on the surface of a polystyrene micro-pore plate through a physical adsorption method, adding a sample to be detected, specifically combining the sample with the antibody coated on a solid-phase carrier, adding an enzyme-labeled secondary antibody, a chromogenic solution, adding a stop solution to terminate the reaction, carrying out quantitative analysis by measuring a specific wavelength absorbance value, wherein the absorbance value of a sample is positively related to the antigen concentration of the sample, and calculating the concentration of the antigen in the sample according to a standard curve. The flow chart of the sandwich ELISA experiment is shown in FIG. 1.

Determination of linear range and working concentration of antibody

(1) Diluting the murine monoclonal antibody (1F 4) to 1 mug/ml according to the conventional coating conditions for coating (coating for 16-24 hours at 2-8 ℃ C., coating liquid formula: naHCO ₃3.068g,Na₂CO₃ 1.435.435 g adding purified water to 1L);

(2) Washing the unbound coating antibody by using PBST (1 x PBST) for the next time, and then adding a sealing liquid for sealing (sealing for 1 hour at 25 ℃) to obtain an ELISA plate coated with the antibody;

(3) When the kit is used, serial diluted standard substances, test substances and diluted horseradish peroxidase-labeled 3B3 monoclonal antibodies are sequentially added, TMB color development liquid of a horseradish peroxidase color development system is added after 25 ℃ reaction, and finally, a specific absorbance value is read after termination by 1M phosphoric acid. The labeling method of the root peroxidase-labeled 3B3 monoclonal antibody is as follows:

(a) Weighing 4.2mgHRP, and dissolving in 420 μl of ultrapure water;

(b) 9.7mgNaIO ₄ was weighed and dissolved in 755. Mu.l of ultrapure water;

(c) Adding 420 μl of the solution (b) into the solution (a), and reacting at 4deg.C for 30min in the dark;

(d) After the reaction is finished, adding 3.78 mu l of ethylene glycol, and reacting for 30min at room temperature in a dark place;

(e) Adding the solution obtained by the reaction in the step (d) and the antibody to be marked into a dialysis bag according to the mass ratio of 1:1, and dialyzing with 1 XCBS buffer solution;

(f) After the dialysis is finished, transferring the liquid in the dialysis bag into a beaker, then adding 45 mu l of NaBH ₄ (5 mg/ml), and carrying out light-shielding reaction for 3 hours at 4 ℃;

(g) After the reaction is finished, adding saturated ammonium sulfate with the total reaction volume of the previous 6 steps, and carrying out light-shielding reaction for 30min at 4 ℃;

(h) After the reaction, taking out the liquid, centrifuging at 11000rpm and 4 ℃ for 15min;

(i) The precipitate was reconstituted with PBS, then added with an equal volume of glycerol, mixed well and stored.

(4) Through experiments, the result shows that the optimal working dilution ratio of the 3B3 enzyme-labeled antibody is 1:400, and the absorbance value and the detection concentration (ng/ml) are highly linearly related (R ² > 0.98) within the range of 240ng/ml to 1.88 ng/ml.

Example 3

The invention performs specificity verification on the sandwich ELISA detection method provided in the embodiment 2.

The experimental design is shown in the following table:

Description:

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

715 Protein is recombinant HPV 16E 6/E7 antigen, and the amino acid sequence of the 715 protein is shown as SEQ ID NO. 19:

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKVSEYRYYCYSLYGTTLEQQYNKPLCDLLIRGINCQKPLCPDEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLgsgsgsgsgsgsgMHGDTPTLHEYMLDLQPETTDLYGYGQLHDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP.

716 is recombinant HPV 18E 6/E7 antigen, and the amino acid sequence of 716 is shown as SEQ ID NO. 20:

MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRGLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVgsgsgsgsgsgsgMHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQ.

the 718 protein is recombinant HPV 16E 1 antigen, and the amino acid sequence of the 718 protein is shown as SEQ ID NO. 21:

MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLYLVSPLSDISGCVDNNEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMVVLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTL

721 protein is recombinant HPV 18E 1 antigen, and the amino acid sequence is shown in SEQ ID NO. 22:

MADPEGTDGEGTGCNGWFYVQAIVDKKTGDVISDDEDENATDTGSDMVDFIDTQGTFCEQAELETAQALFHAQEVHNDAQVLHVLKRKKRRLFTIADSGYGCSEVEATQIQVTTNGEHGGNVCSGGSTEAIDNGGTEGNNSSVDGTSDNSNIENVNPQCTIAQLKDLLKVNNKQGAMLAVFKDTYGLSFTDLVRNFKSDKTTCTDWVTAIFGVNPTIAEGFKTLIQPFILYAHIQCLDCKWGVLILALLRYKCGKSRLTVAKGLSTLLHVPETCMLIQPPKLRSSVAALYWYRTGISNISEVMGDTPEWIQRLTIIQHGIDDSNFDLSEMVQWAFDNELTDESDMAFEYALLADSNSNAAAFLKSNCQAKYLKDCATMCKHYRRAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRYQHIEFITFLGALKSFLKGTPKKNCLVFCGPANTGKSYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCWTYFDTYMRNALDGNPISIDRKHKPLIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFPFDKNGNPVYEINDKNWKCFFERTWSRLDLHEEEEDADTEGNPFGTFKCVAGQNHRPL.

the acceptance criteria are that the recovery rate of each sample is 80% -120%, and detection of the antigen of the sample is not interfered under the condition that other antigens exist.

Recovery (%) = 717 mixed protein actual measurement/717 protein actual measurement x 100%

The measured value of 717 protein is the value detected by the inclusion of 717 protein alone.

Specific verification results

Specificity verification conclusion:

Under the condition that 715, 716, 718 and 721 proteins exist respectively, the content of 717 protein can still be accurately detected (the recovery rate is within the acceptable range of 80% -120%), namely, the detection method has good specificity. And (5) qualified specificity verification.

Example 4

This example demonstrates the precision (reproducibility) of the sandwich ELISA assay provided in example 2.

The experimental design is that 1 batch 717 proteins are selected, diluted with a diluent to three concentrations of 240ng/ml, 120ng/ml and 60 ng/ml, respectively, and then 8 gradients are diluted 2-fold in the plate for detection.

For detection, each concentration was measured 3 times in duplicate. And calculating the CV value of the three repeated detection values.

The standard is accepted, the test sample must have at least 3 dilutions within the linear range of the standard curve, and the CV of the three repeated tests of the test sample in the same batch is less than or equal to 20 percent.

The results of the precision (repeatability) verification are shown in the following table:

precision (reproducibility) conclusions CV between the three replicates of each concentration was less than 20% (5.0%, 1.0%, 0.9%, respectively). And (5) checking repeatability to be qualified.

Example 5

This example demonstrates the precision (intermediate precision) of the sandwich ELISA assay provided in example 2.

Design of experiment three experimental persons on the other day were selected to repeat the "repeatability" experiment and the day and person differences were calculated.

The acceptance criteria are that the CV of the detection values of the samples in the same batch is less than or equal to 20% on different dates and by different personnel.

The results of the precision (intermediate precision) verification are shown in the following table:

The precision (intermediate precision) was concluded that the CV of 717 antigen detection results by different experimenters on different dates was less than 20% (4.2%, 1.7%, 2.7%, respectively). And (5) verifying intermediate precision to be qualified.

Example 6

This example demonstrates the accuracy of the sandwich ELISA detection method provided in example 2.

The experimental design is that 1 batch of 717 stock solution (namely 717 protein) is selected, diluted by the diluent according to the table 1-2, then the standard is added, the standard adding process is shown in the table 3, C5, C6, C7, M4, M5, M6, C1, C2 and C3 are taken for measurement, and the recovery rate is calculated.

Recovery (%) = (addition of standard sample actual measurement value-sample actual measurement value)/standard actual measurement value x 100%.

The recovery rate is 80% -120% according to the acceptance standard.

Table 1 Table 717 stock dilution procedure is as follows

TABLE 2 dilution of reference (i.e., standard) 2.717

TABLE 3 accuracy labeling procedure

The accuracy verification results are shown in the following table:

note that labeling actual measurement = labeling sample actual measurement-sample actual measurement.

The measured value of the sample is the measured value of the concentration of the stock solution, the measured value of the standard sample is the measured value of the reference substance, and the measured value of the standard sample is the measured value of the reference substance.

The accuracy verification conclusion is that the standard adding recovery rate of each concentration is 80% -120% (93%, 98% and 103% respectively), and the accuracy verification is qualified.

Example 7

This example demonstrates the standard curve (linear and range) of the sandwich ELISA assay provided in example 2.

And (3) experimental design, namely summarizing standard curve data in precision, accuracy and specificity verification to obtain linearity and optimal detection range of a standard curve.

The correlation coefficient of the standard curves (four-parameter fitting curves) is not less than 0.98, the CV of R ² of each standard curve is less than or equal to 10%, and the optimal detection range of the standard curves is consistent.

Standard curve (linear and range) validation results are shown in the following table:

The standard curve (linearity and range) verification conclusion shows that the standard curve has good linearity when the first few verifications are combined, R ² is larger than 0.99, CV% of R ² is smaller than 10%, the standard curve meets the verification standard, and the linear range of the standard curve is consistent and is 240-1.88 ng/ml. The standard curve is shown in fig. 2.

Example 8

The embodiment provides a kit comprising a polystyrene microplate on the surface of which a 3B3 monoclonal antibody (coated antibody) is immobilized, and further comprising an enzyme-labeled detection antibody (1F 4), TMB and PBST wash.

(1) According to the conventional coating conditions, 8E6 antibody is diluted in a certain proportion for coating (coating for 16-24 hours at 2-8 ℃ C., coating liquid formula: naHCO ₃3.068g,Na₂CO₃ 1.435.435 g, adding purified water to a constant volume of 1L).

(3) When the kit is used, serial diluted standard substances, test substances and diluted horseradish peroxidase-labeled (1F 4) monoclonal antibodies are sequentially added, TMB color development liquid of a horseradish peroxidase color development system is added after 25 ℃ reaction, and finally, a specific absorbance value is read after termination by 1M phosphoric acid.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein, characterized in that it comprises 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.

2. The antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein according to claim 1, characterized in that 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 AAT.

3. The antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein according to claim 2, characterized in that the antibody or antigen-binding fragment thereof 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, in sequence; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 13-16, in sequence.

4. The antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein according to claim 3, characterized in that 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;

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 against HPV16 and/or HPV18 type E2 protein according to any one of claims 1 to 4 in any of the following:

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

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

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

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

(5) Preparation of HPV18 vaccine quality control products or HPV18 vaccine immune effect evaluation products;

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 against HPV16 and/or HPV18 type E2 protein or its antigen-binding fragment according to any one of claims 1 to 4 is used as a coating antibody or a detection antibody;

When the antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein is used as the 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 the coating antibody;

Alternatively, when the antibody or antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein is the 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 the detection antibody.

7. An antibody conjugate, characterized in that it is formed by coupling the antibody against HPV16 and/or HPV18 type E2 protein or its antigen-binding fragment according to any one of claims 1 to 4 and a marker, and the marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes substrate color development, a radioactive isotope, a chemiluminescent reagent and a nanoparticle marker.

8. A HPV16 and/or HPV18 type E2 protein detection product, characterized in that it comprises the antibody or its antigen-binding fragment against HPV16 and/or HPV18 type E2 protein according to any one of claims 1-4, or comprises 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 against HPV16 and/or HPV18 type E2 protein according to any one of claims 1 to 4.

10. The method for preparing an antibody or an antigen-binding fragment thereof against HPV16 and/or HPV18 type E2 protein according to any one of claims 1 to 4, characterized in that cells comprising the nucleic acid molecule according to claim 9 are cultured.