CN114957452A

CN114957452A - Antibody of human papilloma virus 11 type L1 protein and preparation method thereof

Info

Publication number: CN114957452A
Application number: CN202110575487.6A
Authority: CN
Inventors: 虎丽兴; 闫梅; 张琼月; 李坤; 徐岚; 吴双; 陈丹; 班靖洋; 夏丽; 陈健平; 刘勇
Original assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Current assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Priority date: 2021-02-18
Filing date: 2021-05-26
Publication date: 2022-08-30

Abstract

The invention provides a monoclonal antibody capable of specifically recognizing L1 protein (HPV11L1) and/or VLP (HPV11VLP) of human papilloma virus type 11. The specific monoclonal antibody provided by the invention has no obvious cross reaction with other 8 types of HPV (HPV6, 16, 18, 31, 33, 45, 52 and 58) L1 proteins and/or VLP, and has the advantages of high specificity, high sensitivity, high accuracy and high safety. Can be used for accurately detecting the content of the bioactive vaccine in the L1 protein and/or VLP of the HPV11 in seedlings, and can be widely applied in the clinical detection and vaccine production processes.

Description

Antibody of human papilloma virus 11 type L1 protein and preparation method thereof

Technical Field

The invention relates to the field of molecular virology and immunology, in particular to a human papilloma virus 11 type hybridoma cell line, a preparation method of a monoclonal antibody of human papilloma virus 11 type L1 protein and/or VLP, and a diagnostic kit prepared by the monoclonal antibody.

Background

Human Papilloma Virus (HPV) is a group of small DNA viruses without envelopes, which infect Human skin and mucosal epithelial tissues and can induce the formation of warty hyperplasia and even cause benign or malignant tumors. HPV subtypes are numerous, and more than 130 types have been isolated so far, and different types cause different clinical manifestations. Based on their relationship with tumorigenesis, they can be classified into high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, etc.) and low-risk types (HPV6, 11, 40, 42, 43, 44, 54, etc.).

The low-risk type is not integrated in cell genome, mainly causes wart hyperplasia of skin mucosa and is expressed as benign lesions such as condyloma acuminatum and verruca plana. Condyloma acuminatum is one of eight sexually transmitted diseases which are required to be monitored by the national ministry of health, the incidence rate of the condyloma acuminatum is second to that of gonorrhea, and the condyloma acuminatum is one of common venereal diseases seriously endangering the normal life of people due to the fact that the disease condition is easy to recur and extremely high in infectivity. Lesions caused by HPV such as HPV6, 11 in the anogenital mucosa account for around 90% of the total according to epidemiological investigations. Therefore, the development of HPV11 type vaccine can play a good role in preventing diseases such as condyloma acuminatum and the like.

The major capsid protein L1 of HPV has the characteristic of self-assembling as hollow Virus-Like particles (VLPs). HPVVLP is a 20-sided stereosymmetric structure composed of pentamers of the 72 major capsid proteins L1. The VLP vaccine can express one or more structural proteins of the virus through a molecular biology technology, the structural proteins have natural self-assembly capacity, can form a spatial configuration and an antigenic epitope similar to natural virus particles, but have no virus nucleic acid, strong immunogenicity and no infectivity, have no risk of incomplete inactivation or recovery of virulence, have high-density virus antigens on the surface, preserve conformational epitopes, can be presented to immune cells through the same way as a whole virus vaccine, effectively induce the immune system of the body to generate immune protection reaction, and induce a neutralizing antibody with high titer.

During HPV vaccine development, typing, antigen content detection and in vitro potency assays are required. The method can adopt a double-antibody sandwich ELISA. Therefore, in vaccine research, monoclonal antibodies are important tools for controlling the quality of vaccine antigens, especially antibodies with specificity and neutralizing activity, and have irreplaceable important roles in vaccine development. The invention provides a monoclonal antibody which can aim at the specificity and the neutralization activity of HPV11, an ELISA detection kit prepared by utilizing two strains of the monoclonal antibody can be specifically used for quickly identifying HPV11L1 protein, can be widely applied to clinical detection and quality inspection in the process of producing vaccines by current vaccine manufacturers, and has important significance for the research and development of HPV11 type vaccines and the prevention of disease lesions such as condyloma acuminatum and the like.

Disclosure of Invention

In a first aspect, the present invention provides monoclonal antibodies capable of recognizing the L1 protein of HPV11 and/or VLPs and hybridoma cell lines producing the antibodies.

In a second aspect, the invention provides a method for producing a monoclonal antibody.

The third aspect of the invention provides a preparation method of an antigen detection kit for L1 protein and/or VLP of HPV 11.

In a fourth aspect, the invention provides a double antibody sandwich ELISA kit for detecting HPV 11.

The invention is realized by the following technical scheme:

the monoclonal antibody provided by the invention is directed against HPV11 type L1 protein, HPV11 type L1 protein is coding HPV11 type L1 protein, and the HPV11 type L1 protein nucleic acid base sequence can be inserted into a prokaryotic expression vector by a cloning method of conventional molecular biology. The expressed HPV11 type L1 protein can be polymerized to form soluble multimers. The amino acid sequence of the L1 Protein of HPV11 type as well as the nucleic acid sequence encoding the L1 Protein can be obtained from databases such as GenBank, Swiss-prot, EMBL. PIR Protein Data Base, PDBProtein Data Bank, or from HPV sequence libraries (http:// HPV-web. land. gov). Including but not limited to, the production of HPV11L1 protein by hansenula polymorpha expression system as described in Chinese patent application with application publication No. CN103361280A, and the preparation of virus-like particles (VLP) as immunogen. Specifically, HPV11VLP is used as immunogen to immunize mouse, hybridoma cell strain capable of secreting HPV11VLP continuously and stably is obtained through cell fusion and screening by means of hybridoma technology, and monoclonal antibody is obtained through secretion of each cell strain.

In a preferred embodiment, the antibody 23F11 recognizing the L1 protein and/or VLP of human papillomavirus type 11 is a monoclonal antibody produced by a hybridoma cell line obtained by fusing immunized host spleen cells and myeloma cells, wherein the hybridoma cells have a preservation number of CGMCC No. 21984; and (3) classification and naming: a hybridoma cell; the preservation unit: china general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No.3, Beijing, Chaoyang, North; the preservation date is as follows: 2021, 4 months and 1 day.

The monoclonal antibody produced by the hybridoma cell line with the preservation number of CGMCC No.21984 comprises amino acid sequences of CDRs of heavy chain variable regions of CDRH1, CDRH2 or/and CDRH3 regions shown in SEQ ID NO.1-3 and amino acid sequences of CDRs of light chain variable regions of CDRL1, CDRL2 or/and CDRL3 regions shown in SEQ ID NO. 4-6.

The monoclonal antibody produced by the hybridoma cell strain comprises an amino acid sequence of an FR of a heavy chain variable region of FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID NO.7-10 and an amino acid sequence of an FR of a light chain variable region of FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID NO. 11-14.

The monoclonal antibody produced by the hybridoma cell strain comprises a heavy chain variable region amino acid sequence shown as SEQ ID NO.15 and a light chain variable region amino acid sequence shown as SEQ ID NO. 16.

The monoclonal antibody produced by the hybridoma cell strain comprises nucleotide sequences of CDRs of heavy chain variable regions of CDRH1, CDRH2 or/and CDRH3 regions shown in SEQ ID NO.17-19 and nucleotide sequences of CDRs of light chain variable regions of CDRL1, CDRL2 or/and CDRL3 regions shown in SEQ ID NO. 20-22.

The monoclonal antibody produced by the hybridoma cell strain comprises a nucleotide sequence of an FR of a heavy chain variable region of FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID NO.23-26 and a nucleotide sequence of an FR of a light chain variable region of FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID NO. 27-30.

The monoclonal antibody produced by the hybridoma cell strain comprises a heavy chain variable region nucleotide sequence shown as SEQ ID NO.31 and a light chain variable region nucleotide sequence shown as SEQ ID NO. 32.

In a second aspect, in another preferred embodiment, the second antibody 24C7 recognizing the L1 protein and/or VLP of human papillomavirus type 11 is a monoclonal antibody produced by a hybridoma cell line obtained by fusing another strain of immunized host spleen cells and myeloma cells, wherein the hybridoma cell line has a accession number of CGMCC No. 21985; and (3) classification and naming: a hybridoma cell; the preservation unit: china general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No.3, Beijing, Chaoyang, Beicheng; the preservation date is as follows: 2021, 4 months and 1 day.

The second antibody comprises the amino acid sequence of a CDR of the heavy chain variable region having the region of CDRH1, CDRH2 or/and CDRH3 as shown in SEQ ID NO.33-35 and the amino acid sequence of a CDR of the light chain variable region of the region of CDRL1, CDRL2 or/and CDRL3 as shown in SEQ ID NO. 36-38.

The second antibody comprises an amino acid sequence of FR of a heavy chain variable region having FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID Nos. 39-42, and an amino acid sequence of FR of a light chain variable region having FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID Nos. 43-46.

The second antibody comprises a heavy chain variable region amino acid sequence shown as SEQ ID No.47 and a light chain variable region amino acid sequence shown as SEQ ID No. 48.

The second antibody further comprises the nucleotide sequence of a CDR of the heavy chain variable region having the region of CDRH1, CDRH2 or/and CDRH3 as shown in SEQ ID NO.49-51 and the nucleotide sequence of a CDR of the light chain variable region of the region of CDRL1, CDRL2 or/and CDRL3 as shown in SEQ ID NO. 52-54.

The second antibody comprises a nucleotide sequence of FR of the heavy chain variable region having FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID Nos. 55-58, and a nucleotide sequence of FR of the light chain variable region having FR1, FR2, FR3 or/and FR4 regions shown in SEQ ID Nos. 59-62.

The second antibody comprises a heavy chain variable region nucleotide sequence shown as SEQ ID No.63 and a light chain variable region nucleotide sequence shown as SEQ ID No. 64.

In another aspect, the present invention provides a kit for detecting the L1 protein and/or VLP of HPV11, comprising two monoclonal antibodies of the two preferred embodiments disclosed herein.

The kit for detecting the L1 protein and/or VLP of the HPV11 disclosed by the invention is a double-antibody sandwich ELISA kit for detecting the L1 protein and/or VLP antigen of the HPV11, and further comprises detectable markers: fluorescent substances, colored substances and/or enzymes.

The monoclonal antibody of human papillomavirus type 11L1 protein can be prepared by the following method:

the method comprises the following steps: the prepared human papillomavirus 11 type VLP protein is used as immunogen, female Balb/c mice of 6-8 weeks are immunized after purification, immunization is carried out for three times, serum is collected at each immunization interval of 14 days, the titer of the serum is detected by an indirect ELISA method, the tail veins of the Balb/c female mice with high titer are selected according to the ELISA result, and immune spleen cells are prepared from the bodies of the mice;

step two: preparing hybridoma cells. Firstly, preparing a myeloma cell (SP2/0) suspension, injecting a female Balb/c mouse, preparing myeloma cells after the mouse grows solid tumors, fusing the myeloma cells with the immune spleen cells in the step one to prepare hybridoma cells, detecting and screening hybridoma cell strains with high titer, and carrying out clone expansion culture;

step three: and (3) collecting supernatant from the hybridoma cells subjected to clone and amplification culture in the step two, injecting the supernatant into the abdominal cavity of a mouse, collecting ascites of the mouse, and obtaining the monoclonal antibody of the anti-human papilloma virus 11VLP by specific identification and neutralization activity detection after purification.

The preparation method of the human papillomavirus double-antibody sandwich ELISA antigen detection kit comprises the following steps:

the monoclonal antibody 23F11 with specificity and no cross is used as a capture antibody to coat an enzyme label plate, the monoclonal antibody 24C7 with neutralization activity is used as a detection antibody after being labeled by horseradish peroxidase, and the VLP protein of human papilloma virus 11 type is used as a standard substance to prepare a standard curve contrast. The kit also comprises a concentrated washing solution, a sample diluent, an enzyme-labeled antibody diluent, a substrate solution A, a substrate solution B, a stop solution and the like.

The monoclonal antibody obtained by the invention has good specificity, and experiments show that the monoclonal antibody has no cross reaction with other eight types of HPV, so the monoclonal antibody obtained by the invention can be used for specific detection of HPV11VLP in currently newly marketed nine-valent vaccines and vaccines or compositions comprising HPV11 types.

The invention adopts a double-antibody sandwich method, utilizes two monoclonal antibodies to carry out specific detection and quantification on HPV11VLP, and provides a kit for specific detection and quantification of HPV11VLP, wherein the detection limit is as follows: 0.0039. mu.g/mL, linear range: 0.25-0.0039 mug/mL, which has the advantages of high specificity and high sensitivity, can accurately detect the level of HPV11VLP with biological activity in a sample, and can be widely applied to clinical detection and quality inspection in the vaccine production process of a vaccine manufacturer.

Drawings

FIG. 1: SDS-PAGE analysis detection results show that the purity of each purified monoclonal antibody reaches over 90 percent.

FIG. 2 is a drawing: the standard curve of the pairing identification experiment of each type of monoclonal antibody has the ordinate of OD450 and the abscissa of the standard concentration.

FIG. 3: antibodies 23F11 and 24C7 specifically identified the experimental standard curve with OD450 on the ordinate and standard concentration on the abscissa.

FIG. 4 is a drawing: the ELISA double antibody sandwich assay detects HPV11VLP standard curve with OD450 on the ordinate and standard concentration on the abscissa.

SEQ ID NO.1-3 are the amino acid sequences CDRH1, CDRH2 or/and CDRH3 regions of CDRs of the heavy chain variable region of the antibody of the invention;

SEQ ID NO.4-6 are the amino acid sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region of the antibody of the invention;

SEQ ID Nos. 7 to 10 are regions of the amino acid sequences FR1, FR2, FR3 or/and FR4 of the FRs of the heavy chain variable region of the antibody of the present invention;

SEQ ID Nos. 11 to 14 are regions of the amino acid sequences FR1, FR2, FR3 or/and FR4 of the FRs of the light chain variable region of the antibody of the present invention;

SEQ ID NO.15 is the amino acid sequence of the heavy chain variable region of the antibody of the present invention;

SEQ ID NO.16 is the amino acid sequence of the light chain variable region of the antibody of the present invention;

SEQ ID NO.17-19 are the nucleotide sequences of CDRH1, CDRH2 or/and CDRH3 regions of CDRs of the heavy chain variable region of the antibody of the invention;

SEQ ID NO.20-22 are the nucleotide sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region of the antibody of the invention;

SEQ ID Nos. 23 to 26 are the nucleotide sequences FR1, FR2, FR3 or/and FR4 regions of the FRs of the heavy chain variable region of the antibody of the present invention;

SEQ ID Nos. 27 to 30 are the nucleotide sequences FR1, FR2, FR3 or/and FR4 regions of the FRs of the light chain variable region of the antibody of the present invention;

SEQ ID NO.31 is the heavy chain variable region nucleotide sequence of the antibody of the present invention;

SEQ ID NO.32 is the light chain variable region nucleotide sequence of the antibody of the present invention;

SEQ ID Nos. 33 to 35 are the amino acid sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable region of the second antibody of the present invention;

SEQ ID NO.36-38 are the amino acid sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region of the second antibody of the invention;

SEQ ID Nos. 39 to 42 are the amino acid sequences FR1, FR2, FR3 or/and FR4 region of the FR of the heavy chain variable region of the second antibody of the present invention;

SEQ ID Nos. 43 to 46 are regions of the amino acid sequences FR1, FR2, FR3 or/and FR4 of the FR of the light chain variable region of the second antibody of the present invention;

SEQ ID NO.47 is the heavy chain variable region amino acid sequence of the second antibody of the present invention;

SEQ ID No.48 is the light chain variable region amino acid sequence of the second antibody of the present invention;

SEQ ID Nos. 49 to 51 are the nucleotide sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable region of the second antibody of the present invention;

SEQ ID Nos. 52 to 54 are the nucleotide sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region of the second antibody of the invention;

SEQ ID Nos. 55 to 58 are the nucleotide sequences FR1, FR2, FR3 or/and FR4 region of the FR of the heavy chain variable region of the second antibody of the present invention;

SEQ ID Nos. 59 to 62 are nucleotide sequences FR1, FR2, FR3 or/and FR4 regions of the FRs of the light chain variable region of the second antibody of the present invention;

SEQ ID NO.63 is the heavy chain variable region nucleotide sequence of the second antibody of the present invention;

SEQ ID NO.64 is the light chain variable region nucleotide sequence of the second antibody of the present invention;

detailed description of the invention

The principles, features and aspects of the present invention are further described below in conjunction with the following examples, which are set forth to illustrate, but are not to be construed as limiting the present invention. Unless otherwise defined, the technical means used in the examples are conventional means well known to those skilled in the art, and reagents of which sources are not indicated are conventional reagents in the art or commercially available reagents, and scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above while remaining within the scope of the invention. For a better understanding of the present invention, the following provides definitions and explanations of relevant terms.

The term "antibody" as used herein, refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one light (L) chain and one heavy (H) chain. Within the light and heavy chains, the variable and constant regions are separated by about 12 or more amino acids "The J "regions are joined and the heavy chain also comprises a" D "region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V) _H ) And heavy chain constant region (C) _H ) And (4) forming. The heavy chain constant region consists of 3 domains (C) _H 1、C _H 2 and C _H 3) And (4) forming. Each light chain is composed of a light chain variable region (V) _L ) And light chain constant region (C) _L ) And (4) forming. The light chain constant region consists of a domain C _L And (4) forming. V _H And V _L Regions can also be subdivided into regions of high degeneracy, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V _H And V _L Consisting of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of each heavy/light chain pair form antibody binding sites, respectively. The term "antibody" is not limited by any particular method of producing an antibody.

The term "monoclonal antibody" as used herein refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules. Monoclonal antibodies have high specificity for a single epitope on an antigen.

"monoclonal antibody" and "monoclonal antibody" as used herein have the same meaning and are used interchangeably.

The term "HPV VLP" used in the present invention refers to a virus-like particle assembled from HPV L1 protein expressed in vitro, such as HPV11L 1-VLP, which refers to a virus-like particle assembled from HPV11 type L1 protein expressed in vitro.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1 establishment of hybridoma cell lines

1. Animal immunization

1) Antigen preparation: self-made antigen. HPV11L1 protein was produced using the Hansenula polymorpha expression system as described in the Chinese patent application publication No. CN103361280A, and the purified HPV11L1 protein sample was diluted 3-fold with sterile water and dropped in a small drop onto a wax dish. And (3) taking the copper net to enable the surface with the supporting film to be in contact with the surface of the sample liquid, standing for 1 minute, taking out the copper net, absorbing the redundant liquid drops by using a filter paper strip, and slightly drying. A2% uranium acetate solution was dropped on a wax dish. The copper mesh with the adsorbed sample is placed on the surface of the dye solution (the sample is in contact with the dye solution), and is kept stand for 2 minutes. The copper mesh was removed, excess drops were removed with a filter paper strip, and dried under an incandescent lamp. The virus-like particles (VLPs) that have been self-assembled and formed were observed using a JEOL-1400 model transmission electron microscope, and used as an antigen.

Basic immunity: mixing antigen and Freund's complete adjuvant in equal volume, emulsifying, injecting into abdominal cavity, and immunizing in Balb/c female mouse at dose of 30 μ g/mouse for 6-8 weeks.

2) And (3) boosting immunity: the booster immunization uses an emulsion of antigen with incomplete Freund's adjuvant. The first booster immunization was carried out 14 days after the basic immunization, the second booster immunization was carried out 14 days after the basic immunization, and the third booster immunization was carried out 13 days after the basic immunization, wherein each time, 30 mu g of mixed emulsion of antigen and Freund's incomplete adjuvant was intraperitoneally injected.

3) Tail vein: 0.1 mL/mouse, 3 days later, cell fusion.

2. Preparation of hybridoma cells

Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a ratio of 5:1 with PEG 4000. Culturing with HAT selective medium, fusing for 5-7, taking supernatant, and screening hybridoma cell strains by adopting an indirect ELISA method. The obtained positive clones were subcloned by limiting dilution method. The indirect ELISA method was performed as follows:

1) coating: coating by adding 0.5 mu g/mL of HPV11VLP protein and 50 mu L/hole into an enzyme label plate, and placing in a refrigerator at 4 ℃ for overnight;

2) and (3) sealing: taking out the coated plate, washing for 2 times by using a plate washing machine, beating to dry, adding confining liquid, and incubating for 2h in a constant temperature box at 37 ℃ in a volume of 150 mu L/well;

3) sample adding: and (3) washing the closed enzyme label plate for 1 time by a plate washing machine, drying by beating, adding a sample to be detected, and setting a positive control hole and a negative control hole at the same time, wherein the sample to be detected is 100 mu L/well. Incubating in a constant temperature oven at 37 ℃ for 30 minutes;

4) secondary antibody: and washing the incubated enzyme label plate for 3 times by using a plate washing machine, and patting the plate dry. Goat anti-mouse IgG (H + L) secondary antibody was added and diluted 1:4000 for use. Incubating in a thermostat at 37 ℃ for 30 minutes;

5) color development: washing the ELISA plate for 5 times after the incubation of the secondary antibody in the step, beating to dry, mixing the developing solution A, B according to the proportion of 1:1, adding the developing solution into the ELISA plate by 100 mu L/well, and reacting for 10 minutes;

6) and (4) terminating: adding 50. mu.L/well of stop solution, reading OD value at 450nm of an enzyme-labeling instrument, and taking the cell with OD larger than 1.0 as a positive clone.

3. Establishment of hybridoma cell lines

And (3) continuously cloning the positive clones obtained in the step (2), continuously culturing and subculturing the hybridoma cell line in a culture medium containing 10% fetal calf serum and 10 XHT until the hybridoma cell line still can grow well and can be stably subcultured, thereby obtaining the hybridoma cell line stably secreting the monoclonal antibody.

EXAMPLE 2 preparation and purification of monoclonal antibody against HPV11

1. Paraffin sensitization: balb/c multiparous mice were selected, and the cells were sensitized with paraffin 7 days before inoculation (paraffin oil can reduce the rejection of mice to the inoculated hybridoma cells, which is beneficial to the growth of hybridoma cells). The needle head is taken down from the 2mL syringe, 2.5mL of paraffin oil is sucked, then the needle head is fixed, the inclined plane of the needle head faces to the scale direction of the syringe, and 0.5mL of paraffin oil is slowly injected into the abdominal cavity of each mouse.

2. Cell inoculation: and taking cells in a good state, removing the supernatant, adding a certain amount of DMEM medium to blow off adherent cells, centrifuging at 1000rpm for 5 minutes, removing the supernatant, resuspending with 2mL of the medium, taking 10 mu L of the cells, adding an equal volume of trypan blue reagent, and counting. According to the counting result and the total amount of the required cells, taking a certain volume of cell suspension into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding the supernatant, and adding physiological saline to make the cell density be 0.5 multiplied by 10 ⁶ Mice were injected intraperitoneally at 0.5 mL/mL. The growth of ascites in mice was observed at any time, and ascites was generated 7 days after the inoculation of general cells.

3. Ascites collection: the time for collecting ascites is determined according to the abdominal distension and the state of the mouse, and is generally 7 days after inoculation. The mouse was held vertically, and the abdomen of the mouse was pricked with a 12-gauge needle, the length of the needle was about 1/4, and the position of the needle was controlled with forceps, so that ascites flowed through the needle into the centrifuge tube. Centrifuging at 8000rpm for 10 min, collecting supernatant, and storing in-20 deg.C refrigerator. Ascites is collected every 1 day until the mice die or are in a poor state of sacrifice and have no ascites basically, and the ascites is generally collected for about 3 times.

4. Ascites purification: purifying by octanoic acid-ammonium sulfate method, centrifuging ascites (12000r/min 30min), collecting supernatant, and removing precipitate. Ascites volume was accurately quantified. A50 mM citric acid (pH 4.0) solution 2 times the volume of the original ascites was added to the sample, and the pH after mixing was 4.6 to 4.8. Octanoic acid (60. mu.L/mL ascites, calculated here as initial ascites volume) was added dropwise with stirring to mix the solution evenly and precipitate completely. After standing at room temperature for 0.5 hour, the mixture was centrifuged (50mL centrifuge tube: 10000rpm for 15 min; 2mL centrifuge tube: 10000rpm for 5min), the supernatant was carefully collected, the precipitate was discarded, the volume of the supernatant was recorded, 2M Tris (pH 9.0) was added in an amount of 0.03 times the volume of the supernatant, and the pH was adjusted to neutral. Ammonium sulfate powder was slowly added at 0.277g/mL with stirring to dissolve the ammonium sulfate sufficiently, and the mixture was allowed to stand at room temperature for 0.5 hour to complete the precipitation. The mixture was centrifuged (50mL centrifuge tube: 10000rpm for 15 min; 2mL centrifuge tube: 10000rpm for 5min), the supernatant was discarded, and the precipitate was collected. And (3) fully dissolving the precipitate by using PBS (phosphate buffer solution) with the volume of 0.25 time of the original ascites volume, dialyzing by using PBS, and changing the solution for 2-3 times. Collecting the dialyzate, centrifuging at 12000r/min for 15min, and collecting the supernatant to obtain the monoclonal antibody against HPV11 VLP. Samples were taken for SDS-PAGE and BCA to determine antibody concentration. The results show that the purity of the purified monoclonal antibody reaches more than 90%, and particularly, the figure 1 shows that.

Example 3 identification of specificity of each type of monoclonal antibody (Cross test)

The prepared mixture of HPV11VLP and other HPV type 8 (HPV6, 16, 18, 31, 33, 45, 52, 58) VLP are respectively added into a 96-well enzyme label plate, and the plate is coated overnight at 4 ℃ in a manner of 0.5 mu g/mL and 100 mu L/well, and a multi-well and a negative control well are arranged at the same time. Blocking at 37 ℃ for 2 hours. Diluting each antibody sample with sample diluent 10 respectively ^-3 ～10 ^-6 ，100μL/wellAdding into the enzyme label plate, covering the sealing plate membrane, and putting into a 37 ℃ incubator for incubation for 45 minutes. The ELISA plate was removed from the incubator, the liquid in the well was discarded, and the plate was washed 5 times with 300. mu.L/well of the washing solution. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. 100 mul/well of goat anti-mouse enzyme-labeled secondary antibody solution with the working concentration of 1:4000 is respectively added, a cover plate membrane is covered, and the mixture is put into a 37 ℃ incubator to be incubated for 45 minutes. The ELISA plate was removed from the 37 ℃ incubator, the well liquid was discarded, and the plate was washed 10 times with 300. mu.L/well of wash solution. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. Uniformly mixing the chromogenic substrate A solution and the chromogenic substrate B solution with required volumes according to the proportion of 1:1, adding 100 mu L/well into an enzyme label plate, and developing for 10 minutes at room temperature in a dark place. Finally, adding the stop solution with the required volume into an enzyme label plate, and adding the stop solution into the enzyme label plate at a concentration of 50 mu L/well. And (4) placing the enzyme label plate into an enzyme label instrument, and measuring the OD value at 450 nm. The test results of the samples with OD values greater than 0.5 were judged to be positive, and the test results of the samples with OD values less than or equal to 0.5 were judged to be negative. To observe the cross-over of binding of each monoclonal antibody to HPV11 and other HPV types 8, and thereby select monoclonal antibodies that specifically bind to HPV11 for further experiments as described below.

The results of cross-over experiments with each mab in combination with HPV11 and other HPV type 8 mixtures are shown in table 1. The results showed that of the 23 types of monoclonal antibodies prepared, 2 that could bind to HPV11 and did not cross other HPV types 8, 20 that crossed, and 1 that recognized HPV11 negativity. The monoclonal antibodies 23F11 and 24C7 prepared by the invention are found to have good specificity for recognizing HPV11, have no cross with other types 8, have good binding property with HPV11 in a dilution gradient range, and can be used for further research.

TABLE 1 identification of the specificity of the monoclonal antibodies of each class

All values are mean values of multiple wells

Example 4 monoclonal antibodies 23F11 and 24C7 paired identification assay

The HPV11VLP is used for coating a 96-well plate, the antibody 23F11 finally selected in example 3 is used as a coating antibody, an enzyme-labeled antibody 24C7-HRP is used as a detection antibody, an ELISA experiment is carried out, the detection result is shown in Table 2, a standard curve is shown in figure 2, the result shows that 23F11 and 24C7 have no competitive relation with the binding epitope of the antigen, and the fact that 23F11 and 24C7 do not have the same antigen binding site can form an antibody pair of the coating antibody and the detection antibody.

TABLE 2 identification of monoclonal antibodies 23F11 and 24C7 by pairing

Example 5 ELISA method for detecting reactivity of antibody with HPV11VLP

And (3) verifying whether the reactivity of the purified monoclonal antibody and the HPV11VLP is good or not by an indirect ELISA method, and whether the detection is rapid and convenient or not can be realized. Meanwhile, the reactivity of the monoclonal purified antibody with HPV11VLP at different concentrations is determined, and the affinity of the antibody can be preliminarily reflected.

HPV11 VLPs were coated in a 96-well plate at 100. mu.L/well, and then antibodies at final concentrations of 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL and 0.0625. mu.g/mL were added to each well in 3 replicates of each concentration, while negative controls were set, and the binding strength of monoclonal antibodies 23F11 and 24C7 to HPV11 VLPs was examined by ELISA. The results show that monoclonal antibodies 23F11 and 24C7 both bind to HPV11 VLPs at the concentration range used in this experiment, with signal intensities greater than 1, and the results are shown in table 3.

TABLE 3 ELISA test antibody binding Strength to HPV11VLP

Antibodies	1μg/mL	0.5μg/mL	0.25μg/mL	0.125μg/mL	0.0625μg/mL	NC*
							24C7	2.35	2.33	2.33	2.29	2.25	0.04
23F11	2.38	2.32	2.33	2.25	2.20	0.03

All values are mean values of multiple wells

Example 6 detection of neutralizing Activity of antibody

The neutralizing activity of each antibody strain was tested by a pseudovirus-cell neutralization model. Antibodies 23F11 and 24C7 were first diluted 40, 80, 160, 320, 640, 1280, 2560 fold with complete medium. Then adding the supernatant and the pseudovirus into a 96-well plate in sequence in equal volume, mixing uniformly on a microplate oscillator, incubating for 1 hour at 4 ℃, and then transferring to a cell incubator at 37 ℃ for incubation for 10 minutes. The mixture (100. mu.L) was added to each 96-well plate previously plated with 293FT cells, and cultured in a cell incubator for 72 hours. And then, an enzyme-linked immunosorbent spot analyzer is used for scanning the whole 96-well plate and counting, the pseudovirus contains a GFP reporter gene and is green under a fluorescence microscope, and if the antibody has neutralizing activity, the pseudovirus infection can be prevented, so that cells are not colored.

The results are shown in table 4, and indicate that the inhibition rate was 99% when 23F11 was diluted 40, 160, and 2560 times; the inhibition rate of 24C7 was 99% when diluted 160-and 320-fold, 98% when diluted 1280-fold, and other than 97% when diluted 2560-fold, and both antibodies were 100% at each of the other dilution gradients, indicating that both monoclonal antibodies 23F11 and 24C7 had neutralizing activity.

TABLE 4 inhibition ratio of HPV11 by antibodies 23F11 and 24C7

HPV11	23F11	24C7
			40	99％	100％
80	100％	100％
			160	99％	99％
320	100％	99％
			640	100％	100％
1280	100％	98％
			2560	99％	97％

Example 7 specificity identification of antibodies 23F11 and 24C7

The prepared standard and the sample to be tested (HPV11 and VLP of other 8 types of HPV (HPV6, 16, 18, 31, 33, 45, 52 and 58)) are respectively added into a coating plate, coated overnight at 4 ℃ in a 100 mu L/well manner, and a plurality of wells and a negative control well are arranged at the same time. Blocking at 37 ℃ for 2 hours. Antibody 23F11 was added to the microplate at 100. mu.L/well, covered with the cover plate membrane and incubated in a 37 ℃ incubator for 45 minutes. The ELISA plate was removed from the incubator, the liquid in the well was discarded, and the plate was washed 5 times with 300. mu.L/well of the washing solution. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. The prepared enzyme-labeled antibody 24C7-HRP solution is added in 100 mu L/well, the cover plate membrane is covered, and the mixture is put into a 37 ℃ incubator for incubation for 45 minutes. The ELISA plate was removed from the 37 ℃ incubator, the well liquid was discarded, and the plate was washed 10 times with 300. mu.L/well of wash solution. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. Uniformly mixing the chromogenic substrate A solution and the chromogenic substrate B solution with required volumes according to the proportion of 1:1, adding 100 mu L/well into an enzyme label plate, and developing for 5 minutes at room temperature in a dark place. Finally, adding the stop solution with the required volume into an enzyme label plate, and adding the stop solution into the enzyme label plate at a concentration of 50 mu L/well. And (4) placing the enzyme label plate into an enzyme label instrument, and measuring the OD value at 450 nm. The test result of the sample with the OD value larger than 0.5 is judged to be positive. Thus, it was evaluated whether the antibodies 23F11 and 24C7 have specificity of binding to HPV 11.

The results of the specific detection of the antibodies are shown in Table 5, and the standard curve is shown in FIG. 3. The results show that the prepared monoclonal antibodies recognize HPV11VLP with the value of more than 0.5 for the pairs of 23F11 and 24C7, and the results are obviously positive results. The other type 8 HPVVLP values were identified to be less than 0.5, indicating a negative result. The monoclonal antibodies prepared by the invention have good specificity on 23F11 and 24C7 for recognizing HPV11 VLP.

Table 5 specific ELISA detection of antibodies (OD 450).)

All values are mean values of multiple wells

Example 8 affinity identification of antibodies 23F11 and 24C7

The affinities of antibodies 23F11 and 24C7 were identified by analyzing the kinetics of antigen-antibody binding and dissociation, and the association rate constants Kon, Koff and Kd of monoclonal antibodies 23F11 and 24C7 with HPV11 VLPs were calculated using a GE BIAcore 3000 biomacromolecule interaction analyzer to reflect the strength of the degree of binding of the antibodies to HPV11 VLPs.

The HPV11 VLPs were diluted to 40. mu.g/mL with 10mM pH4.5 sodium acetate buffer and HPV11 VLPs were coupled to the chip surface according to the manufacturer's instructions. The antibody was diluted to the appropriate concentration with PBS buffer. In the detection, the antibody is injected for 60 seconds, then the antibody is combined for 60 seconds, the antibody is dissociated for 500 seconds, and finally the chip is regenerated by 10mM sodium acetate buffer solution with pH 5.0. Kinetic analysis of antigen-antibody binding was performed on the data using BIAcore 3000Evaluation software according to the instructions. The results of the affinity detection of antibodies 23F11 and 24C7 with HPV11 VLPs are shown in table 6. The results indicate that the 23F11 and 24C7 antibodies are specific antibodies, bind only to HPV11 VLPs and do not bind to the other 8 HPVVLPs.

Table 6 affinity identification of antibodies 23F11 and 24C7

Example 9, subtype identification of clone 23F11 and clone 24C7

The IgG subtypes of monoclonal antibodies 23F11 and 24C7 were identified using antibodies against various IgG subtypes of mice using an indirect ELISA method. The results showed that the subtype of monoclonal antibody 23F11 was IgG 2b and the subtype of monoclonal antibody 24C7 was IgG1, and the results are shown in Table 7. (a defined subtype with OD450 > 1)

Table 7 identification of antibody subtypes by ELISA (OD450 values.)

All values are mean values of multiple wells

Example 10, determination of variable region sequences of clone 23F11 and clone 24C7

Before sequencing cell variable region, subtype detection is performed on 2 cell strains by using cell supernatant, frozen cells in logarithmic growth phase are taken after single subtype is determined, the cells are recovered, centrifuged and collected, and each cell is multiplied by 5 to 10 ⁶ After 1mL of Total RNA Extractor (Trizol) was added to each cell, Nanjing Kingsri sequencing unit was assigned for variable region sequencing.

The sequence is shown in a sequence table.

Using the sequences identified above, various genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, single chain antibodies, diabodies, and the like, can be prepared by known antibody engineering techniques, while retaining the biological properties of the monoclonal antibody from which it is derived.

EXAMPLE 11 Assembly of HPV11 detection kit

1. Coating: diluting the antibody 23F11 to 2 mu g/mL by using 1X PBS of coating solution, adding the antibody 100 mu L/well into an enzyme label plate, and placing the enzyme label plate in a refrigerator at 4 ℃ for 18-24 hours;

2. washing the plate: taking out the enzyme label plate from the refrigerator, washing for 2 times by using a plate washing machine, washing liquor of 1X, and washing for 250 mu L/well;

3. and (3) sealing: washing with 1X lotion twice, drying, sealing at 150 μ L/well at 4 deg.C overnight, and standing for 18-24 hr;

4. and (3) drying: sealing the ELISA plate for 18-24 hours, throwing off liquid in the ELISA plate, drying in a drying room (ensuring that the humidity in the drying room is below 30 percent and the temperature is 30 +/-37 ℃, and recording the temperature and humidity at any time) until the ELISA plate is dried completely, and drying for at least 4.5 hours;

5. closing the plate: the dried ELISA plate is arranged in an aluminum foil bag which is pasted with a label (the label needs a name, the production batch number of the ELISA plate and the validity period), a bag of drying agent is matched at the same time, a sealing machine is used for sealing the plate, and the sealed plate is stored in a refrigerator at 4 ℃ with the validity period of 1 year;

6. the 24C7 was labeled with horseradish peroxidase to give 24C7-HRP and stored at-20 ℃.

The monoclonal antibody 23F11 with specificity and no cross is used as a coating antibody, the monoclonal antibody 24C7 with neutralization activity is used as a detection antibody after being marked by horseradish peroxidase, and the recombinant human papilloma virus-11 VLP protein is used as a standard substance to prepare a standard curve control.

Example 12 Linear and reproducible detection of HPV11 detection kit

An ELISA double-antibody sandwich method is adopted, 23F11 is used as a coating antibody, 24C7-HRP is used as a detection antibody, an ELISA detection method is determined, the detection linear range of the kit is shown in a table 8, a standard curve is shown in an attached figure 4, and the linear range of the kit is 0.25-0.0039 mu g/mL.

TABLE 8 kit Linear Range detection

The antigen was diluted sequentially at 0.25. mu.g/mL, 0.125. mu.g/mL, 0.0625. mu.g/mL, 0.03125. mu.g/mL, 0.0156. mu.g/mL, 0.0078125. mu.g/mL and 0.00390625. mu.g/mL, and repeated experiments were carried out according to the above-described procedures for ELISA experiments. The results are shown in Table 9, the standard curves are shown in figure 4B, and the result accuracy falls between 80% and 120%, which indicates that the double-antibody sandwich ELISA antigen detection method established by the invention has good repeatability.

TABLE 9 kit repeatability tests

EXAMPLE 13 HPV11 detection kit determination procedure

1. Preparing samples

1) Preparing a washing liquid: taking out 50mL of 20 times of concentrated washing liquor, adding distilled water, and metering to 1L, and fully and uniformly mixing for later use;

2) a sample diluent;

3) enzyme-labeled antibody diluent;

4) diluting the standard substance by using the sample diluent, and setting a dilution gradient for later use;

5) diluting the enzyme-labeled antibody with an enzyme-labeled antibody diluent, and taking a proper amount of enzyme-labeled antibody to be diluted by 10000 times for later use.

2. Measurement procedure

1) Sample adding: adding the prepared standard substance and a sample to be detected into a coating plate, covering the coating plate with a sealing plate film, and putting the coating plate film into an incubator at 37 ℃ for incubation for 45 minutes;

2) adding an enzyme-labeled antibody: and taking the enzyme label plate out of the incubator, discarding liquid in the hole, adding washing liquid of 300 mu L/well, washing the plate for 5 times, and soaking for 30 seconds each time. And finally, removing liquid in the holes, and then patting the holes on absorbent paper. Adding 100 mu L/well of the prepared enzyme-labeled antibody solution, covering a cover plate membrane, and putting the mixture into a 37 ℃ incubator for incubation for 45 minutes;

3) color development: taking the enzyme label plate out of the 37 ℃ incubator, discarding the liquid in the hole, adding 300 mu L/well of washing liquid, washing the plate for 5 times, and soaking for 30 seconds each time. And finally, removing liquid in the holes, and then patting the holes on absorbent paper. Uniformly mixing the chromogenic substrate A solution and the chromogenic substrate B solution with required volumes according to the proportion of 1:1, adding 100 mu L/well into an enzyme label plate, and placing the enzyme label plate into an incubator at 37 ℃ for incubation for 10 minutes in a dark place;

4) and (3) terminating the reaction: adding the stop solution with the required volume into an enzyme label plate, wherein the volume of the stop solution is 50 mu L/well;

5) and (3) determination: placing the enzyme label plate into an enzyme label instrument, setting 630nm as a reference wavelength, measuring absorbance at the wavelength of 450nm, and recording the measurement result;

6) and (4) judging a result:

four-parameter fitting curve R of standard product ² ≥0.9801；

The linear range of the method is 0.25-0.0039 mug/mL, the detection limit is 0.0039 mug/mL, the result of the test sample higher than 0.0039 mug/mL is judged to be positive, and the negative control OD value is less than or equal to 0.100;

the detection results of different concentration ranges of the test sample must be all in the linear range of the standard curve, and points higher or lower than the linear range have no reference significance. The final assay quantification is averaged over a number of concentration range points.

And fourthly, the three points are required to be simultaneously met except for the stipulations.

Sequence listing

<110> Jiangsu Rike Biotechnology, Inc.; beijing Anbaisheng Biotech Co., Ltd

<120> antibody of human papillomavirus type 11L1 protein and preparation method thereof

<130> 2021

<150> 202110189825.2

<151> 2021-02-18

<160> 64

<170> SIPOSequenceListing 1.0

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Asn Tyr Leu Ile Glu

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<211> 17

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<400> 2

Val Ile Asn Pro Gly Asn Gly Gly Ile Asn Tyr Ser Glu Lys Phe Lys

1 5 10 15

Gly

<210> 3

<211> 12

<212> PRT

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Pro His Tyr Tyr Gly Asn Ile Tyr Ala Met Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

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<400> 4

Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His

1 5 10

<210> 5

<211> 7

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<400> 5

Tyr Ala Ser Gln Ser Ile Ser

1 5

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<211> 9

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Gln Gln Ser Asn Ser Trp Pro Trp Thr

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr

20 25 30

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Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10

<210> 9

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Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

1 5 10 15

Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys Ala Ser

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<210> 10

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Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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<210> 11

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<400> 11

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Ser Val Ser Leu Ser Cys

20

<210> 12

<211> 15

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Trp Tyr Gln Leu Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

1 5 10 15

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Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Ser Ile Asn Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys

20 25 30

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Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

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Met Glu Trp Ser Arg Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe

35 40 45

Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Val Ile Asn Pro Gly Asn Gly Gly Ile Asn Tyr Ser

65 70 75 80

Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

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Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val

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Tyr Phe Cys Ala Ser Pro His Tyr Tyr Gly Asn Ile Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser

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Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Ile Ser Asn Asn Leu His Trp Tyr Gln Leu Lys Ser His Glu Ser Pro

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Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn

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Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn

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Ser Trp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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aattacttga tagag 15

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<210> 19

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<210> 23

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<212> DNA

<213> Artificial sequence

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<210> 27

<211> 69

<212> DNA

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<400> 27

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<212> DNA

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<212> DNA

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<210> 31

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<211> 381

<212> DNA

<213> Artificial sequence

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atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60

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ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca actaaaatca 180

catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 240

aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaatag tgtggagact 300

gaagattttg gaatgtattt ctgtcaacag agtaacagct ggccgtggac gttcggtgga 360

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Ser Asn Trp Leu His

1 5

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Ala Ile Tyr Pro Gly Ser Gly Asp Ser Thr Tyr Thr Gln Lys Phe Lys

1 5 10 15

Asp

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Glu Tyr Asp Lys Asp Trp Tyr Phe Asp Val

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Arg Ser Ser Arg Ser Ile Val Gln Ser Asn Gly Asn Thr Tyr Leu Glu

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Lys Val Ser Asn Arg Phe Ser

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Phe Gln Gly Ser His Val Pro Phe Thr

1 5

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Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Thr Gly Tyr Arg Phe Thr

20 25 30

<210> 40

<211> 14

<212> PRT

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Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10

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<211> 32

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Lys Ala Ile Leu Thr Ala Val Ser Ser Ala Asn Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Val

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<211> 11

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Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

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Asp Val Val Ile Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys

20

<210> 44

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Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 45

<211> 32

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<400> 45

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

20 25 30

<210> 46

<211> 10

<212> PRT

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<400> 46

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 47

<211> 138

<212> PRT

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<400> 47

Met Glu Trp Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Ile Ser Gly

1 5 10 15

Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Thr Gly Tyr Arg Phe

35 40 45

Thr Ser Asn Trp Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Tyr Pro Gly Ser Gly Asp Ser Thr Tyr Thr

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Ala Val Ser Ser Ala Asn

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Val Glu Tyr Asp Lys Asp Trp Tyr Phe Asp Val Trp

115 120 125

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

130 135

<210> 48

<211> 131

<212> PRT

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<400> 48

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Gly

1 5 10 15

Ser Ser Ser Asp Val Val Ile Thr Gln Ile Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Ile

35 40 45

Val Gln Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

100 105 110

Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 49

<211> 15

<212> DNA

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<400> 49

agcaactggc tgcac 15

<210> 50

<211> 51

<212> DNA

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gcgatttatc ctggaagtgg tgatagtact tacacccaga agttcaagga c 51

<210> 51

<211> 30

<212> DNA

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<400> 51

gaatacgaca aggactggta tttcgatgtc 30

<210> 52

<211> 48

<212> DNA

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agatctagtc ggagcattgt acaaagtaat ggaaacacct atttagag 48

<210> 53

<211> 21

<212> DNA

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<400> 53

aaagtttcca accgattttc t 21

<210> 54

<211> 27

<212> DNA

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<400> 54

tttcaaggtt cacatgttcc attcacg 27

<210> 55

<211> 90

<212> DNA

<213> Artificial sequence

<400> 55

gaggttcagc tccagcagtc tgggacagtg ctggcaaggc ctggggcttc cgtgaagatg 60

tcctgtaagg ctactggcta caggtttacc 90

<210> 56

<211> 42

<212> DNA

<213> Artificial sequence

<400> 56

tgggtaaaac agaggcctgg acagggtcta gaatggattg gt 42

<210> 57

<211> 96

<212> DNA

<213> Artificial sequence

<400> 57

aaggccatac tgaccgcagt ctcatccgcc aacactgcct acatggaact cagcagcctg 60

acaaatgagg actctgcggt ctattactgt acagtt 96

<210> 58

<211> 33

<212> DNA

<213> Artificial sequence

<400> 58

tggggcgcag ggaccacggt caccgtctcc tca 33

<210> 59

<211> 69

<212> DNA

<213> Artificial sequence

<400> 59

gatgtcgtga ttacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgc 69

<210> 60

<211> 45

<212> DNA

<213> Artificial sequence

<400> 60

tggtacctgc agaagccagg ccagtctcca aagctcctga tctac 45

<210> 61

<211> 96

<212> DNA

<213> Artificial sequence

<400> 61

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caggatcagt 60

agagtggagg ctgaggatct gggagtttat tactgc 96

<210> 62

<211> 30

<212> DNA

<213> Artificial sequence

<400> 62

ttcggctcgg ggacaaagtt ggaaataaaa 30

<210> 63

<211> 414

<212> DNA

<213> Artificial sequence

<400> 63

atggaatgga actggatact tccttttatt ctgtcggtaa tttcaggggt ctactcagag 60

gttcagctcc agcagtctgg gacagtgctg gcaaggcctg gggcttccgt gaagatgtcc 120

tgtaaggcta ctggctacag gtttaccagc aactggctgc actgggtaaa acagaggcct 180

ggacagggtc tagaatggat tggtgcgatt tatcctggaa gtggtgatag tacttacacc 240

cagaagttca aggacaaggc catactgacc gcagtctcat ccgccaacac tgcctacatg 300

gaactcagca gcctgacaaa tgaggactct gcggtctatt actgtacagt tgaatacgac 360

aaggactggt atttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctca 414

<210> 64

<211> 393

<212> DNA

<213> Artificial sequence

<400> 64

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctggttc cagcagtgat 60

gtcgtgatta cccaaattcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcggag cattgtacaa agtaatggaa acacctattt agagtggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caggatcagt 300

agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 360

acgttcggct cggggacaaa gttggaaata aaa 393

Claims

1. An antibody which binds to the L1 protein and/or VLP of human papillomavirus HPV11, wherein the antibody comprises the amino acid sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable regions shown in SEQ ID nos. 1-3, and the amino acid sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable regions shown in SEQ ID nos. 4-6.

2. The antibody according to claim 1, which binds to L1 protein and/or VLP of human papillomavirus HPV11, wherein the antibody further comprises the regions of amino acid sequences FR1, FR2, FR3 or/and FR4 of the FR of the heavy chain variable region shown in SEQ ID nos. 7-10, and the regions of amino acid sequences FR1, FR2, FR3 or/and FR4 of the FR of the light chain variable region shown in SEQ ID nos. 11-14.

3. The antibody of claims 1-2, comprising the heavy chain variable region amino acid sequence set forth in SEQ ID No.15 and the light chain variable region amino acid sequence set forth in SEQ ID No. 16.

4. An antibody which binds to the L1 protein and/or VLP of human papillomavirus HPV11, wherein the antibody comprises the nucleotide sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable region as set out in SEQ ID nos. 17-19, and the nucleotide sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region as set out in SEQ ID nos. 20-22.

5. The antibody binding to L1 protein and/or VLP of human papillomavirus HPV11 of claim 4, wherein the antibody further comprises the nucleotide sequences FR1, FR2, FR3 or/and FR4 region of the FRs of the heavy chain variable region as set forth in SEQ ID nos. 23-26, and the nucleotide sequences FR1, FR2, FR3 or/and FR4 region of the FRs of the light chain variable region as set forth in SEQ ID nos. 27-30.

6. The antibody of claims 4-5, comprising a heavy chain variable region nucleotide sequence as set forth in SEQ ID No.31 and a light chain variable region nucleotide sequence as set forth in SEQ ID No. 32.

7. The antibody of claims 1-6, wherein said antibody is a monoclonal antibody produced by a hybridoma cell line obtained by fusion of an immunized host spleen cell with a myeloma cell, said hybridoma cell line having a accession number of CGMCC No. 21984.

8. The antibody of claims 1-6, wherein said antibody is a specific monoclonal antibody.

9. A kit for the detection of L1 protein and/or VLP of HPV11, wherein the kit comprises an antibody according to any one of claims 1-8.

10. The kit of claim 9, further comprising a second antibody that binds to the L1 protein of HPV11 and/or the VLP.

11. The kit of claim 10, wherein the second antibody comprises the amino acid sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable region as set forth in SEQ ID nos. 33-35 and the amino acid sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region as set forth in SEQ ID nos. 36-38.

12. The kit of claim 11, wherein the second antibody further comprises the regions of amino acid sequences FR1, FR2, FR3 or/and FR4 of the FR of the heavy chain variable region shown in SEQ ID nos. 39-42 and the regions of amino acid sequences FR1, FR2, FR3 or/and FR4 of the FR of the light chain variable region shown in SEQ ID nos. 43-46.

13. The kit of claims 11-12, wherein the second antibody comprises the heavy chain variable region amino acid sequence set forth in SEQ ID No.47 and the light chain variable region amino acid sequence set forth in SEQ ID No. 48.

14. The kit of claim 10, wherein the second antibody comprises the nucleotide sequences CDRH1, CDRH2 or/and CDRH3 regions of the CDRs of the heavy chain variable region as set forth in SEQ ID nos. 49-51 and the nucleotide sequences CDRL1, CDRL2 or/and CDRL3 regions of the CDRs of the light chain variable region as set forth in SEQ ID nos. 52-54.

15. The kit of claim 14, wherein said second antibody further comprises the nucleotide sequences FR1, FR2, FR3 or/and FR4 region of the FR of the heavy chain variable region shown in SEQ ID nos. 55-58 and the nucleotide sequences FR1, FR2, FR3 or/and FR4 region of the FR of the light chain variable region shown in SEQ ID nos. 59-62.

16. The kit of claims 14-15, wherein the second antibody comprises the heavy chain variable region nucleotide sequence set forth in SEQ ID No.63 and the light chain variable region nucleotide sequence set forth in SEQ ID No. 64.

17. The kit according to claims 9-16, wherein the second antibody is also a monoclonal antibody produced by a hybridoma cell line obtained by fusing immunized host spleen cells and myeloma cells, the hybridoma cell line having a accession number of CGMCC No. 21985.

18. The kit of claims 11-17, wherein the second antibody is a monoclonal antibody having neutralizing activity.

19. Use of the monoclonal antibody of any one of claims 1-8 in the preparation of a reagent for the detection of L1 protein and/or VLP of HPV 11.

20. Use of the kit of any one of claims 9-18 in the preparation of a reagent for the detection of L1 protein and/or VLP of HPV 11.

21. The kit for detecting L1 protein and/or VLP of HPV11 of claims 9-18, further comprising a detectable label: a radioisotope, a fluorescent substance, a luminescent substance, a colored substance and/or an enzyme.

22. The kit according to claims 9-18, wherein the kit is an ELISA kit comprising a capture antibody and a detection antibody, wherein the capture antibody is the monoclonal antibody of any one of claims 1-8 and the detection antibody is the second antibody of claims 10-18.