Antibody to human papillomavirus type 11 L1 protein and preparation method thereof

Abstract

The present invention provides a monoclonal antibody that specifically recognizes the L1 protein (HPV11 L1) and / or VLP (HPV11 VLP) of human papillomavirus type 11. The specific monoclonal antibody provided by the present invention has no significant cross-reactivity with the L1 proteins and / or VLPs of eight other HPV types (HPV6, 16, 18, 31, 33, 45, 52, and 58), and exhibits high specificity, sensitivity, accuracy, and safety. It can be used to accurately detect the bioactive content of HPV11 L1 protein and / or VLP in vaccine strains and is expected to be widely used in clinical testing and vaccine production.

Description

Technical Field

[0001] The present invention relates to the fields of molecular virology and immunology, and more specifically, to a human papillomavirus type 11 hybridoma cell line, a method for preparing a monoclonal antibody against human papillomavirus type 11 L1 protein and / or VLP, and a diagnostic kit prepared therefrom. Background Art

[0002] Human papillomavirus (HPV) is a small, non-enveloped DNA virus that infects human epithelial tissues of the skin and mucous membranes, causing wart-like growths and potentially leading to benign or malignant tumors. There are numerous HPV subtypes, with over 130 identified to date, each causing distinct clinical manifestations. Based on their association with tumorigenesis, HPV can be categorized as high-risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58) and low-risk (HPV6, 11, 40, 42, 43, 44, and 54).

[0003] Low-risk types do not integrate into the cellular genome and primarily cause warty growths on the skin and mucous membranes, manifesting as benign lesions such as genital warts and flat warts. Genital warts are one of the eight sexually transmitted diseases (STDs) monitored by the Ministry of Health. Its incidence is second only to gonorrhea. Due to its high recurrence rate and high infectiousness, it has become a common STD that poses a serious threat to people's normal lives. According to epidemiological studies, HPV types such as HPV 6 and 11 cause approximately 90% of lesions in the anogenital mucosa. Therefore, developing an HPV 11 vaccine would be a valuable tool in preventing genital warts and other diseases.

[0004] HPV's major capsid protein L1 has the characteristic of self-assembling into hollow virus-like particles (VLPs). HPV VLP is a 20-sided symmetrical structure composed of 72 pentamers of the major capsid protein L1. VLP vaccines can express one or more structural proteins of the virus through molecular biology techniques. These structural proteins have natural autonomous assembly capabilities and can form spatial configurations and antigenic epitopes similar to natural virus particles, but without viral nucleic acids, strong immunogenicity, and non-infectiousness. There is no risk of incomplete inactivation or virulence reversion, and there is a high density of viral antigens on the surface, which preserves conformational epitopes and can be presented to immune cells through the same pathway as whole-virus vaccines, effectively inducing the body's immune system to produce an immune protection response and induce high-titer neutralizing antibodies.

[0005] In the process of HPV vaccine development, type identification, antigen content detection and in vitro efficacy determination are required. The methods can all adopt the double antibody sandwich ELISA method. Therefore, in vaccine research, monoclonal antibodies are important tools for vaccine antigen quality control, especially antibodies with specificity and neutralizing activity, which play an irreplaceable and important role in vaccine development. The present invention provides a monoclonal antibody with specificity and neutralizing activity against HPV11. The ELISA detection kit prepared using two strains thereof can be specifically used to quickly identify HPV11 L1 protein, and can be widely used in clinical testing and quality inspection in the current vaccine production process of vaccine manufacturers. It is of great significance to the development of HPV11 vaccine and the prevention of diseases such as genital warts. Summary of the Invention

[0006] The first aspect of the present invention provides a monoclonal antibody capable of recognizing the L1 protein and / or VLP of HPV11 and a hybridoma cell line producing the antibody.

[0007] The second aspect of the present invention provides a method for preparing a monoclonal antibody.

[0008] The third aspect of the present invention provides a method for preparing an antigen detection kit for HPV11 L1 protein and / or VLP.

[0009] A fourth aspect of the present invention provides a double-antibody sandwich ELISA kit for detecting HPV11.

[0010] The present invention is achieved through the following technical solutions:

[0011] The monoclonal antibody provided by the present invention is directed against the HPV11 L1 protein, which refers to the L1 protein encoding the papillomavirus HPV11. The nucleic acid base sequence of the HPV11 L1 protein can be inserted into a prokaryotic expression vector by conventional molecular biological cloning methods. The expressed HPV11 L1 protein can polymerize to form soluble polymers. The amino acid sequence of the HPV11 L1 protein and the nucleic acid sequence encoding the L1 protein can be obtained from databases such as GenBank, Swiss-prot, EMBL.PIR Protein Data Base, PDB Protein Data Bank, or from the HPV sequence library (http: / / hpv-web.lanl.gov). Including but not limited to the production of HPV11 L1 protein using the Hansenula expression system described in the Chinese patent application with application publication number CN103361280A, to prepare virus-like particles (VLPs) as immunogens. Specifically, HPV11 VLP is used as an immunogen to immunize mice, and hybridoma technology is used to obtain hybridoma cell lines that can continuously and stably secrete anti-HPV11 VLP through cell fusion and screening, and monoclonal antibodies are secreted by each cell line.

[0012] In the first aspect, in a preferred embodiment, the antibody 23F11 that recognizes the L1 protein and / or VLP of human papillomavirus type 11 is a monoclonal antibody produced by a hybridoma cell line obtained by fusion of immunized host spleen cells and myeloma cells, and the preservation number of the hybridoma cell is CGMCC No. 21984; classification name: hybridoma cell; preservation unit: General Microbiology Center of China Culture Collection Administration; preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; preservation date: April 1, 2021.

[0013] The monoclonal antibody produced by the hybridoma cell line with the deposit number CGMCC No. 21984 comprises the amino acid sequence of the CDR of the heavy chain variable region having the CDRH1, CDRH2 or / and CDRH3 regions as shown in SEQ ID NOs. 1-3, and the amino acid sequence of the CDR of the light chain variable region having the CDRL1, CDRL2 or / and CDRL3 regions as shown in SEQ ID NOs. 4-6.

[0014] The monoclonal antibody produced by the hybridoma cell line comprises an amino acid sequence of the FR of the heavy chain variable region having the FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 7-10, and an amino acid sequence of the FR of the light chain variable region having the FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 11-14.

[0015] The monoclonal antibody produced by the hybridoma cell line comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO.15 and the light chain variable region amino acid sequence shown in SEQ ID NO.16.

[0016] The monoclonal antibody produced by the above hybridoma cell line comprises a nucleotide sequence of the CDR of the heavy chain variable region having the CDRH1, CDRH2 or / and CDRH3 regions as shown in SEQ ID NOs. 17-19, and a nucleotide sequence of the CDR of the light chain variable region having the CDRL1, CDRL2 or / and CDRL3 regions as shown in SEQ ID NOs. 20-22.

[0017] The monoclonal antibody produced by the above hybridoma cell line comprises a nucleotide sequence of the FR of the heavy chain variable region having the FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 23-26, and a nucleotide sequence of the FR of the light chain variable region having the FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 27-30.

[0018] The monoclonal antibody produced by the hybridoma cell line comprises a heavy chain variable region nucleotide sequence as shown in SEQ ID NO.31 and a light chain variable region nucleotide sequence as shown in SEQ ID NO.32.

[0019] In the second aspect, in another preferred embodiment, the second antibody 24C7 that recognizes the L1 protein and / or VLP of human papillomavirus type 11 is a monoclonal antibody produced by a hybridoma cell line obtained by fusion of another immunized host spleen cell and myeloma cell, and the preservation number of the hybridoma cell is CGMCC No. 21985; classification name: hybridoma cell; preservation unit: General Microbiology Center of China Culture Collection Administration; preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; preservation date: April 1, 2021.

[0020] The second antibody comprises the amino acid sequence of the CDR of the heavy chain variable region of CDRH1, CDRH2 or / and CDRH3 region as shown in SEQ ID NOs. 33-35, and the amino acid sequence of the CDR of the light chain variable region of CDRL1, CDRL2 or / and CDRL3 region as shown in SEQ ID NOs. 36-38.

[0021] The second antibody comprises an amino acid sequence of FR of a heavy chain variable region having FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 39-42, and an amino acid sequence of FR of a light chain variable region having FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 43-46.

[0022] The second antibody comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO.47 and a light chain variable region amino acid sequence as shown in SEQ ID NO.48.

[0023] The second antibody further comprises a nucleotide sequence of CDRs of the heavy chain variable region comprising CDRH1, CDRH2 or / and CDRH3 regions as shown in SEQ ID NOs. 49-51, and a nucleotide sequence of CDRs of the light chain variable region comprising CDRL1, CDRL2 or / and CDRL3 regions as shown in SEQ ID NOs. 52-54.

[0024] The second antibody comprises a nucleotide sequence of FR of a heavy chain variable region having FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 55-58, and a nucleotide sequence of FR of a light chain variable region having FR1, FR2, FR3 and / or FR4 regions as shown in SEQ ID NOs. 59-62.

[0025] The second antibody comprises a heavy chain variable region nucleotide sequence as shown in SEQ ID NO.63 and a light chain variable region nucleotide sequence as shown in SEQ ID NO.64.

[0026] On the other hand, the present invention provides a kit for detecting L1 protein and / or VLP of HPV11, comprising two monoclonal antibodies according to the two preferred embodiments disclosed in the present invention.

[0027] The kit for detecting L1 protein and / or VLP of HPV11 disclosed in the present invention is a double antibody sandwich ELISA kit for detecting L1 protein and / or VLP antigen of HPV11, and further comprises a detectable marker: a fluorescent substance, a colored substance and / or an enzyme.

[0028] Monoclonal antibodies to human papillomavirus type 11 L1 protein can be prepared as follows:

[0029] Step 1: Using the prepared human papillomavirus type 11 VLP protein as an immunogen, the purified protein is used to immunize 6-8 week old female Balb / c mice three times, with an interval of 14 days between each immunization. Serum is collected and the serum titer is tested by indirect ELISA. Based on the ELISA results, the tail vein of female Balb / c mice with high titer are selected, and immune spleen cells are prepared from the mice.

[0030] Step 2: Prepare hybridoma cells. First, prepare a myeloma cell (SP2 / 0) suspension and inject it into female Balb / c mice. After the mice develop solid tumors, prepare myeloma cells. Fuse the myeloma cells with the immune spleen cells described in Step 1 to prepare hybridoma cells. Test and screen for high-titer hybridoma cell lines and clone and expand them for culture.

[0031] Step 3: Collect the supernatant from the hybridoma cells cloned and expanded in step 2 and inject it into mice via the peritoneal cavity. Collect the mouse ascites, purify it, and then conduct specific identification and neutralization activity detection to obtain monoclonal antibodies against human papillomavirus type 11 VLP.

[0032] Preparation method of human papillomavirus double antibody sandwich ELISA antigen detection kit:

[0033] The ELISA plate is coated with the specific, cross-link-free monoclonal antibody 23F11 as the capture antibody, and the neutralizing monoclonal antibody 24C7, labeled with horseradish peroxidase, is used as the detection antibody. A standard curve control is prepared using human papillomavirus type 11 VLP protein as the standard. The kit also includes concentrated wash buffer, sample diluent, enzyme-labeled antibody diluent, substrate solution A, substrate solution B, and stop solution.

[0034] The monoclonal antibody obtained in the present invention has good specificity. Experiments have shown that it has no cross-reaction with the other eight HPV types. Therefore, the monoclonal antibody obtained in the present invention can be used for the specific detection of HPV11 VLP in the latest nine-valent vaccine on the market and vaccines or compositions including HPV11 types.

[0035] The present invention adopts a double antibody sandwich method and utilizes two monoclonal antibodies to specifically detect and quantify HPV11 VLP. The present invention also provides a kit for the specific detection and quantification of HPV11 VLP, with a detection limit of 0.0039 μg / mL and a linear range of 0.25-0.0039 μg / mL. It has the advantages of high specificity and high sensitivity, and can accurately detect the level of bioactive HPV11 VLP in samples. It will be widely used in clinical testing and quality inspection during vaccine production by vaccine manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Attachment Figure 1 : SDS-PAGE analysis results showed that the purity of each purified monoclonal antibody reached more than 90%.

[0037] Attachment Figure 2 : Standard curve of paired identification experiment for each type of monoclonal antibody, the ordinate is OD450, and the abscissa is the concentration of the standard.

[0038] Attachment Figure 3 : Standard curve of the specific identification experiment of antibodies 23F11 and 24C7, the ordinate is OD450, and the abscissa is the concentration of the standard.

[0039] Attachment Figure 4 : ELISA double antibody sandwich method to detect HPV11 VLP standard curve, the ordinate is OD450, and the abscissa is the standard concentration.

[0040] SEQ ID NO. 1-3 are the amino acid sequences of the CDRs of the heavy chain variable regions of the antibodies of the present invention, CDRH1, CDRH2, and / or CDRH3;

[0041] SEQ ID NO. 4-6 are the amino acid sequences of the CDRs of the light chain variable regions of the antibodies of the present invention, CDRL1, CDRL2, and / or CDRL3 regions;

[0042] SEQ ID NO. 7-10 are the amino acid sequences FR1, FR2, FR3 and / or FR4 of the heavy chain variable region of the antibody of the present invention;

[0043] SEQ ID NOs. 11-14 are the amino acid sequences FR1, FR2, FR3 and / or FR4 of the light chain variable region of the antibody of the present invention;

[0044] SEQ ID NO.15 is the amino acid sequence of the heavy chain variable region of the antibody of the present invention;

[0045] SEQ ID NO.16 is the amino acid sequence of the light chain variable region of the antibody of the present invention;

[0046] SEQ ID NO. 17-19 are the nucleotide sequences of the CDRs of the heavy chain variable regions of the antibodies of the present invention, CDRH1, CDRH2 and / or CDRH3;

[0047] SEQ ID NO. 20-22 are the nucleotide sequences of the CDRs of the light chain variable region of the antibody of the present invention, CDRL1, CDRL2, and / or CDRL3 regions;

[0048] SEQ ID NO. 23-26 are the nucleotide sequences of FR1, FR2, FR3 and / or FR4 regions of the heavy chain variable region of the antibody of the present invention;

[0049] SEQ ID NO. 27-30 are the nucleotide sequences FR1, FR2, FR3 and / or FR4 of the light chain variable region of the antibody of the present invention;

[0050] SEQ ID NO.31 is the nucleotide sequence of the heavy chain variable region of the antibody of the present invention;

[0051] SEQ ID NO.32 is the nucleotide sequence of the light chain variable region of the antibody of the present invention;

[0052] SEQ ID NOs. 33-35 are the amino acid sequences CDRH1, CDRH2, and / or CDRH3 of the CDRs of the heavy chain variable region of the second antibody of the present invention;

[0053] SEQ ID NOs. 36-38 are the amino acid sequences CDRL1, CDRL2, and / or CDRL3 of the CDRs of the light chain variable region of the second antibody of the present invention;

[0054] SEQ ID NOs. 39-42 are the amino acid sequences FR1, FR2, FR3, and / or FR4 of the heavy chain variable region of the second antibody of the present invention;

[0055] SEQ ID NOs. 43-46 are the amino acid sequences FR1, FR2, FR3, and / or FR4 of the light chain variable region of the second antibody of the present invention;

[0056] SEQ ID NO.47 is the amino acid sequence of the heavy chain variable region of the second antibody of the present invention;

[0057] SEQ ID NO.48 is the amino acid sequence of the light chain variable region of the second antibody of the present invention;

[0058] SEQ ID NOs. 49-51 are the nucleotide sequences CDRH1, CDRH2, and / or CDRH3 of the CDRs of the heavy chain variable region of the second antibody of the present invention;

[0059] SEQ ID NOs. 52-54 are the nucleotide sequences CDRL1, CDRL2, and / or CDRL3 of the CDRs of the light chain variable region of the second antibody of the present invention;

[0060] SEQ ID NOs. 55-58 are the nucleotide sequences FR1, FR2, FR3 and / or FR4 of the heavy chain variable region of the second antibody of the present invention;

[0061] SEQ ID NOs. 59-62 are the nucleotide sequences FR1, FR2, FR3 and / or FR4 of the light chain variable region of the second antibody of the present invention;

[0062] SEQ ID NO.63 is the nucleotide sequence of the heavy chain variable region of the second antibody of the present invention;

[0063] SEQ ID NO.64 is the nucleotide sequence of the light chain variable region of the second antibody of the present invention; Specific implementation methods

[0064] The principles, features and contents of the present invention are further described below in conjunction with the examples, and the examples are only used to explain the present invention and are not to be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents without indicating the source are all conventional reagents in the art or commercially available reagents, and the scientific and technical terms used in the text have the meanings generally understood by those skilled in the art. Those skilled in the art will carry out non-essential improvements and adjustments to the embodiments according to the foregoing invention content, which still fall within the scope of protection of the present invention. In order to better understand the present invention, the definition and explanation of the relevant terms are provided below.

[0065] The term "antibody" as used herein refers to an immunoglobulin molecule generally 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 connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L Composition. H and V L The V domain can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain / light chain pair form the antibody binding site. The term "antibody" is not limited to any particular method for producing antibodies.

[0066] The term "monoclonal antibody" as used in the present invention refers to an antibody or an antibody fragment derived from a population of highly homologous antibody molecules. A monoclonal antibody has high specificity for a single epitope on an antigen.

[0067] As used herein, "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably.

[0068] The term "HPVVLP" used in the present invention refers to virus-like particles assembled from HPV L1 protein expressed in vitro, such as HPV11 L1-VLP refers to virus-like particles assembled from HPV11 L1 protein expressed in vitro.

[0069] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0070] Example 1. Establishment of hybridoma cell lines

[0071] 1. Animal immunization

[0072] 1) Antigen preparation: homemade antigen. HPV11 L1 protein was produced using the Hansenula expression system described in the Chinese patent application with application publication number CN103361280A. The purified HPV11 L1 protein sample was diluted 3 times with sterile water and a small drop was placed on a wax plate. A copper mesh was placed so that the surface with the support membrane was in contact with the surface of the sample liquid. The sample was allowed to stand for 1 minute and then removed. The copper mesh was removed and excess droplets were removed with a filter paper strip and allowed to dry slightly. A small drop of 2% uranyl acetate solution was placed on the wax plate. The copper mesh adsorbed with the sample was placed on the surface of the dye solution (the sample was in contact with the dye solution) and allowed to stand for 2 minutes. The copper mesh was removed and excess droplets were removed with a filter paper strip and allowed to dry under an incandescent lamp. A JEOL-1400 transmission electron microscope was used to observe the self-assembled virus-like particles (VLPs), which were used as antigens.

[0073] Primary immunization: Mix equal volumes of antigen and Freund's complete adjuvant and fully emulsify, then inject intraperitoneally into 6-8 week old Balb / c female mice at a dose of 30 μg / mouse.

[0074] 2) Booster Immunization: Booster immunizations are administered with an emulsion of antigen and Freund's incomplete adjuvant. The first booster immunization is administered 14 days after the primary immunization, followed by a second booster immunization 14 days later, and a third booster immunization 13 days later. Each booster immunization consists of an intraperitoneal injection of 30 μg of the antigen and Freund's incomplete adjuvant emulsion.

[0075] 3) Tail vein: 0.1 mL / mouse, cell fusion was performed 3 days later.

[0076] 2. Preparation of hybridoma cells

[0077] Mouse spleen cells were harvested as per conventional methods and fused with SP2 / 0 cells at a ratio of 5:1 using PEG4000. Cultured in HAT selection medium, after 5-7 days of fusion, the supernatant was collected and screened for hybridoma cell lines using indirect ELISA. Positive clones were subcloned using limiting dilution. The indirect ELISA procedure is as follows:

[0078] 1) Coating: Add 0.5 μg / mL HPV11 VLP protein, 50 μL / well, to the ELISA plate for coating and place in a 4°C refrigerator overnight;

[0079] 2) Blocking: Remove the coated plate, wash twice with a plate washer, pat dry, add blocking solution (150 μL / well), and incubate at 37°C for 2 h.

[0080] 3) Sample Addition: Wash the blocked ELISA plate once with a plate washer, pat dry, and add the sample to be tested at 100 μL / well. Also set up positive and negative control wells. Incubate in a 37°C incubator for 30 minutes.

[0081] 4) Secondary Antibody: Wash the incubated plate three times with a plate washer and pat dry. Add goat anti-mouse IgG (H+L) secondary antibody at a 1:4000 dilution. Incubate at 37°C for 30 minutes.

[0082] 5) Color development: After incubation with the secondary antibody in the previous step, wash the plate five times, pat dry, and add 100 μL / well of color development solution A and B mixed in a 1:1 ratio to the plate and react for 10 minutes.

[0083] 6) Stop: Add 50 μL / well of stop solution and read the OD value at 450 nm using a microplate reader. A clone with an OD greater than 1.0 is considered a positive clone.

[0084] 3. Establishment of hybridoma cell lines

[0085] The positive clones obtained in step 2 are further cloned, and the hybridoma cell lines are further cultured and passaged in a culture medium containing 10% fetal bovine serum and 10×HT until the hybridoma cell lines can still grow well and be stably passaged, thereby obtaining hybridoma cell lines that stably secrete monoclonal antibodies.

[0086] Example 2: Preparation and purification of monoclonal antibodies against HPV11

[0087] 1. Paraffin Sensitization: Select Balb / c multiparous mice and sensitize them with paraffin 7 days before cell inoculation (paraffin oil can reduce mouse rejection of inoculated hybridoma cells and promote hybridoma cell growth). Remove the needle from a 2mL syringe, draw up 2.5mL of paraffin oil, and secure the needle with the bevel of the needle facing the syringe scale. Slowly inject 0.5mL into the peritoneal cavity of each mouse.

[0088] 2. Cell inoculation: Take cells in good condition, discard the supernatant, add a certain amount of DMEM medium to disperse the adherent cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend with 2 mL of culture medium, take 10 μL of cells and add an equal volume of trypan blue reagent for counting. Based on the counting results and the required total number of cells, take a certain volume of cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add physiological saline to make the cell density 0.5×10 6 Cells / mL were injected intraperitoneally into mice at a rate of 0.5 mL / mouse. The growth of ascites in the mice was observed at all times. Ascites generally formed 7 days after cell inoculation.

[0089] 3. Ascites Collection: The timing of ascites collection will be determined based on the distension of the mouse's abdomen and its condition, generally 7 days after inoculation. Hold the mouse upright and pierce the abdomen with a 12-gauge needle, approximately 1 / 4 of the way into the needle. Use forceps to control the needle's position, allowing the ascites to flow through the needle into a centrifuge tube. Centrifuge at 8000 rpm for 10 minutes, collect the ascites supernatant, and store in a -20°C refrigerator. Ascites should be collected every other day until the mouse dies or is in poor condition after being sacrificed, with virtually no ascites present. This is typically done approximately three times.

[0090] 4. Ascites Purification: Purify using the octanoic acid-ammonium sulfate method. Centrifuge the ascites (12,000 rpm for 30 minutes), collect the supernatant, and discard the precipitate. Accurately quantify the ascites volume. Add 2 times the original volume of the ascites to the sample with a 50 mM citric acid solution (pH 4.0) to adjust the pH after mixing to 4.6-4.8. Add octanoic acid dropwise (60 μL / mL ascites, calculated based on the initial ascites volume), stirring to ensure uniform mixing and complete precipitation. After standing at room temperature for 0.5 hours, centrifuge the mixture (50 mL centrifuge tube: 10,000 rpm for 15 minutes; 2 mL centrifuge tube: 10,000 rpm for 5 minutes). Carefully collect the supernatant, discard the precipitate, and record the supernatant volume. Add 2 M Tris (pH 9.0) to a volume equal to 0.03 times the original supernatant volume to adjust the pH to neutral. Slowly add ammonium sulfate powder at 0.277g / mL, stirring while adding to fully dissolve the ammonium sulfate. Let it stand at room temperature for 0.5 hours until the precipitation is complete. Centrifuge the mixed solution (50mL centrifuge tube: 10000rpm centrifugation for 15min; 2mL centrifuge tube: 10000rpm centrifugation for 5min), discard the supernatant, and collect the precipitate. The precipitate was fully dissolved with PBS with 0.25 times the original ascites volume, dialyzed with PBS, and the solution was changed 2 to 3 times. The dialysate was collected, centrifuged at 12000r / min for 15min, and the supernatant was taken to obtain the monoclonal antibody against HPV11 VLP. Samples were taken for SDS-PAGE and BCA method to measure the antibody concentration. The results showed that the purity of the purified monoclonal antibodies reached more than 90%, see the attached for details. Figure 1 .

[0091] Example 3: Specificity Identification of Various Types of Monoclonal Antibodies (Cross-Test)

[0092] The prepared HPV11 VLP and other 8 types of HPV (HPV6, 16, 18, 31, 33, 45, 52, 58) VLP mixtures were added to 96-well ELISA plates at 0.5 μg / mL, 100 μL / well, and coated overnight at 4°C. Duplicate wells and negative control wells were also set up. Block at 37°C for 2 hours. Dilute each antibody sample 10% with sample diluent. -3 ~10 -6Add 100 μL / well of the solution to the ELISA plate, cover with film, and incubate in a 37°C incubator for 45 minutes. Remove the plate from the incubator, discard all liquid, add 300 μL / well of wash solution, and wash the plate five times. Discard all liquid in the wells for the final time and pat dry on tissue paper. Add 100 μL / well of a goat anti-mouse enzyme-linked secondary antibody solution at a working concentration of 1:4000, cover with film, and incubate in a 37°C incubator for 45 minutes. Remove the plate from the 37°C incubator, discard all liquid in the wells, add 300 μL / well of wash solution, and wash the plate ten times. Discard all liquid in the wells and pat dry on tissue paper. Mix the required volume of chromogenic substrate solution A and chromogenic substrate solution B in a 1:1 ratio, add 100 μL / well of the plate, and develop at room temperature in the dark for 10 minutes. Finally, add the required volume of stop solution to the plate at 50 μL / well. Place the microplate in a microplate reader and measure the OD value at 450nm. Samples with an OD value greater than 0.5 are considered positive, while samples with an OD value less than or equal to 0.5 are considered negative. This allows for cross-binding analysis of each monoclonal antibody with HPV11 and the other eight HPV types, allowing selection of antibodies that specifically bind to HPV11 for further testing.

[0093] Table 1 shows the results of cross-binding experiments for each monoclonal antibody against a mixture of HPV11 and eight other HPV types. The results show that among the 23 monoclonal antibodies prepared, two bind to HPV11 and show no cross-binding activity with the other eight HPV types, 20 show cross-binding activity, and one shows negative recognition of HPV11. Comprehensive consideration revealed that the monoclonal antibodies 23F11 and 24C7 prepared in the present invention exhibit excellent specificity for HPV11 recognition, show no cross-binding activity with the other eight HPV types, and exhibit good binding to HPV11 across a dilution gradient, making them suitable for further research in the present invention.

[0094] Table 1 Specificity identification results of various types of monoclonal antibodies

[0095]

[0096] *All values are the average of duplicate wells

[0097] Example 4: Paired Identification Test of Monoclonal Antibodies 23F11 and 24C7

[0098] HPV11 VLP was coated on a 96-well plate, the antibody 23F11 selected in Example 3 was used as the coating antibody, and the enzyme-labeled antibody 24C7-HRP was used as the detection antibody. The ELISA experiment was performed. The test results are shown in Table 2, and the standard curve is shown in the attached figure. Figure 2As shown, the results show that 23F11 and 24C7 have no competitive relationship with the antigen binding epitope, indicating that 23F11 and 24C7 do not have the same antigen binding site and can form the antibody pair of the coating antibody and the detection antibody of the present invention.

[0099] Table 2 Pairing identification results of monoclonal antibodies 23F11 and 24C7

[0100]

[0101] Example 5: ELISA to detect the reactivity of antibodies with HPV11 VLPs

[0102] The indirect ELISA method is used to verify whether the purified monoclonal antibody has good reactivity with HPV11 VLPs and whether it can be detected quickly and conveniently. At the same time, measuring the reactivity of different concentrations of monoclonal purified antibodies with HPV11 VLPs can also preliminarily reflect the affinity of the antibody.

[0103] HPV11 VLPs were coated onto 96-well plates at 100 μL / well. Antibodies were then added to each well at final concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL. Three replicates were run for each concentration, along with a negative control. ELISA was used to determine the binding strength of monoclonal antibodies 23F11 and 24C7 to HPV11 VLPs. The results showed that both monoclonal antibodies 23F11 and 24C7 bound to HPV11 VLPs within the concentration range used in this experiment, with signal intensities greater than 1. The results are shown in Table 3.

[0104] Table 3 ELISA detection of antibody binding strength to HPV11 VLP

[0105] Antibody 1 μg / mL 0.5 μg / mL 0.25 μg / mL 0.125 μg / mL 0.0625 μg / 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

[0106] *All values are the average of duplicate wells

[0107] Example 6: Neutralizing activity detection of antibodies

[0108] The neutralizing activity of each antibody strain was tested using a pseudovirus-cell neutralization model. Antibodies 23F11 and 24C7 were first diluted 40-, 80-, 160-, 320-, 640-, 1280-, and 2560-fold in complete culture medium. Equal volumes of the supernatant and pseudovirus were then added to a 96-well plate, mixed on a microplate shaker, and incubated at 4°C for 1 hour. The cells were then transferred to a 37°C cell culture incubator and incubated for 10 minutes. 100 μL of the mixture was then added to a 96-well plate pre-seeded with 293FT cells and incubated in a cell culture incubator for 72 hours. The entire 96-well plate was then scanned and counted using an ELISpot analyzer. The pseudovirus contains a GFP reporter gene and appears green under a fluorescence microscope. Neutralizing antibodies can prevent pseudovirus infection and prevent cell coloration.

[0109] The results are shown in Table 4. The results indicate that, except for 99% inhibition rate of 23F11 when diluted 40, 160, and 2560 times; 99% inhibition rate of 24C7 when diluted 160 and 320 times, 98% inhibition rate when diluted 1280 times, and 97% inhibition rate when diluted 2560 times, the inhibition rates of the two antibodies at other dilution gradients were all 100%, indicating that both monoclonal antibodies 23F11 and 24C7 have neutralizing activity.

[0110] Table 4 Inhibition rate of antibodies 23F11 and 24C7 against HPV11

[0111] HPV11 23F11 24C7 40 99％ 100％ 80 100％ 100％ 160 99％ 99％ 320 100％ 99％ 640 100％ 100％ 1280 100％ 98％ 2560 99％ 97％

[0112] Example 7: Specificity Identification of Antibodies 23F11 and 24C7

[0113] Add the prepared standards and test samples (HPV11 and other 8 types of HPV (HPV6, 16, 18, 31, 33, 45, 52, 58) VLPs) to the coated plate at 100 μL / well, coat overnight at 4°C, and set up duplicate wells and negative control wells. Block at 37°C for 2 hours. Add the antibody 23F11 to the ELISA plate at 100 μL / well, cover with a sealing film and incubate in a 37°C incubator for 45 minutes. Remove the ELISA plate from the incubator, discard the liquid in the well, add 300 μL / well of washing solution, and wash the plate 5 times. After discarding the liquid in the well for the last time, pat dry on toilet paper. Add 100 μL / well of the prepared enzyme-labeled antibody 24C7-HRP solution, cover with a cover film and incubate in a 37°C incubator for 45 minutes. Remove the ELISA plate from the 37°C incubator, discard the liquid in the wells, add 300 μL / well of wash solution, and wash the plate 10 times. Discard the liquid in the wells for the final time and pat dry on toilet paper. Mix the required volume of chromogenic substrate solution A and chromogenic substrate solution B in a 1:1 ratio, add 100 μL / well to the ELISA plate, and develop at room temperature in the dark for 5 minutes. Finally, add the required volume of stop solution to the plate at 50 μL / well. Place the ELISA plate in a microplate reader and measure the OD value at 450 nm. Samples with an OD value greater than 0.5 are considered positive. This is used to evaluate the specificity of antibodies 23F11 and 24C7 for HPV11 binding.

[0114] The specificity test results of the antibodies are shown in Table 5, and the standard curve is shown in the attached Figure 3 The results showed that the prepared monoclonal antibody pairs 23F11 and 24C7 had recognition values greater than 0.5 for HPV11 VLPs, indicating a clear positive result. The values for the other eight HPV VLPs were all less than 0.5, indicating a negative result. This demonstrates that the monoclonal antibody pairs 23F11 and 24C7 prepared by the present invention have good specificity for recognizing HPV11 VLPs.

[0115] Table 5 Specificity of Antibodies by ELISA (OD450)*

[0116]

[0117] *All values are the average of duplicate wells

[0118] Example 8. Affinity Identification of Antibodies 23F11 and 24C7

[0119] The affinity of antibodies 23F11 and 24C7 was identified by analyzing the kinetics of antigen-antibody binding and dissociation. The association rate constant Kon, dissociation rate constant Koff and dissociation constant Kd of monoclonal antibodies 23F11 and 24C7 with HPV11 VLP were calculated using the GE BIAcore 3000 biomacromolecule interaction analyzer to reflect the strength of the binding between the antibody and HPV11 VLP.

[0120] HPV11 VLP was diluted to 40 μg / mL with 10 mM sodium acetate buffer at pH 4.5 and coupled to the chip surface according to the manufacturer's instructions. The antibody was diluted to an appropriate concentration with PBS buffer. During the detection, the antibody was first injected for 60 seconds, then bound for 60 seconds, dissociated for 500 seconds, and finally the chip was regenerated with 10 mM sodium acetate buffer at pH 5.0. The data were subjected to kinetic analysis of antigen-antibody binding using BIAcore 3000 Evaluation software according to the instructions. The affinity test results of antibodies 23F11 and 24C7 with HPV11 VLP are shown in Table 6. The results show that 23F11 and 24C7 antibodies are specific antibodies that only bind to HPV11 VLP and do not bind to the other 8 types of HPV VLP.

[0121] Table 6 Affinity identification of antibodies 23F11 and 24C7

[0122]

[0123] Example 9: Subtype Identification of Clone 23F11 and Clone 24C7

[0124] The IgG subtypes of monoclonal antibodies 23F11 and 24C7 were identified using indirect ELISA using antibodies raised against various mouse IgG subtypes. The results showed that the subtype of monoclonal antibody 23F11 was IgG 2b, and the subtype of monoclonal antibody 24C7 was IgG1. The results are shown in Table 7. (An OD450 greater than 1 indicates a confirmed subtype.)

[0125] Table 7 ELISA identification of antibody subtypes (OD450 values*)

[0126]

[0127] *All values are the average of duplicate wells

[0128] Example 10: Determination of variable region sequences of clone 23F11 and clone 24C7

[0129] Before sequencing the variable regions of cells, the cell supernatants of the two cell lines were used for subtype detection. After confirming that they were of a single subtype, the frozen cells in the logarithmic growth phase were taken, the cells were revived by centrifugation and collected, and 5×106 After adding 1 mL of Total RNA Extractor (Trizol) to each cell, the variable region sequencing was commissioned to Nanjing GenScript Sequencing Unit.

[0130] See the sequence listing for the sequence.

[0131] Using the above-identified sequences, various genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, single-chain antibodies, and diabodies, can be prepared through known antibody engineering techniques, while retaining the biological properties of the monoclonal antibodies from which they are derived.

[0132] Example 11: Assembly of HPV11 Detection Kit

[0133] 1. Coating: Dilute the antibody 23F11 to 2 μg / mL with 1X PBS coating solution, add 100 μL / well to the ELISA plate, and place in a 4°C refrigerator for 18-24 hours;

[0134] 2. Wash the plate: Take out the ELISA plate from the refrigerator and wash it twice using a plate washer with 1X wash buffer, 250 μL / well;

[0135] 3. Blocking: Wash twice with 1X washing solution, pat dry, block overnight at 150μL / well at 4℃ for 18-24 hours;

[0136] 4. Drying: After sealing for 18-24 hours, shake off the liquid in the plate, pat dry, and place in a drying room (make sure the humidity in the drying room is below 30%, the temperature is 30℃±37℃, and record the temperature and humidity at any time during the drying process) until the plate is completely dry, which should take at least 4.5 hours.

[0137] 5. Sealing the plate: Place the dried ELISA plate in an aluminum foil bag with a label (the label must contain the name, ELISA plate production batch number, and expiration date), and a bag of desiccant. Seal the plate with a sealing machine and store it in a refrigerator at 4°C. The shelf life is 1 year.

[0138] 6. Label 24C7 with horseradish peroxidase to obtain 24C7-HRP and store at -20℃.

[0139] The monoclonal antibody 23F11 with specificity and no cross-reactivity was used as the coating antibody, the monoclonal antibody 24C7 with neutralizing activity was labeled with horseradish peroxidase and used as the detection antibody. The recombinant human papillomavirus-11 VLP protein was used as the standard to prepare the standard curve control.

[0140] Example 12: Linearity and Repeatability Testing of HPV11 Detection Kit

[0141] The ELISA double antibody sandwich method was used, with 23F11 as the coating antibody and 24C7-HRP as the detection antibody. The ELISA detection method was determined. The linear range of the kit is shown in Table 8, and the standard curve is shown in the attached figure. Figure 4 As shown, the linear range of the kit of the present invention is 0.25-0.0039 μg / mL.

[0142] Table 8 Linear range detection of the kit

[0143]

[0144]

[0145] The antigen was diluted in the order of 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.0156 μg / mL, 0.0078125 μg / mL and 0.00390625 μg / mL, and the ELISA experimental procedure was repeated. The results are shown in Table 9 and the standard curve is shown in the attached figure. Figure 4 As shown in Figure B, the accuracy of the results fell between 80% and 120%, indicating that the double-antibody sandwich ELISA antigen detection method established in the present invention has good repeatability.

[0146] Table 9 Kit Repeatability Test

[0147]

[0148] Example 13, HPV11 Detection Kit Assay Procedure

[0149] 1. Prepare samples

[0150] 1) Preparation of washing solution: Take out 50 mL of 20-fold concentrated washing solution, add distilled water, dilute to 1 L and mix thoroughly for later use;

[0151] 2) Sample diluent;

[0152] 3) Enzyme-labeled antibody diluent;

[0153] 4) Dilute the standard with sample diluent, set up a dilution gradient, and set aside;

[0154] 5) Dilute the enzyme-labeled antibody with enzyme-labeled antibody diluent, take an appropriate amount of enzyme-labeled antibody and dilute it 10,000 times, and set aside.

[0155] 2. Measurement steps

[0156] 1) Sample addition: Add the prepared standard and test sample to the coated plate at 100 μL / well, cover with sealing film, and incubate in a 37°C incubator for 45 minutes;

[0157] 2) Add enzyme-labeled antibody: Remove the plate from the incubator, discard the liquid in the wells, add 300 μL / well of wash solution, and wash the plate five times, soaking for 30 seconds each time. After the final wash, discard the liquid in the wells and pat dry on absorbent paper. Add 100 μL / well of the prepared enzyme-labeled antibody solution, cover with a cover film, and incubate in a 37°C incubator for 45 minutes.

[0158] 3) Color development: Remove the ELISA plate from the 37°C incubator, discard the liquid in the wells, add 300 μL / well of wash solution, and wash the plate five times, soaking for 30 seconds each time. Discard the liquid in the wells for the final time and pat dry on absorbent paper. Mix the required volume of chromogenic substrate solution A and chromogenic substrate solution B in a 1:1 ratio, add 100 μL / well to the ELISA plate, and incubate in a 37°C incubator protected from light for 10 minutes.

[0159] 4) Stop the reaction: Add the required volume of stop solution to the ELISA plate, 50 μL / well;

[0160] 5) Measurement: Place the microplate into a microplate reader, set 630 nm as the reference wavelength, measure the absorbance at 450 nm, and record the measurement results;

[0161] 6) Result determination:

[0162] ① Four-parameter fitting curve R of the standard 2 ≥0.9801;

[0163] ②The linear range of the method is 0.25-0.0039 μg / mL, and the detection limit is 0.0039 μg / mL. The test results above 0.0039 μg / mL are judged as positive, and the OD value of the negative control is ≤0.100;

[0164] ③ The test results of the different concentration ranges of the test sample must all be within the linear range of the standard curve. Points above or below the linear range are not meaningful. The final quantitative result of the test sample is the average of the points within the multiple concentration ranges.

[0165] ④ The above three points must be met simultaneously, unless otherwise specified. Sequence Listing <110> Jiangsu Ruike Biotechnology Co., Ltd.; Beijing Anbaisheng Biotechnology Co., Ltd. <120> Antibody to human papillomavirus type 11 L1 protein and preparation method thereof <130> 2021 <150> 202110189825.2 <151> 2021-02-18 <160> 64 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <400> 1 Asn Tyr Leu Ile Glu 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <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 <213> Artificial sequence <400> 3 Pro His Tyr Tyr Gly Asn Ile Tyr Ala Met Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <400> 4 Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <400> 5 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <400> 6 Gln Gln Ser Asn Ser Trp Pro Trp Thr 1 5 <210> 7 <211> 30 <212> PRT <213> Artificial sequence <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr 20 25 30 <210> 8 <211> 14 <212> PRT <213> Artificial sequence <400> 8 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 9 <211> 32 <212> PRT <213> Artificial sequence <400> 9 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 20 25 30 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <400> 10 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 11 <211> twenty three <212> PRT <213> Artificial sequence <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 <212> PRT <213> Artificial sequence <400> 12 Trp Tyr Gln Leu Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys 1 5 10 15 <210> 13 <211> 32 <212> PRT <213> Artificial sequence <400> 13 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 <210> 14 <211> 10 <212> PRT <213> Artificial sequence <400> 14 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 15 <211> 140 <212> PRT <213> Artificial sequence <400> 15 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 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 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 130 135 140 <210> 16 <211> 127 <212> PRT <213> Artificial sequence <400> 16 Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 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 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Ser Trp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 <210> 17 <211> 15 <212> DNA <213> Artificial sequence <400> 17 aattacttga tagag 15 <210> 18 <211> 51 <212> DNA <213> Artificial sequence <400> 18 gtgattaatc ctggaaatgg aggtattaat tatagtgaga agttcaaggg c 51 <210> 19 <211> 36 <212> DNA <213> Artificial sequence <400> 19 ccccattact acggtaatat ctatgctatg gactac 36 <210> 20 <211> 33 <212> DNA <213> Artificial sequence <400> 20 agggccagcc aaagtattag caacaaccta cac 33 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <400> twenty one tatgcttccc agtccatctc t 21 <210> twenty two <211> 27 <212> DNA <213> Artificial sequence <400> twenty two caacagagta acagctggcc gtggacg 27 <210> twenty three <211> 90 <212> DNA <213> Artificial sequence <400> twenty three caggtccagt tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact 90 <210> twenty four <211> 42 <212> DNA <213> Artificial sequence <400> twenty four tgggtaaagc agaggcctgg acagggcctt gagtggattg gg 42 <210> 25 <211> 96 <212> DNA <213> Artificial sequence <400> 25 aaggcaacac tgactgcaga caaatcctcc agcactgcct acatgcagct cagcagcctg 60 acatctgatg actctgcggt ctatttctgt gcaagc 96 <210> 26 <211> 33 <212> DNA <213> Artificial sequence <400> 26 tggggtcaag gaacctcagt caccgtctcc tca 33 <210> 27 <211> 69 <212> DNA <213> Artificial sequence <400> 27 gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 60 ctttcctgc 69 <210> 28 <211> 45 <212> DNA <213> Artificial sequence <400> 28 tggtatcaac taaaatcaca tgagtctcca aggcttctca tcaag 45 <210> 29 <211> 96 <212> DNA <213> Artificial sequence <400> 29 gggatcccct ccaggttcag tggcagtgga tcagggacag atttcactct cagtatcaat 60 agtgtggaga ctgaagattt tggaatgtat ttctgt 96 <210> 30 <211> 30 <212> DNA <213> Artificial sequence <400> 30 ttcggtggag gcaccaagct ggaaatcaaa 30 <210> 31 <211> 420 <212> DNA <213> Artificial sequence <400> 31 atggaatgga gcagagtctt tatctttctc ctatcagtaa ctgcaggtgt tcactcccag 60 gtccagttgc agcagtctgg agctgagctg gtaaggcctg ggacttcagt gaaggtgtcc 120 tgcaaggctt ctggatacgc cttcactaat tacttgatag agtgggtaaa gcagaggcct 180 ggacagggcc ttgagtggat tggggtgatt aatcctggaa atggaggtat taattatagt 240 gagaagttca agggcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg 300 cagctcagca gcctgacatc tgatgactct gcggtctatt tctgtgcaag cccccattac 360 tacggtaata tctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 420 <210> 32 <211> 381 <212> DNA <213> Artificial sequence <400> 32 atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60 gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120 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 ggcaccaagc tggaaatcaa a 381 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <400> 33 Ser Asn Trp Leu His 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <400> 34 Ala Ile Tyr Pro Gly Ser Gly Asp Ser Thr Tyr Thr Gln Lys Phe Lys 1 5 10 15 Asp <210> 35 <211> 10 <212> PRT <213> Artificial sequence <400> 35 Glu Tyr Asp Lys Asp Trp Tyr Phe Asp Val 1 5 10 <210> 36 <211> 16 <212> PRT <213> Artificial sequence <400> 36 Arg Ser Ser Arg Ser Ile Val Gln Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <400> 37 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <400> 38 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 39 <211> 30 <212> PRT <213> Artificial sequence <400> 39 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 <213> Artificial sequence (artificial sequence) <400> 40 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 41 <211> 32 <212> PRT <213> Artificial sequence <400> 41 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 20 25 30 <210> 42 <211> 11 <212> PRT <213> Artificial sequence <400> 42 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 43 <211> twenty three <212> PRT <213> Artificial sequence <400> 43 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 <211> 15 <212> PRT <213> Artificial sequence <400> 44 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 45 <211> 32 <212> PRT <213> Artificial sequence <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 <213> Artificial sequence <400> 46 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 47 <211> 138 <212> PRT <213> Artificial sequence <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 <213> Artificial Sequence <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 Light 130 <210> 49 <211> 15 <212> DNA <213> artificial sequence <400> 49 agcaactggc tgcac 15 <210> 50 <211> 51 <212> DNA <213> Artificial sequence <400> 50 gcgatttatc ctggaagtgg tgatagtact tacacccaga agttcaagga c 51 <210> 51 <211> 30 <212> DNA <213> Artificial sequence <400> 51 gaatacgaca aggactggta tttcgatgtc 30 <210> 52 <211> 48 <212> DNA <213> Artificial sequence <400> 52 agatctagtc ggagcattgt acaaagtaat ggaaacacct atttagag 48 <210> 53 <211> twenty one <212> DNA <213> Artificial sequence <400> 53 aaagtttcca accgattttc t 21 <210> 54 <211> 27 <212> DNA <213> Artificial sequence <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 ggacccacggt 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 that binds to the L1 protein and / or VLP of human papillomavirus HPV11, characterized in that: The amino acid sequences of CDRH1, CDRH2 and CDRH3 in the CDR of the antibody heavy chain variable region are shown in SEQ ID NOs. 1-3, and the amino acid sequences of CDRL1, CDRL2 and CDRL3 in the CDR of the antibody light chain variable region are shown in SEQ ID NOs. 4-6.

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

3. The antibody according to claim 2, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

4. The antibody according to claim 3, wherein The antibody is a monoclonal antibody produced by a hybridoma cell line obtained by fusion of immunized host spleen cells and myeloma cells. The deposit number of the hybridoma cell is CGMCC No. 21984.

5. A kit for detecting L1 protein and / or VLP of HPV11, characterized in that: The kit comprises the antibody according to any one of claims 1 to 4.

6. The kit according to claim 5, wherein The kit further comprises a second antibody that binds to the L1 protein and / or VLP of HPV11.

7. The kit according to claim 6, wherein The amino acid sequences of CDRH1, CDRH2 and CDRH3 in the CDR of the heavy chain variable region of the second antibody are shown in SEQ ID NOs. 33-35, and the amino acid sequences of CDRL1, CDRL2 and CDRL3 in the CDR of the light chain variable region of the second antibody are shown in SEQ ID NOs. 36-38.

8. The kit according to claim 7, wherein The amino acid sequences of FR1, FR2, FR3 and FR4 in the FR of the heavy chain variable region of the second antibody are shown in SEQ ID NOs. 39-42, and the amino acid sequences of FR1, FR2, FR3 and FR4 in the FR of the light chain variable region of the second antibody are shown in SEQ ID NOs. 43-46.

9. The kit according to claim 8, wherein The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.47, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

48.

10. The kit according to claim 9, wherein The second antibody is a monoclonal antibody produced by a hybridoma cell line obtained by fusion of immunized host spleen cells and myeloma cells. The deposit number of the hybridoma cell is CGMCC No. 21985.

11. The kit according to any one of claims 5 to 10, further comprising a detectable label: a radioisotope, a luminescent substance, a colored substance and / or an enzyme.

12. The kit according to any one of claims 5 to 10, wherein The kit is an ELISA kit, comprising a capture antibody and a detection antibody, wherein the capture antibody is the antibody according to any one of claims 1 to 4, and the detection antibody is the second antibody according to any one of claims 6 to 10.

13. Use of the antibody according to any one of claims 1 to 4, or the kit according to any one of claims 5 to 10, in the preparation of a reagent for detecting HPV11 L1 protein and / or VLP.

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