CN117466995B

CN117466995B - Antibodies or antigen binding fragments thereof specifically binding to HPV45 type capsid protein L1 and application thereof

Info

Publication number: CN117466995B
Application number: CN202311174930.4A
Authority: CN
Inventors: 严嘉成; 阮宝阳; 张雯苑; 曹玉锋; 孙晓东; 朱俊郦; 任柳铭; 史力
Original assignee: Immune Path Biotechnology Suzhou Co Ltd
Current assignee: Immune Path Biotechnology Suzhou Co Ltd
Priority date: 2023-09-12
Filing date: 2023-09-12
Publication date: 2024-04-05
Anticipated expiration: 2043-09-12
Also published as: CN117466995A

Abstract

The invention provides an antibody specifically combined with HPV45 type capsid protein L1 or an antigen-binding fragment thereof and application thereof, and relates to the technical field of antibodies. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a VHCDR1 with an amino acid sequence shown as SEQ ID NO.1, a VHCDR2 with an amino acid sequence shown as SEQ ID NO.2 or 3, and a VHCDR3 with an amino acid sequence shown as SEQ ID NO.4 or 5; the light chain variable region comprises VLCDR1 having an amino acid sequence as shown in SEQ ID NO.6, VLCDR2 having an amino acid sequence as shown in SEQ ID NO.7, and VLCDR3 having an amino acid sequence as shown in SEQ ID NO. 8. The antibody has high specificity and neutralization activity on HPV45 type capsid protein L1, and has important value on HPV vaccine production and prevention and control of cervical cancer.

Description

Antibodies or antigen binding fragments thereof specifically binding to HPV45 type capsid protein L1 and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an antibody specifically combined with HPV45 type capsid protein L1 or an antigen-binding fragment thereof and application thereof.

Background

Human papillomaviruses (Human Papillomavirus, HPV) are small non-enveloped DNA viruses, about 200 types exist, more than 40 types of HPV mainly infect skin and mucosal tissues, and more than 40 types of HPV infection can cause human diseases, and HPV can be classified into high-risk types and low-risk types according to the relation between HPV infection and occurrence of cancers, wherein HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and the like are high-risk types, and persistent infection of HPV of high-risk types can cause malignant tumors such as cervical cancer, anal cancer, vaginal cancer and the like. Other HPV subtypes are low-risk types, mainly cause wart hyperplasia of skin mucous membrane, are represented by condyloma acuminatum, flat wart and other lesions, and have no good treatment method clinically at present.

The most effective method for preventing HPV infection is to inoculate HPV vaccine, at present, 5 cervical cancer vaccines which are marketed at home and abroad all take HPV main capsid protein L1 as effective antigen, and the HPV main capsid protein L1 is assembled into Virus-like particles (VLPs) under certain conditions by a gene recombination technology, and then different adjuvants are used for preparing the vaccine. Based on successful marketing and wide application of recombinant HPV L1VLPs vaccine, the L1VLPs fully show that the structure of the natural HPV is highly reduced, key neutralizing epitopes of the natural virus are reserved, the recombinant HPV L1VLPs have antigenicity and immunogenicity which are the same as or similar to those of the wild homotype virus, and high-titer neutralizing antibodies can be induced, so that HPV persistent infection, cervical cancer and other related diseases can be well prevented. The HPV vaccines which are currently marketed, the tetravalent vaccine 'Gardasil' and the nine-valent vaccine 'Gardasil 9' of Merck company, are mostly more than twice developed at home and abroad and comprise HPV45 type vaccine components.

The correct neutralizing epitope and stable VLPs structure are maintained in the whole process of HPV vaccine production, and are the preconditions for guaranteeing the quality and effectiveness of the vaccine. The establishment of accurate, sensitive and specific detection methods in each link of vaccine production process for whole-course monitoring of antigen structure and quantification is not only a technical requirement, but also a guiding principle of regulations. The classical antigen-antibody reaction principle is utilized, the neutralizing antibody is adopted to identify neutralizing epitopes of the antigen, which is an effective means for vaccine quality control, and the antigen components in HPV vaccine stock solution, semi-finished products and finished products are subjected to identification experiments, antigen content detection and in-vitro effectiveness determination by using Western-blot, ELISA, IVRP and other technical means, so that a basis can be provided for vaccine detection release. Therefore, monoclonal neutralizing antibodies are important reagents for quality control of vaccine antigens, and monoclonal antibodies with specificity and neutralization activity have irreplaceable roles in the quality control process of vaccines. In addition, vaccine efficacy detection is an important index for the release of vaccine finished products, at present, HPV vaccine efficacy is evaluated by collecting serum after vaccine immunization of animals and detecting the neutralization titer of the serum by adopting a pseudo-virus neutralization experiment, and although the method can better reflect the neutralization antibody level of the vaccine finished products, the method also has the defects of more animal consumption, larger fluctuation of detection values, long detection period and higher cost, and simultaneously breaks the 3R principle of animal experiments, and some enterprises at home and abroad respond to the advocacy of International organization and gradually eliminate the method for detecting the vaccine finished product efficacy by animal experiments. It has been recognized and used in part practice that quality control and characterization based on neutralizing antibodies against neutralizing epitopes and in vitro activity of vaccine antigens has been an efficient, economical, and viable alternative, such as the merck HPV vaccine, chinese hepatitis a and b vaccine, all have been partially or fully utilized with neutralizing antibodies rather than animal-test-release vaccine products. Thus, it is currently commercially desirable to provide more neutralizing monoclonal antibodies capable of binding HPV.

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

Disclosure of Invention

The present invention aims to provide an antibody or antigen-binding fragment thereof specifically binding to HPV45 type capsid protein L1, monoclonal antibodies obtained by screening with HPV45 as antigen, CDR region sequences thereof determined by cloning, identification and analysis of genetic structure, and a biological material related to the antibodies or antigen-binding fragments thereof and their use.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention there is provided an antibody or antigen binding fragment thereof which specifically binds HPV type 45 capsid protein L1, comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises a VHCDR1 with an amino acid sequence shown as SEQ ID NO.1, a VHCDR2 with an amino acid sequence shown as SEQ ID NO.2 or 3, and a VHCDR3 with an amino acid sequence shown as SEQ ID NO.4 or 5;

the light chain variable region comprises VLCDR1 with an amino acid sequence shown as SEQ ID NO.6, VLCDR2 with an amino acid sequence shown as SEQ ID NO.7 and VLCDR3 with an amino acid sequence shown as SEQ ID NO. 8.

According to another aspect of the present invention, there is also provided a biomaterial selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof which specifically binds HPV type 45 capsid protein L1 as described above;

(ii) a vector carrying the polynucleotide of (i) above;

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing an antibody or antigen-binding fragment thereof which specifically binds HPV type 45 capsid protein L1 as described above.

According to another aspect of the present invention, there is also provided a hybridoma cell secreting a monoclonal antibody specifically binding to HPV45 type capsid protein L1, which is murine hybridoma cell line 7A5 with a collection number of CGMCC NO:45706, or with a collection number of CGMCC NO:45707 a murine hybridoma cell line 11G1.

According to another aspect of the present invention there is also provided the use of an antibody or antigen binding fragment thereof as described above which specifically binds to HPV type 45 capsid protein L1, or a biological material as described above, or a hybridoma as described above, for the detection of HPV type 45 capsid protein L1 for non-diagnostic and therapeutic purposes, or in the preparation of a product for the detection of HPV type 45 capsid protein L1.

According to another aspect of the present invention there is also provided the use of an antibody or antigen binding fragment thereof as described above which specifically binds HPV type 45 capsid protein L1, or a biological material as described above, or a hybridoma as described above, in vaccine production or quality control.

According to another aspect of the present invention there is also provided a reagent or kit for detecting HPV type 45 capsid protein L1, the reagent or kit comprising an antibody or antigen-binding fragment thereof as described above that specifically binds HPV type 45 capsid protein L1.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens and obtains the murine monoclonal antibody and hybridoma cell aiming at the type 45 HPV by the hybridoma technology, and the antibody has high specificity and neutralization activity for HPV 45L 1VLPs and pseudoviruses, which indicates that the antibody is the antibody aiming at HPV 45L 1 protein neutralization epitope. The specificity of the monoclonal antibody is detected by a direct ELISA method, and the antibody has no cross reaction with antigens such as HPV 6, HPV11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 52, HPV 58 and the like, and has good specificity. The neutralizing activity of the antibody is detected by an ELISPOT pseudovirus neutralization experiment method, and the neutralizing activity of the antibody on HPV45 type pseudovirus is high. Meanwhile, the heavy chain variable region and the light chain variable region genes of the antibody are sequenced, the CDR regions of the heavy chain variable region and the light chain variable region genes are analyzed, the antibody is comprehensively characterized, and the antibody or antigen binding fragment thereof which has the CDR regions and specifically binds with HPV45 type capsid protein L1 is obtained.

Based on the antibody or antigen binding fragment thereof, the invention also establishes a method for detecting the content of the HPV45 type capsid protein L1 antigen, and results show that the method has good specificity, sensitivity, linearity and accuracy, can specifically and rapidly identify and quantify the HPV45 type capsid protein L1 antigen, evaluate the content and activity of effective antigen components of the vaccine, is widely applied to the production quality control, clinical etiology detection and epidemic investigation of HPV vaccine, and has important value for the production of the HPV vaccine and the prevention and control of cervical cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a standard curve of detection antigen after HPV45 type rabbit polyclonal antibody and murine monoclonal antibody 7A5 are diluted 500 and 20000 fold, respectively;

FIG. 2 shows standard curves of detection of antigens after 500 and 20000 times dilution of HPV45 type rabbit polyclonal antibody and murine monoclonal antibody 11G1, respectively.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

An "antibody or antigen-binding fragment thereof" as used herein refers to a protein that binds a particular antigen, which broadly refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" also includes naturally occurring antibodies as well as non-naturally occurring antibodies. An "antigen binding fragment" in this context is a substance comprising the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. The antigen binding fragment has the capability of specifically recognizing and binding to HPV45 type capsid protein L1.

"variable region" or "variable domain" of an antibody or antigen binding fragment thereof refers to the domain of an antibody that recognizes and binds to an antigen at the amino terminus of the heavy or light chain of the antibody, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) connected by 4 Frameworks (FR). The framework and CDR ranges have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any CDR determination method well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, typically the variable regions VL/VH of the heavy and light chains are obtained by ligating the CDRs numbered FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combination arrangement with the FRs.

The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide comprises a portion encoding the antibody or antigen binding fragment thereof described above, optionally encoding the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. The offspring may not necessarily be identical to the primary cells due to natural, accidental or deliberate mutation, e.g. are morphologically and/or differ from the primary cells in genomic DNA.

The HPV45 type capsid protein L1 in the "detection HPV45 type capsid protein L1" herein may be a naturally occurring, recombinantly expressed by genetic engineering, chemically and/or biochemically modified, non-naturally occurring or derived protein or polypeptide, or any combination thereof. Also included are detection of HPV type 45 viruses, VLPs containing HPV type 45 capsid protein L1, and pseudoviruses. The expression "HPV 45-type capsid protein L1", "HPV 45L 1" or their similar expressions may be used interchangeably herein.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising" or the like will be understood to include the stated element or component without excluding other elements or components.

According to one aspect of the present invention there is provided an antibody or antigen binding fragment thereof which specifically binds to HPV type 45 capsid protein L1. The invention screens and obtains the murine monoclonal antibody aiming at the HPV45 through the hybridoma technology, and the antibody has high specificity and neutralization activity on HPV 45L 1VLPs and pseudoviruses, which indicates that the antibody is the antibody aiming at the HPV45 capsid protein L1 neutralization epitope. According to the murine monoclonal antibody, the CDR region with good binding capacity to HPV45 type capsid protein L1 is obtained.

The epitopes of the antibodies and the antigens are in one-to-one correspondence, one antigen epitope corresponds to one corresponding antibody, the branching epitopes and the space epitopes of the antigen epitope, most of the neutralizing epitopes are space epitopes, and the neutralizing epitopes of the antigen are structural bases for generating neutralizing antibodies and are also used as the precondition that vaccine candidate antigens excite effective immune responses. Because of the heterogeneity and diversity of antibody production, antibodies having different CDR regions are produced even against the same antigen, and the probability of screening to obtain a high-quality antibody having neutralizing activity is low, the antibody has uniqueness.

The invention provides antibodies or antigen-binding fragments thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VHCDR1, VHCDR2, and VHCDR3 as follows:

VHCDR1：SYWMH(SEQ ID NO.1)；

VHCDR2: EINPSNGLTNYNEKFKS (SEQ ID NO. 2); or EINPSNGHTNYNEKFKS (SEQ ID NO. 3);

VHCDR3: FHGSDLYAMDY (SEQ ID NO. 4); or CYGSSYDYYAMDY (SEQ ID NO. 5).

In an alternative embodiment, the heavy chain variable region comprises a VHCDR1 having an amino acid sequence of SEQ ID NO.1, a VHCDR2 having an amino acid sequence of SEQ ID NO.2, and a VHCDR3 having an amino acid sequence of SEQ ID NO. 4; or, the heavy chain variable region comprises VHCDR1 with an amino acid sequence shown as SEQ ID NO.1, VHCDR2 with an amino acid sequence shown as SEQ ID NO.3, and VHCDR3 with an amino acid sequence shown as SEQ ID NO. 5.

The light chain variable region comprises the following VLCDR1, VLCDR2, and VLCDR3:

VLCDR1：RASQSVSTSSYSYMH(SEQ ID NO.6)；

VLCDR2：YASNLES(SEQ ID NO.7)；

VLCDR3：QHSWEIPYT(SEQ ID NO.8)。

the heavy chain variable region comprises the following structure: VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4. In alternative embodiments, any one of the framework regions of the heavy chain variable region is selected from the framework regions of the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.9 or 13, i.e., one or more of VHFR1, VHFR2, VHFR3 and VHFR4 is selected from the corresponding framework regions of the heavy chain variable region shown in SEQ ID NO.9 or 13. In alternative embodiments, any one or more of VHFRs 1 to 4 is selected from the corresponding framework regions of the heavy chain variable region shown in SEQ ID NO. 9; in alternative embodiments, any one or more of VHFRs 1 to 4 is selected from the corresponding framework regions of the heavy chain variable region shown in SEQ ID NO. 13; in alternative embodiments, any one or more of VHFRs 1 to 4 is selected from the corresponding framework regions of the heavy chain variable region shown in SEQ ID NO.9, and the remaining framework regions are selected from the corresponding framework regions of the heavy chain variable region shown in SEQ ID NO. 13.

The light chain variable region comprises the following structure: VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4. In alternative embodiments, any one of the framework regions of the light chain variable region is selected from the framework regions of the light chain variable region having the amino acid sequence shown in SEQ ID NO.10 or 14, i.e., one or more of VLFR1, VLFR2, VLFR3 and VLFR4 is selected from the corresponding framework regions of the light chain variable region shown in SEQ ID NO.10 or 14. In alternative embodiments, any one or more of VLFR 1-4 is selected from the corresponding framework regions of the light chain variable region shown in SEQ ID No. 10; in alternative embodiments, any one or more of VLFR 1-4 is selected from the group consisting of the corresponding framework regions of the light chain variable region shown in SEQ ID No. 14; in alternative embodiments, any one or more of VLFR 1-4 is selected from the corresponding framework regions of the light chain variable region shown in SEQ ID No.10, and the remaining framework regions are selected from the corresponding framework regions of the light chain variable region shown in SEQ ID No. 14.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10; or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14.

In alternative embodiments, the antibody or antigen binding fragment thereof further comprises the sequence of a portion or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD.

In alternative embodiments, the antigen binding fragment comprises F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units.

In alternative embodiments, the antibody or antigen binding fragment thereof removes CDR regions, and the remaining sequence-derived species include one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

In alternative embodiments, the antibody is selected from the group consisting of an intact antibody molecule, further comprising a heavy chain constant region and a light chain constant region, and the antibody is an IgG1 antibody or an IgG2a antibody, and the light chain is a kappa chain.

In an alternative embodiment, the antibody is a monoclonal antibody secreted by hybridoma cell line 7A5 with a collection number of CGMCC NO:45706 or secreted by a collection number of CGMCC NO:45707 hybridoma cell line 11G1.

a polynucleotide comprising a nucleotide sequence encoding the aforementioned antibody or antigen binding fragment thereof; in an alternative embodiment, the polynucleotide encodes a heavy chain variable region, and the nucleotide sequence is shown in SEQ ID NO.11 or 15; in alternative embodiments, the polynucleotide encodes a light chain variable region and the nucleotide sequence is set forth in SEQ ID NO.12 or 16.

(ii) a vector carrying the polynucleotide of (i) above.

(iii) a cell carrying said polynucleotide (i), or comprising said vector (ii), or expressing an antibody or antigen-binding fragment thereof as hereinbefore described which specifically binds to HPV type 45 capsid protein L1.

According to another aspect of the present invention, there is also provided a hybridoma cell secreting a monoclonal antibody specifically binding to HPV type 45 capsid protein L1, which is murine hybridoma cell line 7A5 with a collection number of CGMCC NO:45706, or with a collection number of CGMCCNO:45707 a murine hybridoma cell line 11G1.

The preservation number of the hybridoma cell strain 7A5 is CGMCC NO:45706, the preservation unit is China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation address is China academy of sciences of China, national institute of microbiology, no.1, and No.3 of North Chen West Lu, the area of Korea of Beijing city. The secreted monoclonal antibody has VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown as SEQ ID NO.1, 2 and 4 and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO. 6-8.

The preservation number of the hybridoma cell strain 11G1 is CGMCC NO:45707 the preservation unit is China general microbiological culture Collection center, and the preservation address is China national academy of sciences of China, including national institute of microbiology, national institute of sciences, no.1, no.3, north Chen West Lu, korea, beijing, city. The secreted monoclonal antibody has VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown as SEQ ID NO.1, 3 and 5 and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO. 6-8.

According to another aspect of the present invention there is also provided the use of an antibody or antigen binding fragment thereof which specifically binds to HPV type 45 capsid protein L1, or a biological material related to an antibody or antigen binding fragment thereof which specifically binds to HPV type 45 capsid protein L1, or a hybridoma cell as described above, for the detection of HPV type 45 capsid protein L1 for non-diagnostic and therapeutic purposes, or in the preparation of a product for the detection of HPV type 45 capsid protein L1.

In alternative embodiments, where antigens are to be detected for non-diagnostic and therapeutic purposes, detection may be selected by conventional means known in the art, for example by immunodetection techniques, using the antibodies or antigen-binding fragments thereof described above as solid phase carriers for capturing the antigen in combination with solid phase carriers, or as antibodies that bind to the captured antigen. The antibody or antigen binding fragment thereof is also optionally conjugated to a label to provide a detectable signal, including but not limited to one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin. Specific detection means include, but are not limited to, immunoblotting, immunohistochemistry, immunochromatography, ELISA, etc., and the present invention is not limited thereto.

According to another aspect of the present invention there is also provided the use of an antibody or antigen-binding fragment thereof which specifically binds to HPV type 45 capsid protein L1, or a biological material related to an antibody or antigen-binding fragment thereof which specifically binds to HPV type 45 capsid protein L1, or a hybridoma cell as described above, in vaccine production or quality control.

According to another aspect of the present invention there is also provided a reagent or kit for detecting HPV type 45 capsid protein L1, the reagent or kit comprising the antibody or antigen-binding fragment thereof described above.

In alternative embodiments, the reagent or kit comprises a label linked to the antibody or antigen binding fragment thereof described above or separately packaged. The label includes, but is not limited to, one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a color microsphere, colloidal gold, biotin, or streptavidin.

In alternative embodiments, the reagent or kit comprises a reagent or kit for immunodetection. The reagent or kit includes, but is not limited to, an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoblotting detection reagent or kit. Those skilled in the art can select the corresponding reagent composition according to the specific detection means of the reagent or the kit, and the present invention is not limited thereto.

In alternative embodiments, the kit comprises a double antibody sandwich indirect ELISA detection kit.

In an alternative embodiment, the double antibody sandwich indirect ELISA detection kit comprises a solid phase antibody, a detection antibody combined with an antigen captured by the solid phase antibody and a labeled secondary antibody, wherein the labeled secondary antibody is specifically combined with the detection antibody; the labeled secondary antibody provides a detection signal; wherein the solid phase antibody is selected from the group consisting of HPV45 polyclonal antibodies; the solid phase antibody is selected from the antibody or antigen binding fragment thereof which specifically binds to HPV45 type capsid protein L1; in alternative embodiments, the solid phase antibody is selected from the monoclonal antibody secreted by hybridoma cell line 7A5, or from the monoclonal antibody secreted by hybridoma cell line 11G1.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

EXAMPLE 1 preparation of monoclonal antibodies

1. Immunization of mice

HPV45 type stock solution was mixed with adjuvants CFA and AD11.15, balb/c mice were immunized, mouse tail blood was taken on day 14, and the evaluation of antibody titer in tail blood was performed using an indirect ELISA method. Coating an ELISA plate (1:100 dilution) with HPV45 type stock solution, adding 100 mu L of the ELISA plate into each hole, and reacting at 4 ℃ overnight; the plates were washed 3 times with PBS and blocked with 5% milk-PBS for 1hr at room temperature; washing the plate with PBS solution for 1 time, adding gradient diluted mouse tail blood, and reacting at room temperature for 1hr; washing the plate for 3 times by using a PBS solution, beating, adding an HRP-marked goat anti-mouse Fc secondary antibody diluted 1:2000, reacting for 1hr at room temperature, washing the plate for 5 times by using the PBS solution, beating, adding substrate A solution and substrate B solution with equal volumes, and reacting for 20min under the conditions of light shielding and room temperature; then 50 mu L of stop solution is added, and after even mixing, OD is read on an enzyme labeling instrument ₄₅₀ And OD (optical density) ₆₃₀ Value, output od=od ₄₅₀ -OD ₆₃₀ . Mice with tail blood titers exceeding 1/50000 were taken for subsequent experiments.

2. Cell fusion and monoclonal screening

The splenocytes from the mice were sacrificed and fused with SP2/0 cells, and were cultured in selection medium as monoclonal cell lines and then cloned, and 9 clones were picked up in 96 well plates. The HPV45 stock solution is used for coating an ELISA plate (1:100 dilution) for monoclonal screening, and the method is the same. A total of 1128 clones from 12 96-well plates were initially screened to retain 104 positive clones with OD.gtoreq.1.0. And (3) performing amplification culture on the primary screening positive clone from a 96-well plate to a 48-well plate, performing rescreening after 2-3 days of culture, and retaining 37 strains of strong positive clone with the rescreening OD being more than or equal to 2.0.

3. Clone-specific screening

The supernatants of 34 positive clone cells were specifically tested, wherein 13 of 10H6, 7A5, 11G1, 12B7, 7A2, 11B11, 9E7, 7B5, 7E7, 7E4, 7C12, 7E6, and 10H1 were positive clones specifically recognizing HPV45, and the test results are shown in Table 1. The method comprises the following steps: VLP antigens of HPV of subtypes HPV 6, HPV11, HPV 16, HPV 18, HPV 31, HPV 33, HPV45, HPV 52, HPV 58 total 9 were diluted 1: 500. Mu.L of each well was added and reacted overnight at 4 ℃; the plates were washed 3 times with PBS and blocked with 5% milk-PBS for 1hr at room temperature; then washing the plate with PBS solution for 1 time, adding hybridoma cell supernatants of different clones diluted by 20 times, and reacting for 1hr at room temperature; washing the plate for 3 times by using PBS solution, beating, adding HRP-marked goat anti-mouse Fc secondary antibody diluted by 1:10000, reacting for 1hr at room temperature, washing the plate for 5 times by using PBS solution, beating, adding substrate A solution and substrate B solution with equal volumes, and reacting for 20min under the conditions of light shielding and room temperature; then 50 mu L of stop solution is added, and after even mixing, OD is read on an enzyme labeling instrument ₄₅₀ And OD (optical density) ₆₃₀ Value, output od=od ₄₅₀ -OD ₆₃₀ 。

TABLE 1 specific ELISA detection of antibodies (OD value. Times.)

* All values are average values of complex pores; virus Like Particle: a virus-like particle; naN represents OD >4.0;

4. neutralization activity assay

The cell supernatants of 13 positive clones specifically recognizing HPV45 were assayed for neutralization activity, and the results are shown in table 2. The neutralization experiment of pseudoviruses proves that cell supernatants of 5 clones of 10H6, 7E7, 7A5, 11G1 and 7C12 have higher neutralization activity. The method comprises the following steps: 1) 293FT cells were diluted with DMEM complete medium (containing 10% fetal bovine serum, 1% diabody, 1% L-Glu and 1% G418) and added to 96-well cell culture plates for overnight incubation at 37 ℃; 2) The HPV45-GFP pseudovirus was diluted to the desired concentration (about 400 fluorescent spots per well) with a DMEM complete medium doubling ratio; 3) Diluting the supernatant of HPV45 type hybridoma cells to be detected by using DEME complete medium in a multiple ratio; 4) Diluting commercial HPV45 type monoclonal antibodies by DEME complete medium multiple ratio; 5) Mixing 60 mu L of diluted cell supernatant and pseudovirus uniformly, and then placing the mixture into a temperature of 4 ℃ for reaction for 1 hour; 6) Slowly adding 100 μl of the mixed solution into a 96-well cell culture plate paved with 293FT cells, and culturing at 37deg.C for 60-96 hr; 6) Cell culture media was discarded and read with ELSPOT (AID). The detection result shows that the received HPV pseudoviruses meet the expected requirements, the number of fluorescent spots in each hole meets the requirements (about 400), the number of the fluorescent spots is reduced along with the increase of the dilution multiple of the pseudoviruses, and the HPV45 type cell supernatants 11B11, 7E7, 7A5, 11G1 and 7C12 have higher neutralization activity and still have neutralization activity after dilution by 320 times.

TABLE 2 neutralization activity assay

Clone number	Pseudovirus neutralization Activity	Neutralization potency
			7E7	Is that	320
11B11	Is that	1280
			7A5	Is that	5120
11G1	Is that	5120
			7C12	Is that	1280

Note that: neutralization activity positive determination: the number of the fluorescent spots is less than or equal to the average value of the number of the positive fluorescent spots per 2.

5. Ascites preparation and antibody purification

Clones 7A5 and 11G1 are selected for ascites preparation according to the affinity, specificity and neutralization activity results of the supernatant, 4 Balb/c mice are inoculated with hybridoma cells respectively, the ascites is collected on days 10-14, and the ascites is purified by protein G to obtain purified antibodies.

Example 2 antibody sensitivity detection

Clones 7A5 and 11G1 were selected for ascites production and antibody purification according to the affinity, specificity and neutralization activity results of the supernatant, and the detection method was the same as in the clone-specific screening step of example 1, and the detection antigen was VLP antigen of HPV45 subtype, and the sensitivity of each of antibodies 7A5 and 11G1 was 0.0005. Mu.g/mL, as shown in Table 3.

TABLE 3 antibody sensitivity detection

Note that: NC is negative control 5% milk-PBS, positive judgment standard is OD value > 2.1 times NC value, naN is exceeding maximum detection limit.

EXAMPLE 3 identification of antibody type

Subtype identification of antibodies 7A5 and 11G1 was performed using the antibody subtype identification kit, and the detection results are shown in Table 4. Antibody 7A5 is of the IgG1 subtype, the light chain is of the kappa type, 11G1 is of the IgG2a subtype and the light chain is of the kappa type.

TABLE 4 identification of antibody subtypes

Note that: naN is the maximum detection limit exceeded.

Example 4 antibody Gene sequencing and analysis

Total RNA of 11G1 and 7A5 hybridoma cell lines was extracted, reverse transcribed by PrimeScript 1st Strand cDNA Synthesis Kit (Takara, cat#6110A), antibody VH and VL genes were amplified by RT-PCR, cloned into pUC-19T vector, and sequenced by M13 universal primer on the vector, with the following results:

1.11G1：

1.1VH Region-11G 1 (Variable Region)

1) DNA sequence 360bp

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCGGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGACTTACTAACTACAATGAGAAGTTCAAGAGCAAGGGCACACTGACTGTAGACAAATCCTCCAGCGCAGCCTTCATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTCCACGGTAGTGACCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.11)。

2) Amino acid sequence 120aa

QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKRRPGQGLEWIGEINPSNGLTNYN EKFKSKGTLTVDKSSSAAFMHLSSLTSEDSAVYYCARFHGSDLYAMDYWGQGTSVTVSS(SEQ ID NO.9)。

VHCDR1：SYWMH(SEQ ID NO.1)；

VHCDR2：EINPSNGLTNYNEKFKS(SEQ ID NO.2)；

VHCDR3：FHGSDLYAMDY(SEQ ID NO.4)。

1.2VL domain-11G 1 (Variable Region)

1) DNA sequence 333bp

GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTAGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGGAGATTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO.12)。

2) Amino acid sequence 111aa

DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLESGVPA RFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK(SEQ ID NO.10)。

VLCDR1：RASQSVSTSSYSYMH(SEQ ID NO.6)；

VLCDR2：YASNLES(SEQ ID NO.7)；

VLCDR3：QHSWEIPYT(SEQ ID NO.8)。

2.7A5：

2.1VH Region-7A 5 (Leader sequence-Variable Region)

1) DNA sequence 351bp

CAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCATACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCCCTTGCTACGGTAGTAGCTACGATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.15)。

2) 117aa amino acid sequence

QPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGHTNYNEKFKS KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAPCYGSSYDYYAMDYWGQGTSVTVSS(SEQ ID NO.13)。

VHCDR1：SYWMH(SEQ ID NO.1)；

VHCDR2：EINPSNGHTNYNEKFKS(SEQ ID NO.3)；

VHCDR3：CYGSSYDYYAMDY(SEQ ID NO.5)。

2.2VL Region-7A 5 (Leader sequence-Variable Region)

1) DNA sequence 333bp

GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTAGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGGAGATTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO.16)。

2) Amino acid sequence 111aa

DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK(SEQ ID NO.14)。

VLCDR1：RASQSVSTSSYSYMH(SEQ ID NO.6)；

VLCDR2：YASNLES(SEQ ID NO.7)；

VLCDR3：QHSWEIPYT(SEQ ID NO.8)。

Example 5 establishment of double antibody sandwich indirect ELISA method

The method comprises the following specific steps: 1) Diluting the HPV45 (R163) rabbit polyclonal antibody 500-fold with coating solution (carbonate buffer), 100 μl/well, coating (carbonate buffer) at 4 ℃, overnight; 2) Adding 5% skimmed milk (PBS), 200 μl/well, and sealing at 37deg.C for 2 hr; 3) 2% skim milk dilutes HPV45 stock solution to 13.3. Mu.g/ml, then 2.5 fold ratio to 11 gradients, 100. Mu.L/well, 37℃for 1h; 4) Diluting the corresponding HPV45 (11G 1 or 7A 5) murine mab with 20000 times 2% skim milk at 100. Mu.L/well, 37℃for 1h; 5) 2% skim milk 10000-fold diluted goat anti-murine lgG-HRP secondary antibody (BIO-RAD, 100. Mu.L/well, 37 ℃ C., 1h; 6) TMB color development liquid is added, 100 mu L/ml is added, and color development is carried out for 10min at 37 ℃; 7) The reaction was terminated by adding 50. Mu.L/Kong Nong sulfuric acid, and the read values at 450nm and reference wavelength of 620nm were measured by an ELISA reader, and the results are shown in Table 5 below. The results show that the 4-parameter curve shows that the HPV45 type rabbit polyclonal antibody and mouse monoclonal antibody have good linear relation under the condition of the following antibody reaction concentration, namely dilution factors of 500 and 20000 respectively, the initial antigen dilution concentration is 13.3ug/ml, and specific detection results are shown in figures 1 and 2.

Table 5: OD value of detection antigen after dilution of HPV45 type polyclonal antibody and murine monoclonal antibody by 500 and 20000 times respectively

1	2	3	4	5	6	7	8	9	10	11	12
												0.004	0.006	0.005	0.014	0.005	0.005	0.004	0.005	0.005	0.004	0.004	0.004
3.097	3.027	2.669	1.825	0.922	0.364	0.218	0.115	0.084	0.062	0.050	0.045
												3.010	3.090	2.649	1.983	1.021	0.447	0.206	0.108	0.007	0.051	0.046	0.050
3.124	3.047	2.899	2.336	1.545	0.614	0.379	0.175	0.106	0.068	0.066	0.067
												3.124	3.002	2.779	2.367	1.563	0.773	0.358	0.189	0.106	0.072	0.056	0.064

Note that: row 1 is a blank control; rows 2 and 3 are detection results of 20000 times dilution of murine monoclonal antibody secreted by hybridoma cell 11G 1; rows 4 and 5 are detection results of 20000-fold dilution of murine monoclonal antibody secreted by hybridoma cell 7 A5.

In summary, the invention screens by hybridoma technology to obtain murine monoclonal antibodies against HPV type 45, which have high specificity and neutralization activity to HPV 45L 1VLPs and pseudoviruses, indicating that the antibodies are antibodies against HPV45 capsid protein L1 neutralization epitopes. The identification and characterization of neutralizing epitopes of vaccine antigens by using suitable neutralizing antibodies is an important matter of quality control and release in the vaccine industry, which requires good specificity and neutralizing activity of the antibodies. In order to fully prove the specificity of the obtained antibody, the ELISA method is firstly adopted to carry out the cross reaction verification of HPV 6, HPV 31, HPV11, HPV 16, HPV 18, HPV 33, HPV45, HPV 52 and HPV 58 antigens, which shows that the antibody only reacts with HPV45 type antigens in a specific way, and the excellent specificity is shown. In order to confirm the neutralizing activity of the antibody, a pseudo virus neutralization experiment is adopted to verify that the neutralizing activity of the antibody secreted by the hybridoma cell 11G1 can reach 5120 and the neutralizing activity of the antibody secreted by the cell 7A5 can reach 5120.

In the above embodiment, a method for detecting the content of the HPV 45L 1 antigen by double-antibody sandwich indirect ELISA is also established, the HPV 45L 1 rabbit polyclonal antibody is coated, the antigen to be detected is added for incubation, the monoclonal antibody is added for incubation, and the anti-mouse IgG enzyme-labeled complex is added for display detection. The method has good specificity, sensitivity, linearity and accuracy, can rapidly identify and quantify the HPV 45L 1 antigen, and evaluates the content and activity of the effective antigen component of the vaccine. Because the detection antibody is the neutralizing monoclonal antibody, the method can perform good quality control and characterization on the neutralizing epitope of the vaccine antigen, and indicates the immunogenicity of a vaccine finished product to a certain extent, so the method has the potential advantages of evaluating the neutralizing activity of the vaccine by replacing an animal experiment, avoids the problems of large detection fluctuation, long detection period and high cost caused by the animal experiment, and also accords with the animal experiment 3R principle advocated by the international society. The method adopts HPV 45L 1 rabbit polyclonal antibody to carry out plate wrapping, can capture antigen to be detected to the greatest extent, improves sensitivity, adds the antigen to be detected, then adds HPV45 type monoclonal antibody (11G 1 or 7A 5), and finally adds anti-mouse IgG enzyme-labeled antibody to carry out color development and reading. The traditional double-antibody sandwich method needs to label one monoclonal antibody, especially when the double-antibody sandwich method is applied to multivalent HPV vaccine, a plurality of types of HPV monoclonal antibodies need to be labeled respectively, so that the workload and the cost are increased, and when the double-antibody sandwich method is used for quantitatively detecting HPV antigens of different types of multivalent HPV vaccine, the anti-mouse IgG enzyme-labeled antibody is adopted as a universal chromogenic antibody, so that the convenience of detection is greatly improved, and the cost is reduced.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds HPV type 45 capsid protein L1, comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises a VHCDR1 with an amino acid sequence shown as SEQ ID NO.1, a VHCDR2 with an amino acid sequence shown as SEQ ID NO.2 and a VHCDR3 with an amino acid sequence shown as SEQ ID NO. 4; the light chain variable region comprises VLCDR1 with an amino acid sequence shown as SEQ ID NO.6, VLCDR2 with an amino acid sequence shown as SEQ ID NO.7 and VLCDR3 with an amino acid sequence shown as SEQ ID NO. 8;

or, the heavy chain variable region comprises VHCDR1 with an amino acid sequence shown as SEQ ID NO.1, VHCDR2 with an amino acid sequence shown as SEQ ID NO.3 and VHCDR3 with an amino acid sequence shown as SEQ ID NO. 5; the light chain variable region comprises VLCDR1 with an amino acid sequence shown as SEQ ID NO.6, VLCDR2 with an amino acid sequence shown as SEQ ID NO.7 and VLCDR3 with an amino acid sequence shown as SEQ ID NO. 8.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.9 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 10;

or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising the sequence of a part or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding fragment is F (ab') ₂ Fab', fab, fv, scFv, dsFv, bispecific antibody or antibody minimal recognition unit.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof removes CDR regions and the remaining sequence-derived species comprises one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and a light chain constant region; the antibody is an IgG1 antibody or an IgG2a antibody, and the light chain is a kappa chain.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is secreted by hybridoma cell line 7A5 having a collection number of CGMCC No. 45706 or by a cell line having a collection number of CGMCC NO:45707 hybridoma cell line 11G1.

8. A biomaterial, characterized in that the biomaterial is selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the antibody or antigen binding fragment thereof of any one of claims 1 to 7;

(ii) a vector carrying the polynucleotide of (i) above;

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

9. The biomaterial according to claim 8, wherein the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No.11 or 15.

10. The biomaterial according to claim 8, wherein the nucleotide sequence of the light chain variable region is as set forth in SEQ ID No.12 or 16.

11. The hybridoma cell secreting the monoclonal antibody specifically combined with HPV45 type capsid protein L1 is a murine hybridoma cell line 7A5 with the preservation number of CGMCC NO:45706 or a hybridoma cell line with the preservation number of CGMCC NO:45707 a murine hybridoma cell line 11G1.

12. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 7 that specifically binds HPV type 45 capsid protein L1, or a biological material according to any one of claims 8 to 10, or a hybridoma cell according to claim 11 for the detection of HPV type 45 capsid protein L1 for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of HPV type 45 capsid protein L1.

13. The use of an antibody or antigen binding fragment thereof that specifically binds HPV type 45 capsid protein L1 of any one of claims 1-7, or the biomaterial of any one of claims 8-10, or the hybridoma cell of claim 11 in vaccine production or quality control.

14. A reagent or kit for detecting HPV type 45 capsid protein L1, comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, which specifically binds HPV type 45 capsid protein L1.

15. The reagent or kit according to claim 14, wherein the kit is a double antibody sandwich indirect ELISA detection kit.

16. The reagent or kit according to claim 15, wherein the double-antibody sandwich indirect ELISA detection kit comprises a solid phase antibody, a detection antibody bound to an antigen captured by the solid phase antibody and a labeled secondary antibody that specifically binds to the detection antibody; the labeled secondary antibody provides a detection signal;

the solid phase antibody is selected from HPV45 polyclonal antibody; the solid-phase antibody is selected from the group consisting of the antibody or antigen-binding fragment thereof specifically binding to HPV type 45 capsid protein L1 according to any one of claims 1 to 7.

17. The reagent or kit according to claim 16, wherein the solid phase antibody is selected from the monoclonal antibodies secreted by the hybridoma cell line 7A5 or hybridoma cell line 11G1.