Monoclonal antibody for resisting HPV59 L1 protein as well as preparation method and application of monoclonal antibody

Abstract

The invention provides an anti-HPV59L1 protein monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of biology. The monoclonal antibodies 13E1 and 23E11 provided by the invention can be specifically combined with the HPV59 subtype, have the characteristics of neutralizing the HPV59 pseudovirus and blocking the infection of the HPV59 pseudovirus, and can be used for detecting the HPV59 antigen. Wherein the epitope recognized by 13E1 is the epitope targeted by a dominant neutralizing antibody in serum after vaccine immunization, so that the epitope not only can be used for antigen detection, but also can be directly used for evaluating the neutralizing antibody generation capability induced by the HPV vaccine. The invention further provides a double-antibody sandwich ELISA kit, the kit can specifically recognize and quantitatively detect the HPV59LI protein which is complete in conformation and has immunogenicity in a sample, the kit can be stably produced in batch, rapid evaluation of the in-vitro activity of the HPV vaccine can be achieved, and the kit has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against HPV59 L1 protein, its preparation method, and its uses. Background Technology

[0002] Human papillomavirus (HPV) is a common epitheliotropic DNA virus that causes various malignant tumors, including cervical cancer, anal cancer, and oropharyngeal cancer, through persistent infection of human skin and mucous membranes. Based on the virus's carcinogenic risk, the World Health Organization (WHO) classifies HPV into high-risk and low-risk types. HPV type 59 is classified as a high-risk carcinogenic type and is closely related to the development of cervical squamous cell carcinoma and adenocarcinoma. Epidemiological surveys show that the infection rate of HPV type 59 in cervical cancer cases among women in my country is approximately 2.6%. Therefore, prevention of high-risk HPV types such as HPV 59 is crucial, and HPV vaccination is the most effective means of preventing infection with this type of virus.

[0003] Currently, all marketed and investigational HPV vaccines use recombinantly expressed HPV L1 protein as the antigen. Under specific conditions, the HPV L1 protein can self-assemble into virus-like particles (VLPs), whose structure is highly similar to that of natural HPV virus particles and retains most of the key neutralizing epitopes. Based on this, HPV vaccination can induce the body to produce protective neutralizing antibodies targeting HPV VLPs. These neutralizing antibodies specifically bind to key epitopes of the HPV L1 protein, blocking the binding sites of the virus and cell surface receptors, thereby blocking viral infection and establishing effective immune protection. Therefore, detecting the activity of neutralizing antibodies induced by the vaccine is an important standard for evaluating the immunoprotective efficacy of HPV vaccines.

[0004] The World Health Organization (WHO), in its "Guiding Principles for the Evaluation of the Quality, Safety and Efficacy of HPV VLP Vaccines," states that neutralization testing is the "gold standard" for evaluating whether HPV vaccine-induced antibodies have a protective effect. However, this method relies on complex in vivo animal experiments, and is characterized by long testing cycles, high costs, and high variability, making it difficult to meet the needs of rapid antigen quality evaluation during large-scale vaccine production. Given that the immunogenicity of HPV vaccines fundamentally depends on the correct conformation of VLPs and their key neutralizing epitopes, developing tool antibodies that specifically recognize these epitopes and establishing efficient in vitro detection methods based on this has significant application value and prospects. This strategy aims to quantitatively detect intact and correctly conformed VLPs in the vaccine through the specific binding of neutralizing monoclonal antibodies to VLPs, achieving in vitro determination of vaccine immunogenicity and active antigen content, and thus indirectly and rapidly predicting its ability to induce a protective immune response.

[0005] In summary, further research and development of more types of HPV59-specific monoclonal antibodies targeting different epitopes will not only deepen our understanding of the biological characteristics of HPV, but will also provide crucial tools for the development and quality control of multivalent HPV vaccines. Furthermore, these types of specific antibodies also have broad translational application prospects in the diagnosis, prevention, and treatment of HPV. Summary of the Invention

[0006] The purpose of this invention is to provide a monoclonal antibody against HPV59 L1 protein, its preparation method, and its uses.

[0007] This invention provides a monoclonal antibody against HPV59 L1 protein. The monoclonal antibody is composed of a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.5, the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO.6, the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.11, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.12.

[0008] Preferably, the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.4, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO.10.

[0009] The present invention also provides a gene fragment encoding the aforementioned monoclonal antibody, comprising a gene fragment encoding a heavy chain with a nucleotide sequence as shown in SEQ ID NO.1, and a gene fragment encoding a light chain with a nucleotide sequence as shown in SEQ ID NO.7.

[0010] The present invention also provides a hybridoma cell line 13E1 for producing the aforementioned monoclonal antibody. The hybridoma cell line 13E1 was deposited on November 7, 2025 at the China Center for Type Culture Collection (CCTCC) located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: C2025318.

[0011] The present invention also provides a monoclonal antibody against HPV59 L1 protein, the monoclonal antibody being composed of a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.17, the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO.18, the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.23, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.24.

[0012] Preferably, the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.16, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO.22.

[0013] The present invention also provides a gene fragment encoding the aforementioned monoclonal antibody, comprising a gene fragment encoding a heavy chain with a nucleotide sequence as shown in SEQ ID NO.13, and a gene fragment encoding a light chain with a nucleotide sequence as shown in SEQ ID NO.19.

[0014] The present invention also provides a hybridoma cell line 23E11 for producing the aforementioned monoclonal antibody. The hybridoma cell line 23E11 was deposited on November 7, 2025 at the China Center for Type Culture Collection (CCTCC) located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: C2025319.

[0015] The present invention also provides a method for preparing the aforementioned monoclonal antibody, comprising the following steps:

[0016] (1) The aforementioned hybridoma cells were injected into the peritoneal cavity of mice that had been pre-injected with IFA adjuvant, and the ascites was aspirated.

[0017] (2) Centrifuge the ascites obtained in step (1), collect the supernatant and purify it with a Protein G affinity chromatography column to obtain the final product.

[0018] The present invention also provides the use of monoclonal antibodies with the full-length heavy chain amino acid sequence shown in SEQ ID NO.4 and the full-length light chain amino acid sequence shown in SEQ ID NO.10 and / or monoclonal antibodies with the full-length heavy chain amino acid sequence shown in SEQ ID NO.16 and the full-length light chain amino acid sequence shown in SEQ ID NO.22 in the preparation of kits for detecting HPV59 antigen.

[0019] The present invention also provides the use of monoclonal antibodies with the full-length heavy chain amino acid sequence shown in SEQ ID NO.4 and the full-length light chain amino acid sequence shown in SEQ ID NO.10, and monoclonal antibodies with the full-length heavy chain amino acid sequence shown in SEQ ID NO.16 and the full-length light chain amino acid sequence shown in SEQ ID NO.22 in the preparation of a kit for detecting the in vitro activity of HPV vaccines.

[0020] The present invention also provides a kit for detecting the in vitro activity of HPV vaccines, the kit comprising a monoclonal antibody with a heavy chain full-length amino acid sequence as shown in SEQ ID NO.4 and a light chain full-length amino acid sequence as shown in SEQ ID NO.10, and a monoclonal antibody with a heavy chain full-length amino acid sequence as shown in SEQ ID NO.16 and a light chain full-length amino acid sequence as shown in SEQ ID NO.22.

[0021] Preferably, the monoclonal antibody with the full-length amino acid sequence of the heavy chain as shown in SEQ ID NO.4 and the full-length amino acid sequence of the light chain as shown in SEQ ID NO.10 is an enzyme-labeled antibody; the monoclonal antibody with the full-length amino acid sequence of the heavy chain as shown in SEQ ID NO.16 and the full-length amino acid sequence of the light chain as shown in SEQ ID NO.22 is a coating antibody.

[0022] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0023] This invention provides monoclonal antibodies 13E1 and 23E11 against HPV59 L1 protein. These two antibodies specifically bind to HPV subtype 59 (without cross-reactivity with most other HPV subtypes such as HPV6, 11, 16, 18, 31, 33, 45, 52, 58, and 68), exhibiting high specificity and neutralizing activity, and can be used to detect HPV59 antigen. Monoclonal antibody 13E1 recognizes the epitope targeted by the dominant neutralizing antibody in serum after vaccine immunization; therefore, it can be used not only for antigen detection but also directly for evaluating the ability of HPV vaccines to induce neutralizing antibody production. Based on the above antibodies, this invention also provides a double-antibody sandwich ELISA kit. This kit can specifically identify and quantify conformationally intact and immunogenic HPV59 L1 protein in samples. The kit can be stably mass-produced and enables rapid in vitro evaluation of HPV vaccine activity, showing promising application prospects.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1The image shows the SDS-PAGE results of monoclonal antibodies 13E1 and 23E11. The loading amount for both was 10 μg. The samples in each lane were: 1, marker; 6, HPV59-13E1 reduced; 7, HPV59-13E1 non-reduced; 8, HPV59-23E11 reduced; 9, HPV59-23E11 non-reduced; 10, antibody standard 4G1 non-reduced. Reduction refers to loading buffer with added reducing agent, heated at 95°C for 10 min, followed by gel running. Non-reduction refers to loading buffer without added reducing agent, mixed thoroughly, and then directly run on the gel.

[0027] Figure 2 The figure shows the results of the inhibition of the binding reaction of 13E1 and 23E11 antibodies in rat serum after immunization.

[0028] Figure 3 The titers of 13E1 and 23E11 antibodies in the serum of immunized rats (BID) 50 ) and serum total neutralizing antibody titer (ID) 50 The correlation analysis results are shown in the figure.

[0029] Figure 4 This is a graph showing the comparison of binding activity of the kit to conformationally intact HPV59 VLPs and those denatured by DTT. Detailed Implementation

[0030] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0031] The experiments involved in the specific implementation were supported by the "Juyuan Xingchuan" Human Papillomavirus (HPV) Vaccine Industrialization Project 2024ZHCG0187 of the Sichuan Provincial Department of Science and Technology.

[0032] 1. Main reagents

[0033] Table 1. Main Reagents

[0034]

[0035]

[0036] 2. Main Instruments

[0037] Table 2. Main Instruments

[0038]

[0039] 3. Laboratory animals, cell lines, and pseudoviruses

[0040] Laboratory animals: BALB / c mice: SPF (Specific Pathogen Free) grade BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., female, 5 mice, aged 35-41 days.

[0041] Cell lines: SP2 / 0 myeloma cells (from Beijing Tiancheng Biotechnology Co., Ltd.), and 293FT cells (from Sinopharm Biotechnology Research Institute Co., Ltd.).

[0042] Pseudovirus: HPV59 pseudovirus, batch number: HPV59G-20200424, Chengdu Institute of Biological Products Co., Ltd.

[0043] Example 1: Preparation and Screening of Monoclonal Antibodies

[0044] 1. Preparation of immunogens

[0045] The immunogen used to prepare the monoclonal antibody against HPV59 L1 protein was HPV59 L1-VLP protein with a protein concentration of 174 μg / mL. Immunogen A was prepared by mixing the above HPV59 L1-VLP protein with an equal volume of Freund's complete adjuvant (CFA) and then ultrasonically emulsifying the mixture. Immunogen B was prepared by mixing the above HPV59 L1-VLP protein with an equal volume of AD11.15 adjuvant.

[0046] 2. Mouse immunization

[0047] Five BALB / c mice were immunized using the following schedule: On day 0, the primary immunization was performed by subcutaneous injection of immunogen A (0.17 mL / 14.8 μg / mouse) at multiple sites on the back, and intramuscular injection of immunogen B (0.2 mL / 17.4 μg / mouse) at multiple sites on the leg. On day 3 post-primary immunization, a first booster immunization was performed by intramuscular injection of immunogen B (0.2 mL / 17.4 μg / mouse) at multiple sites on the leg. On day 7 post-primary immunization, a second booster immunization was performed using immunogen B (0.2 mL / 3.66 μg / mouse, further diluted in a 1:1 mixture of AD11.15 adjuvant and water). The animals were given a third booster immunization on day 10 after the initial immunization, with immunogen A (0.17 mL / 14.8 μg / animal) administered subcutaneously at multiple points on the back and immunogen B (0.2 mL / 3.66 μg / animal, further diluted in a 1:1 mixture of AD11.15 adjuvant and water) administered subcutaneously at multiple points on the leg. On day 12 after the initial immunization, a fourth booster immunization was given, with immunogen B (0.2 mL / 3.66 μg / animal, further diluted in a 1:1 mixture of AD11.15 adjuvant and water) administered subcutaneously at multiple points on the leg. Subcutaneous injection sites on the back included: two points on the neck, two points on the waist, and two points on the abdomen. Intramuscular injection sites on the legs included: two points in the forelimb muscles and two points in the hindlimb muscles.

[0048] 3. Indirect ELISA screening

[0049] Use HPV59 L1-VLP to coat the microplate, 100 ng / well, and react overnight at 4°C; discard the liquid in the plate, wash the plate 3 times with PBS solution, add 200 μL / well of PBS solution containing 5% skim milk powder, block at room temperature for 1 hour, and wash the plate once with PBS solution. Add mouse tail vein blood or hybridoma cell culture supernatant or purified antibody (mouse tail vein blood serially diluted from 1:500 to 1:50000; hybridoma cell culture supernatant diluted 1:1; purified antibody serially diluted from 1 μg / mL to 0.005 μg / mL, with 5% milk-PBS) to the coated ELISA plate, 100 μL / well, and incubate at room temperature for 1 hour. Discard the liquid in the plate, wash the plate 3 times with PBS solution and blot dry. Add 1:2000 diluted HRP-labeled goat anti-mouse IgG (Fc) secondary antibody and incubate at room temperature for 1 hour. Wash the plate 5 times with PBS solution, blot dry, add 100 μL of TMB for color development, and react for 20 min in the dark at room temperature. Then add 50 μL of stop solution and read the OD on an ELISA reader. 450 Value, in OD 450(Positive) > 2.1×OD 450 (NC) is used as the criterion.

[0050] 4. Preparation of hybridoma cells and screening of monoclonal antibody cell lines

[0051] Blood was collected from the tail vein of mice on day 14 after the initial immunization, serum was separated, and antibody titers were detected using an indirect ELISA method. The results are shown in Table 3.

[0052] Table 3. Results of mouse serum antibody ELISA detection

[0053]

[0054] Note: NC is the negative control, which is a diluted solution of 5% milk-PBS. NaN indicates that the OD value exceeds the upper limit of the instrument's detection range.

[0055] Based on the ELISA evaluation results in Table 3, three mice with high serum antibody titers (1#, 3#, and 4#) were selected for cell fusion. Splenic cells from these mice were fused with SP2 / 0 myeloma cells. First, individual spleen cells were isolated, and then fused with SP2 / 0 cells at a ratio of 10:1 using PEG4000. The fusion was performed using recovery medium (ClonaCell). TM -HY Medium C), after incubation in a petri dish at 37°C and 5% CO2 for 48 hours, was transferred to a selective semi-solid medium containing HAT (ClonaCell). TM -HY Medium D). After 10 days of culture, 1222 monoclonal cell colonies were selected and transferred to 96-well plates and cultured in ClonaCell medium. TM -HY Medium E) cells were cultured for 7 days, and the cell supernatant was collected for indirect ELISA. 54 relatively strong positive clones were selected and transferred to 48-well plates for further expansion. The supernatant was collected and re-screened using indirect ELISA. The results are shown in Table 4. Finally, 36 cell lines were selected that could stably secrete anti-HPV59 L1-VLP protein antibodies. The other 18 initially positive cell lines lost their antibody-secreting ability, and their cell supernatant tests were negative or weakly positive.

[0056] Table 4. ELISA results of cell line supernatants with stable secretion of anti-HPV59 L1-VLP protein antibodies.

[0057]

[0058]

[0059] Note: NaN indicates that the OD value exceeds the upper limit of the device's detection range.

[0060] 5. Specific detection of antibodies in the supernatant secreted by hybridoma cells

[0061] Thirty-six hybridoma cell lines were coated with 100 ng / well of L1-VLP protein for 11 HPV subtypes (HPV6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68). The antibodies secreted in the cell supernatant by these cells were specifically detected using an indirect ELISA method. The results showed that 15 hybridoma cell lines (clone13C11, clone13E1, clone13G4, clone13G8, clone13H1, clone13H10, clone16C9, clone17D11, clone17F7, clone18E11, clone18H2, clone18G11, clone23E11, clone23G8, and clone23H2) were positive only for HPV59 L1-VLP protein, and negative for other types. Therefore, the antibodies secreted by these 15 hybridoma cell lines were determined to be type-specific antibodies.

[0062] 6. Preparation of ascites

[0063] Ascites was prepared from the above hybridoma cells, and approximately 10 cells were extracted from each positive clone. 7 Two BALB / c mice pre-injected with IFA adjuvant were injected into the peritoneal cavity. Ten days later, ascites fluid was aspirated, and the supernatant was collected by centrifugation. The ascites fluid was purified with Protein G to obtain purified antibodies. The A280 value was measured by UV spectrophotometer, the antibody concentration was calculated and adjusted, and the binding ability of the purified antibodies to HPV59 L1-VLP was evaluated using an indirect ELISA method. The results are shown in Table 5. Among them, antibody 23E11 had the highest titer and the best affinity, with a sensitivity of 0.0005 μg / mL. The sensitivities of other antibodies were between 0.5 and 0.05 μg / mL.

[0064] Table 5. ELISA results of purified antibodies

[0065]

[0066]

[0067] Note: NaN indicates that the OD value exceeds the upper limit of the device's detection range.

[0068] 7. Antibody neutralization activity detection

[0069] The neutralizing activity and antibody titer of antibodies were detected using the HPV pseudovirus neutralization assay, as detailed below:

[0070] (1) Seed 293FT cells into 96-well cell culture plates, with 1.5 × 10⁻⁶ cells per well. 4 Incubate at 37°C and 5.5% CO2 for 6 hours using 100 μl of the sample.

[0071] (2) Perform serial dilutions of the monoclonal antibody or serum to be tested, with a final volume of 120 μL / well.

[0072] (3) In a 96-well round-bottom plate, mix equal volumes of pseudovirus working solution and monoclonal antibody or serum diluent (120 μl + 120 μl), and set up a negative control (diluent + pseudovirus) and a blank control (DMEM complete medium). After shaking to mix, incubate the 96-well round-bottom plate at 4°C for 1 hour.

[0073] (4) Take 100 μl of pseudovirus-antibody / serum (or culture medium) mixture from each well of the 96-well round-bottom plate and slowly add it to the corresponding well of the culture plate that has been pre-coated with cells. Incubate the cell culture plate at 37°C and 5.5% CO2 for 72–96 h.

[0074] (5) Discard the supernatant from the cell culture plate, pat dry, and place it in an ELISA (fluorescent) immunospot analyzer to detect the number of fluorescent spots in the corresponding wells. Calculate the infection inhibition rate based on the number of fluorescent spots in each well: Infection inhibition rate = {1 - (number of fluorescent spots in sample wells - mean number of fluorescent spots in blank control group) / (mean number of fluorescent spots in negative control group - mean number of fluorescent spots in blank control group)} × 100%. Wells with an infection inhibition rate greater than 50% are considered positive. Calculate the neutralizing titer (IC50) of the monoclonal antibody using the Reed-Muench method. 50 Or serum neutralizing titer ID 50 .

[0075] The results (Table 6) showed that the antibodies secreted by the 13E1 and 23E11 monoclonal cell lines both exhibited neutralizing activity against HPV59 pseudovirus. Among them, the IC50 of 13E1 was [missing information]. 50 The concentration was 9 ng / ml, and the IC50 of 23E11 was [missing information]. 50 The concentration was 31 ng / ml, while the other hybridoma cell lines showed no neutralizing activity.

[0076] Table 6. Neutralizing activity of monoclonal antibodies against HPV59 pseudovirus

[0077]

[0078] 8. Passaging and preservation of hybridoma cells

[0079] The hybridoma cells (13E1 and 23E11) were passaged in DMEM high-glucose medium (containing sodium pyruvate and L-glutamine) containing 10% heat-inactivated fetal bovine serum. When the cell density reached 60%-80%, the cells were gently resuspended by pipetting and passaged at a ratio of 1:3. During long-term passage, cells may undergo genetic mutations or chromosomal drift, leading to changes in characteristics, and there is also a risk of contamination; therefore, a standardized cryopreservation procedure must be established. The specific method is as follows: Remove the cell culture medium, add DMEM basal medium, gently pipette to prepare a cell suspension, centrifuge at 1000 rpm / min for 5 minutes to collect the cell pellet. Resuspend the cells in pre-chilled 2× cryopreservation solution (40% fetal bovine serum, 20% DMSO, 40% DMEM), aliquot the cell suspension into labeled cryovials, 1 mL per tube, tighten the cap, and gently mix. Immediately transfer the cryovials to a programmed cooling box and freeze them at -80°C for 24 hours before finally transferring them to liquid nitrogen for long-term storage.

[0080] The aforementioned hybridoma cell lines 23E11 and 13E1, exhibiting neutralizing activity, were deposited at the China Center for Type Culture Collection (CCTCC). Hybridoma cell line 23E11 was deposited on November 7, 2025, at the CCTCC, located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: C2025319. Hybridoma cell line 13E1 was deposited on November 7, 2025, at the CCTCC, located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: C2025318.

[0081] Example 2: Identification of 13E1 Antibody

[0082] 1. Antibody Acquisition

[0083] Approximately 10 7 One hybridoma cell line 13E1 was injected into the peritoneal cavity of two BALB / c mice that had been pre-injected with IFA adjuvant, and ascites fluid was aspirated 10 days later.

[0084] 2. Antibody purification

[0085] The ascites fluid was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected and purified using a Protein G affinity chromatography column. The resulting monoclonal antibody 13E1 against HPV59 L1 protein was obtained at a concentration above 1 mg / ml and stored at -20°C.

[0086] 3. Antibody purity detection

[0087] The purified monoclonal antibody 13E1 was subjected to SDS-PAGE electrophoresis. The SDS-PAGE results are as follows: Figure 1 As shown, the results indicate that its purity is above 95%.

[0088] 4. Identification of antibody types and subclasses

[0089] The 13E1 subtype was classified using the indirect ELISA method described in Example 1. Except for the addition of mouse monoclonal antibody at a concentration of 1 μg / ml and the addition of subtype identification reagent diluted 1:2000, the rest of the method was the same as in step 3 of Example 1. The results showed that the heavy chain subtype of the monoclonal antibody produced by the hybridoma cell line 13E1 was IgG1, and the light chain subtype was κ.

[0090] 5. Sequencing of antibody light chain and heavy chain variable region genes

[0091] mRNA was extracted from hybridoma cell line 13E1, reverse transcribed into cDNA, and amplified by PCR using universal primers for the variable region. The PCR product fragment was then inserted into a T vector for DNA sequencing. The sequence information of the HPV59 monoclonal antibody 13E1 is as follows:

[0092] The full-length heavy chain nucleotide sequence of 13E1 (SEQ ID NO.1) is shown below:

[0093]

[0094] The nucleotide sequence of the heavy chain variable region of 13E1 (SEQ ID NO.2) is shown below:

[0095] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTATATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTTATGGTGGTAGTTACACCTACTATC CAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACTTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGATGGGTATGGTAACTACGTTACTTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0096] The nucleotide sequence of the heavy chain constant region of 13E1 (SEQ ID NO.3) is shown below:

[0097] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA

[0098] The full-length amino acid sequence of the heavy chain of the 13E1 antibody (SEQ ID NO.4) is shown below:

[0099] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISYGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARDGYGNYVTWFAYW GQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD CGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0100] The amino acid sequence of the heavy chain variable region of the 13E1 antibody (SEQ ID NO.5) is shown below:

[0101] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISYGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARDGYGNYVTWFAYWGQGTLVTVSA

[0102] The amino acid sequence of the heavy chain constant region of the 13E1 antibody (SEQ ID NO.6) is shown below:

[0103] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0104] The full-length nucleotide sequence of the light chain of 13E1 (SEQ ID NO.7) is shown below:

[0105] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0106] The nucleotide sequence of the light chain variable region of 13E1 (SEQ ID NO.8) is shown below:

[0107] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0108] The nucleotide sequence of the light chain constant region of 13E1 (SEQ ID NO.9) is as follows:

[0109] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0110] The full-length amino acid sequence of the light chain of the 13E1 antibody (SEQ ID NO.10) is as follows:

[0111] DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0112] The amino acid sequence of the light chain variable region of the 13E1 antibody (SEQ ID NO.11) is shown below:

[0113] DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK

[0114] The amino acid sequence of the light chain constant region of the 13E1 antibody (SEQ ID NO.12) is shown below:

[0115] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW TDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0116] 6. Antibody type specificity detection

[0117] As shown in the antibody-specific detection results in the supernatant secreted by 5 hybridoma cells in Example 1, the 13E1 antibody only reacted positively for HPV59 L1-VLP protein, and the detection results for other types were negative, thus confirming that the 13E1 antibody is a type-specific monoclonal antibody.

[0118] 7. Identification of antibody neutralizing activity

[0119] As shown in the antibody neutralization activity test results in Example 1, 13E1 is a monoclonal antibody with neutralizing activity.

[0120] 8. Epitope dominance analysis of antibodies

[0121] Immunodominance analysis of 13E1 antibodies was performed using competitive inhibition ELISA. The titer (BID) of 13E1 class antibody in the serum of 16 HPV-vaccinated rats was detected by competitive inhibition ELISA. 50 (It can inhibit 50% of the serum dilution when the monoclonal antibody binds to HPV59 LI-VLP). Results are as follows... Figure 2 As shown, the inhibitory effect of 13E1 antibody on 16 rat sera was dose-dependent, indicating that the neutralizing epitope recognized by 13E1 antibody is the same as that recognized by most antibodies in the serum of immunized rats. That is, the recognized epitope is the dominant neutralizing epitope of HPV59 LI-VLP and is the dominant antibody in the serum of immunized rats.

[0122] The total neutralizing antibody titer (ID) of these 16 rat serum samples was determined using a pseudovirus neutralization assay. 50 Analyze BID 50 With ID 50 The correlation between them, the results are as follows Figure 3 As shown in the figure. The results showed that the 13E1 class antibody in the serum of immunized rats was significantly positively correlated with the total neutralizing antibody (r = 0.6582, P = 0.0056), indicating that the 13E1 antibody can represent most of the neutralizing antibodies in the serum of HPV-immunized rats.

[0123] Example 3: Identification of 23E11 Antibody

[0124] 1. Antibody Acquisition

[0125] The procedure was performed as described in Example 2, except that hybridoma cell 13E1 was replaced with hybridoma cell 23E11.

[0126] 2. Antibody purification

[0127] Following the method described in Example 2, the monoclonal antibody 23E11 of HPV59 L1 protein was finally obtained at a concentration of 1 mg / ml or higher.

[0128] 3. Antibody purity detection

[0129] Following the method described in Example 2, Section 3, the SDS-PAGE electrophoresis results are as follows: Figure 1 As shown, the results indicate that the purity of the monoclonal antibody 23E11 is above 95%.

[0130] 4. Identification of antibody types and subclasses

[0131] Following the method described in Example 2, Section 4, the results showed that the monoclonal antibody produced by hybridoma cell line 23E11 had a heavy chain subtype of IgG1 and a light chain subtype of κ.

[0132] 5. Sequencing of antibody light chain and heavy chain variable region genes

[0133] mRNA was extracted from hybridoma cell line 23E11, reverse transcribed into cDNA, and amplified by PCR using universal primers for the variable region. The PCR product fragment was then inserted into a T vector for DNA sequencing. The sequence information of the HPV59 monoclonal antibody 23E11 is as follows:

[0134] The full-length heavy chain nucleotide sequence of 23E11 (SEQ ID NO.13) is shown below:

[0135]

[0136] The nucleotide sequence of the heavy chain variable region of 23E11 (SEQ ID NO.14) is shown below:

[0137] CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATAACCTGCACAGTCTCTGGTTTCTCATTAACTGACTTTAGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGAGGTGGAAGTACAGACT ACAATGCAGCTTTCATGTCCAGACTGAGCATCACCAGGACAACTCCAAGAGCCAAGTTTTCTTAAATGAACAGTCTGCAAGCTGATGACTCTGCCATATATTACTGTGCCCTGCATTACTACGGCTATGTTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCG

[0138] The nucleotide sequence of the heavy chain constant region of 23E11 (SEQ ID NO.15) is shown below:

[0139] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA

[0140] The full-length amino acid sequence of the heavy chain of the 23E11 antibody (SEQ ID NO.16) is shown below:

[0141] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTDFSVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDSAIYYCALHYYGYVLDYWGQG TSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0142] The amino acid sequence of the heavy chain variable region of the 23E11 antibody (SEQ ID NO.17) is shown below:

[0143] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTDFSVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDSAIYYCALHYYGYVLDYWGQGTSVTVSS

[0144] The amino acid sequence of the heavy chain constant region of the 23E11 antibody (SEQ ID NO.18) is shown below:

[0145] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0146] The full-length nucleotide sequence of the light chain of 23E11 (SEQ ID NO.19) is shown below:

[0147] AACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCAGTATGACCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTACTTGGCCTGGTATCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATACCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0148] The nucleotide sequence of the light chain variable region of 23E11 (SEQ ID NO.20) is shown below:

[0149] AACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCAGTATGACCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTACTTGGCCTGGTATCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATACCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0150] The nucleotide sequence of the light chain constant region of 23E11 (SEQ ID NO.21) is shown below:

[0151] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0152] The full-length amino acid sequence of the light chain of the 23E11 antibody (SEQ ID NO.22) is shown below:

[0153] NIMMTQSPSSLAVSAGEKVSMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0154] The amino acid sequence of the light chain variable region of the 23E11 antibody (SEQ ID NO.23) is shown below:

[0155] NIMMTQSPSSLAVSAGEKVSMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKLEIK

[0156] The amino acid sequence of the light chain constant region of the 23E11 antibody (SEQ ID NO.24) is shown below:

[0157] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW TDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0158] 6. Antibody type specificity detection

[0159] As shown in the antibody-specific detection results in the supernatant secreted by 5 hybridoma cells in Example 1, the 23E11 antibody only reacted positively for HPV59 L1-VLP protein, and the detection results for other types were negative, thus confirming that the 23E11 antibody is a type-specific monoclonal antibody.

[0160] 7. Identification of antibody neutralizing activity

[0161] As shown in the antibody neutralization activity test results in Example 1, 23E11 is a monoclonal antibody with neutralizing activity.

[0162] 8. Epitope dominance analysis of antibodies

[0163] The same method was used as in Example 2, and the results are as follows: Figure 2As shown, the inhibitory effect of 23E11 antibody on 16 rat sera was dose-dependent, indicating that the neutralizing epitope recognized by 23E11 antibody is the same as that recognized by most antibodies in the serum of immunized rats. That is, the recognized epitope is the dominant neutralizing epitope of HPV59 LI-VLP and is the dominant antibody in the serum of immunized rats.

[0164] BID 50 With ID 50 The correlation analysis results between them are as follows Figure 3 As shown in the figure. The results showed that there was no significant correlation between the 23E11 class antibody and the total neutralizing antibody in the serum of immunized rats (r = 0.4540, P = 0.0773).

[0165] The nucleotide and amino acid sequences of the 13E1 antibody and 23E11 antibody in Examples 2 and 3 have been clearly described. Therefore, in addition to the methods described above, they can also be obtained by synthesis.

[0166] Example 4: HPV59 L1 Antigen Detection Kit

[0167] 1. Antibody pairing and type-specific detection in the kit

[0168] This invention constructs an HPV59 double-antibody sandwich ELISA detection kit based on the obtained monoclonal antibodies to achieve highly sensitive and specific detection of intact HPV59 L1-VLP. This method, through two steps of specific antibody recognition and binding, can accurately capture HPV59 L1-VLP with its complete spatial conformation, effectively reducing non-specific interference and significantly improving the accuracy and reliability of the detection.

[0169] To construct a high-performance double-antibody sandwich ELISA kit, this invention performed pairing screening on the obtained monoclonal antibodies. Since both 13E1 and 23E11 are monoclonal antibodies with strong neutralizing activity, they were selected as the core pairing antibodies. The results are shown in Table 7. The double-antibody sandwich ELISA kit established using 23E11 as the coating antibody and 13E1 as the enzyme-labeled antibody exhibited good binding activity with the target antigen, and the standard curve showed good linearity. The linear equation of the standard curve was y = 0.0009x + 0.1287, R0. 2 =1.

[0170] Table 7.23E11 and 13E1-HRP pairing test results

[0171]

[0172] Furthermore, the paired antibody-based double-antibody sandwich ELISA kit exhibited low background signal in the blank matrix (P20 buffer and dilution II), and the kit only specifically reacted with the HPV59 L1 antigen, showing no cross-reactivity with antigens of other HPV types (such as HPV6, 11, 16, 18, 31, 33, 45, 52, 58, 68, etc.). This indicates that the kit possesses strong specificity and type identification capabilities, making it suitable for quality control of multivalent vaccines. The results are shown in Table 8. Therefore, this invention established an HPV59 double-antibody sandwich ELISA kit using 23E11 as the coating antibody and 13E1 as the enzyme-labeled antibody.

[0173] Table 8. Results of reagent kit specificity and type specificity testing

[0174]

[0175] 2. Reagent Kit Principle

[0176] The two monoclonal antibodies, 23E11 and 13E1, obtained by screening in this invention are both HPV59 type-specific antibodies. Among them, antibody 13E1 not only has strong neutralizing activity but also represents the dominant antibody in rat serum after antigen immunization. Based on these characteristics, this invention constructs a double-antibody sandwich ELISA detection kit using 23E11 as the coating antibody and 13E1 as the enzyme-labeled antibody. In this detection system, HPV59 L1 protein in the sample or standard is first captured by the pre-coated 23E11 antibody; then, 13E1-HRP enzyme-labeled antibody is added for specific binding, forming an "antibody-antigen-enzyme-labeled antibody" immune complex. After colorimetric reaction, within a linear concentration range, the absorbance value of the sample is positively correlated with the content of active antigen of HPV59 L1 protein. By comparing with a standard curve, accurate quantification of HPV59 L1 protein with complete conformation in the sample can be achieved.

[0177] 3. Components of the reagent kit

[0178] (1) ELISA plate coated with coating antibody: The coating antibody was the 23E11-type specific monoclonal antibody produced in Example 3, secreted by the hybridoma cell line 23E11 in Example 1. The coating antibody was diluted to 5 μg / ml with coating buffer and coated onto a 96-well ELISA plate, 100 μl per well, and incubated overnight at 2–8°C. The liquid in the wells was discarded, and the plate was washed three times with washing buffer and patted dry. Then, blocking buffer was added to the ELISA plate, 100 μl per well, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, and the plate was washed twice with washing buffer and patted dry. Subsequently, sucrose protectant was added, 100 μl per well, and the plate was incubated at 37°C for 0.5 hours. The liquid in the wells was discarded, and the plate was patted dry. Finally, the plate was dried and vacuum-sealed with aluminum foil for storage. The coating buffer used was a carbonate buffer containing 0.015 mol / L sodium carbonate and 0.035 mol / L sodium bicarbonate at a final concentration in the coating solution. The blocking solution used was a hydrochloric acid buffer containing 3% (w / w) bovine serum albumin at a final concentration in the blocking solution. The sucrose protectant used was an aqueous solution containing 10% (w / w) sucrose at a final concentration in the sucrose protectant.

[0179] (2) Enzyme-labeled antibody: The labeling enzyme is horseradish peroxidase, the labeling method is sodium periodate method, and the enzyme-labeled antibody is the 13E1 type specific neutralizing monoclonal antibody produced in Example 2, which is secreted by the hybridoma cell line 13E1 in Example 1. The working concentration of the enzyme-labeled antibody is 3 μg / ml.

[0180] (3) Standard: HPV59 L1 protein lyophilized standard, 25μg / 0.5ml / bottle.

[0181] (4) Substrate colorimetric solution: 3,3',5,5'-tetramethylbenzidine (TMB) solution.

[0182] (5) Termination solution: 2 mol / L sulfuric acid solution.

[0183] (6) Washing buffer (PBST): Phosphate buffer with a final concentration of 0.5‰ (volume percentage) Tween-20.

[0184] (7) Diluent II: A mixture containing 20% ​​(volume percentage) fetal bovine serum and 0.2% (mass percentage) yeast extract in a final concentration of 20% (volume percentage) in the diluent, and made up to the required volume with PBST solution.

[0185] (8) Kit instructions.

[0186] 4. The binding of the kit to HPV59 VLP depends on the complete VLP conformation.

[0187] HPV59 VLPs were reductively denatured using dithiothreitol (DTT) solution and detected by a double-antibody sandwich ELISA method to verify whether the binding of this kit to HPV59 VLPs depends on their intact conformation. Results are as follows: Figure 4 As shown, after DTT denaturation treatment, the binding signal of the kit to HPV59 VLP was significantly reduced. Furthermore, when an equal volume of untreated intact VLP was added to the denatured VLP solution for detection, the OD value was approximately 50% of that of a sample containing only intact VLP. This result confirms that the binding of the kit to HPV59 VLP is highly dependent on the integrity of the VLP conformation, thereby ensuring its specificity in detecting VLPs with the correct spatial structure.

[0188] 5. Reagent kit sensitivity

[0189] The HPV59 standard was serially diluted from 8000 ng / mL to 7.813 ng / mL in 2-fold increments, resulting in 11 concentration gradients. Each concentration point was tested in duplicate, and a negative control was included. The kit was used to repeat the detection three times to determine the limit of detection (LOD) for the antigen. The average absorbance of the duplicate wells was compared with the cut-off value to determine whether the result was positive or negative (a mean absorbance greater than or equal to the cut-off value was considered positive, and vice versa. When the mean OD value of the negative control was ≥0.05, the cut-off value was calculated as 2.1 × NC; otherwise, NC was calculated as 0.05, i.e., the cut-off value was 2.1 × NC). The average of the lowest concentrations that yielded a positive result was used as the sensitivity of the kit. The results of three repeated tests showed that the detection sensitivity of the HPV59 kit was 15.6 ng / mL.

[0190] 6. Quantitative range of the reagent kit

[0191] The HPV59 standard was serially diluted from 8000 ng / mL to 7.813 ng / mL in 2-fold increments, resulting in 11 concentration gradients. Each concentration point was run in duplicate, and a negative control was included. The assay was repeated three times using the kit described above. The absorbance (OD) values ​​of the standard antigen concentrations were plotted on the X-axis. 450nm / 630nm Using the Y-axis, fit a four-parameter logistic curve, with the regression equation (y = (AD) / (1 + (x / C)^B) + D). Repeat the experiment three times, and calculate the coefficient of determination (R²) of the standard curve. 2 To verify its linear correlation, the measured values ​​at each concentration point of the standard curve were back-calculated, and the recovery rate and the relative standard deviation (RSD) of the measured values ​​at the same concentration points in the three repeated tests were calculated. The R-value of the standard curve equation for three repeated tests was calculated. 2The values ​​were 0.999, 0.999, and 0.998, respectively. Within the concentration range of 31.25–1000 ng / mL, the recoveries at each point were between 80% and 120%, and the RSDs of the three repeated measurements were all ≤20%, meeting the quantification requirements.

[0192] 7. Precision verification of the reagent kit

[0193] 1) Precision verification – intra-plate differences

[0194] HPV59 L1 protein was diluted to five different antigen concentrations, and five samples (n=5) were taken from each antigen concentration. Each sample was replicated. The coefficient of variation (CV) of the five samples at the same antigen concentration was calculated to determine the intra-plate difference, thereby performing precision analysis on the kit. The results are shown in Table 9. The intra-plate CV (%) values ​​of the five different antigen concentration samples were all less than 10%, indicating that the kit has good intra-plate precision.

[0195] Table 9. Intraplate coefficient of variation for HPV59 reagent kit

[0196]

[0197] 2) Precision verification – inter-plate differences

[0198] HPV59 L1 protein was diluted to five different antigen concentrations, and five samples (n=5) were taken from each antigen concentration. Each sample was replicated, and the antigen content of the samples was measured on two ELISA plates. The coefficient of variation (CV) of 10 samples with the same antigen concentration on both ELISA plates was calculated to determine the inter-plate differences, thereby performing precision analysis of the kit. The results are shown in Table 10. The inter-plate CV (%) values ​​for different antigen concentrations were all less than 10%, indicating that the kit has good inter-plate precision.

[0199] Table 10. Interplate variation coefficient of HPV59 reagent kit

[0200]

[0201] 8. Reagent kit accuracy verification

[0202] The HPV59 standard was diluted to three concentrations: high (1000 ng / ml), medium (500 ng / ml), and low (250 ng / ml). Each sample was run in duplicate, and the recovery rate was calculated. The results showed that the recovery rate was between 82.0% and 89.2%, indicating good recovery of the kit.

[0203] In summary, this invention provides monoclonal antibodies 13E1 and 23E11 against HPV59 L1 protein. These two antibodies specifically bind to HPV subtype 59 (without cross-reactivity with most other HPV subtypes such as HPV6, 11, 16, 18, 31, 33, 45, 52, 58, and 68), exhibiting high specificity and neutralizing activity, and can be used to detect HPV59 antigen. Monoclonal antibody 13E1 recognizes the epitope targeted by the dominant neutralizing antibody in serum after vaccine immunization; therefore, it can be used not only for antigen detection but also directly for evaluating the ability of HPV vaccines to induce neutralizing antibody production. Based on the above antibodies, this invention also provides a double-antibody sandwich ELISA kit. This kit can specifically identify and quantify conformationally intact and immunogenic HPV59 L1 protein in samples. This kit can be stably mass-produced and enables rapid in vitro evaluation of HPV vaccine activity, showing promising application prospects.

Claims

1. A monoclonal antibody against HPV59 L1 protein, characterized in that: The monoclonal antibody is composed of a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.5, the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO.6, the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.11, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.

12.

2. A gene fragment encoding the monoclonal antibody of claim 1, characterized in that: This includes gene fragments encoding the heavy chain with nucleotide sequences as shown in SEQ ID NO.1, and gene fragments encoding the light chain with nucleotide sequences as shown in SEQ ID NO.

7.

3. A hybridoma cell line for producing the monoclonal antibody of claim 1, characterized in that: The hybridoma cell line is a cell line preserved at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025318.

4. A monoclonal antibody against HPV59 L1 protein, characterized in that: The monoclonal antibody is composed of a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.17, the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO.18, the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.23, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.

24.

5. A gene fragment encoding the monoclonal antibody of claim 4, characterized in that: This includes gene fragments encoding the heavy chain with nucleotide sequences as shown in SEQ ID NO.13, and gene fragments encoding the light chain with nucleotide sequences as shown in SEQ ID NO.

19.

6. A hybridoma cell line for producing the monoclonal antibody of claim 4, characterized in that: The hybridoma cell line is a cell line preserved at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025319.

7. The method for preparing the monoclonal antibody according to claim 1 or 4, characterized in that: Includes the following steps: (1) Inject the hybridoma cells of claim 3 or claim 6 into the peritoneal cavity of a mouse that has been pre-injected with IFA adjuvant, and aspirate the ascites. (2) Centrifuge the ascites obtained in step (1), collect the supernatant and purify it with a Protein G affinity chromatography column to obtain the final product.

8. Use of the monoclonal antibody according to claim 1 and / or claim 4 in the preparation of a kit for detecting HPV59 antigen.

9. Use of the monoclonal antibody according to claims 1 and 4 in the preparation of a kit for detecting the in vitro activity of HPV vaccines.

10. A kit for detecting the in vitro activity of an HPV vaccine, characterized in that: The kit includes the monoclonal antibody of claim 1 and the monoclonal antibody of claim 4; Preferably, the monoclonal antibody of claim 1 is an enzyme-labeled antibody; the monoclonal antibody of claim 4 is a coated antibody.

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