An anti-hpv 68 antibody and methods of making and uses thereof

Abstract

The present application relates to the field of medicine, particularly to an anti-HPV 68 antibody and a preparation method and application thereof, the anti-HPV 68 antibody has the following technical features: <1> the heavy chain variable region includes CDR-H1 of the amino acid sequence shown in SEQ ID No. 1; <2> the heavy chain variable region includes CDR-H2 of the amino acid sequence shown in SEQ ID No. 2; <3> the heavy chain variable region includes CDR-H3 of the amino acid sequence shown in SEQ ID No. 3; <4> the light chain variable region includes CDR-L1 of the amino acid sequence shown in SEQ ID No. 4; <5> the light chain variable region includes CDR-L2 of the amino acid sequence shown in SEQ ID No. 5; <6> the light chain variable region includes CDR-L3 of the amino acid sequence shown in SEQ ID No. 6. The anti-HPV 68 antibody has no cross reaction with other 16 subtypes of HPV, and the detection sensitivity is high.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to an anti-HPV 68 antibody, its preparation method, and its uses. Background Technology

[0002] Cervical cancer and precancerous lesions are a significant threat to women's health worldwide. Clinical, molecular biological, and epidemiological surveys have proven that human papillomavirus (HPV) is the leading cause of cervical cancer and cervical dysplasia. Currently, over 200 HPV subtypes have been identified, classified according to pathogenicity into high-risk types (including HPV16, 18, 31, 33, 35, 39, and 45) and low-risk types (including HPV6 and 11). HPV is a non-enveloped circular double-stranded DNA virus. Its capsid is an icosahedral structure composed of 72 major capsid proteins (L1 protein pentamers), and each viral particle contains 360 L1 protein monomers. Currently, three cervical cancer vaccines are approved for marketing in China: GlaxoSmithKline's (GSK) bivalent HPV vaccine Cervarix (subtypes HPV 16 and 18), Merck's quadrivalent HPV vaccine Gardasil®, and the nine-valent vaccine Gardasil® 9 (subtypes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58). The International Agency for Research on Cancer (IARC) of the World Health Organization has identified 13 high-risk HPV types that can cause cervical cancer and high-grade precancerous cervical intraepithelial lesions (HR-HPV): HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68. Of these, only HPV68 is classified as Group 2A in the IARC carcinogen classification; the other 12 are classified as Group I carcinogens. Since currently available vaccines cover a maximum of nine HPV subtypes, more and more companies are continuously developing HPV vaccines with multiple valences (such as including HPV68). According to the World Health Organization (WHO) guidelines on the quality, safety, and efficacy of HPV vaccines, and the relevant regulations of the China Food and Drug Administration (CFDA), the development and production of multivalent HPV vaccines require the establishment of methods for identification tests, antigen content determination, adsorption completeness, and in vitro relative potency assays. These methods can all employ enzyme-linked immunosorbent assay (ELISA). Therefore, the type specificity and high neutralizing activity of monoclonal antibodies in the ELISA method are crucial for the quality control of HPV vaccines. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an anti-HPV 68 antibody, its preparation method and uses, to solve the problems in the prior art.

[0004] To achieve the above and other related objectives, the present invention provides an anti-HPV 68 antibody, which includes a heavy chain variable region and a light chain variable region, and has the following technical features: <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1; <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2; <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3; <4> The light chain variable region includes the amino acid sequence CDR-L1 as shown in SEQ ID No. 4; <5> The light chain variable region includes the amino acid sequence CDR-L2 as shown in SEQ ID No. 5; <6> The light chain variable region includes the amino acid sequence CDR-L3 as shown in SEQ ID No. 6.

[0005] Another aspect of the present invention provides an isolated polynucleotide encoding the heavy chain variable region and / or light chain variable region or full-length amino acid of the anti-HPV 68 antibody.

[0006] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0007] The present invention also provides an antibody expression system, wherein the expression system contains the aforementioned nucleic acid construct or genome in which exogenous polynucleotides are integrated.

[0008] The present invention also provides a method for preparing the anti-HPV 68 antibody, comprising the following steps: culturing the expression system of the antibody under suitable conditions for expressing the anti-HPV 68 antibody, thereby expressing the anti-HPV 68 antibody, and isolating the anti-HPV 68 antibody.

[0009] Another aspect of the present invention provides a kit for detecting HPV 68 antigen, comprising the anti-HPV 68 antibody or its immunoconjugate.

[0010] Another aspect of the present invention provides the use of the anti-HPV 68 antibody and the kit in the preparation of screening or quality control products for tumor diagnostic drugs, tumor therapeutic drugs, and tumor prevention drugs, or in the preparation of diagnostic or therapeutic drugs.

[0011] As described above, the anti-HPV 68 antibody, its preparation method, and its uses of the present invention have the following beneficial effects: The human papillomavirus 68 (HPV 68) specific monoclonal antibody of the present invention has no cross-reactivity with the other 16 HPV subtypes, and has the advantage of high specificity in the identification test of HPV vaccines. It has neutralizing antibody activity and can accurately detect the content and adsorption completeness of HPV 68 antigen with neutralizing activity in HPV multivalent vaccines, and has the advantage of high detection sensitivity. It will be widely used in the quality control of HPV vaccines. Attached Figure Description

[0012] Figure 1 The image shown is a pseudovirus control diagram in the HPV68L1 neutralization activity test of this invention.

[0013] Figure 2 The image shown is a chromatogram of HPV 68 L1 monoclonal antibody determined by the immunoblotting method of the present invention.

[0014] Figure 3 This is one of the results of cross-specific identification between antibody subtypes.

[0015] Figure 4 This is one of the results of cross-specific identification between antibody subtypes.

[0016] Figure 5 This is one of the results of cross-specific identification between antibody subtypes.

[0017] Figure 6 The result shows the neutralizing antibody titer in the cell supernatant.

[0018] Figure 7 This is a screening monoclonal antibody for determining the relative potency of HPV 68 L1 in vitro.

[0019] Figure 8 This is a screening monoclonal antibody for determining the relative potency of HPV 68 L1 in vitro.

[0020] Figure 9 This is a screening monoclonal antibody for determining the relative potency of HPV 68 L1 in vitro.

[0021] Figure 10 The table shows a summary of EC50 values ​​for monoclonal antibodies (unit: μg / ml).

[0022] Figure 11 The graph shows the relative potency of HPV 68 L1 stock antigen in vitro. Detailed Implementation

[0023] This invention provides an anti-HPV 68 antibody, which includes a heavy chain variable region and a light chain variable region, and has one or more of the following technical features; <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1; <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2; <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3; <4> The light chain variable region includes the amino acid sequence CDR-L1 as shown in SEQ ID No. 4; <5> The light chain variable region includes the amino acid sequence CDR-L2 as shown in SEQ ID No. 5; <6> The light chain variable region includes the amino acid sequence CDR-L3 as shown in SEQ ID No. 6.

[0024] The anti-HPV 68 antibody recognizes the L1 protein of human papillomavirus type 68. The anti-HPV 68 antibody can neutralize HPV type 68.

[0025] The CDR (complementarity determining region) generally refers to the region in an antibody that is spatially complementary to the antigenic determinant. The variability in an antibody is usually not uniformly distributed throughout its variable regions. Both the heavy chain and light chain variable regions of a monoclonal antibody typically have three hypervariable regions (HVRs). These regions are spatially complementary to the antigenic determinant, hence the term complementarity determining region (CDR). Specifically, the heavy chain variable region typically includes three CDRs: CDR-H1, CDR-H2, and CDR-H3, while the light chain variable region typically includes three CDRs: CDR-L1, CDR-L2, and CDR-L3.

[0026] In some embodiments of the present invention, the complementarity-determining region of the heavy chain variable region of the anti-HPV 68 antibody includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1, the amino acid sequence CDR-H2 as shown in SEQ ID No. 2, and the amino acid sequence CDR-H3 as shown in SEQ ID No. 3.

[0027] In some embodiments of the present invention, the complementarity-determining region of the light chain variable region of the anti-HPV 68 antibody includes CDR-L1 with an amino acid sequence as shown in SEQ ID No. 4, CDR-L2 with an amino acid sequence as shown in SEQ ID No. 5, and CDR-L3 with an amino acid sequence as shown in SEQ ID No. 6.

[0028] In some embodiments of the present invention, the complementarity-determining region of the heavy chain variable region includes amino acid sequences such as CDR-H1 as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2, and CDR-H3 as shown in SEQ ID No. 3, and the complementarity-determining region of the light chain variable region includes amino acid sequences such as CDR-L1 as shown in SEQ ID No. 4, CDR-L2 as shown in SEQ ID No. 5, and CDR-L3 as shown in SEQ ID No. 6.

[0029] The anti-HPV 68 antibody is an antibody fragment and / or a monoclonal antibody. Further, the monoclonal antibody is an IgG1 antibody.

[0030] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region or variable region. For example, antibody fragments include single-chain antibodies (scFv), Fab, Fab', F(ab'), or F(ab')2.

[0031] In some embodiments of the present invention, the anti-HPV 68 antibody is a single-chain antibody (single-chain Fv, scFv). A single-chain antibody can typically be a V containing an antibody. H (heavy chain variable region) and V L A polypeptide chain (light chain variable region). Typically, single-chain antibodies may also include a linker, which is usually located in the V... H and V L Between these elements, so that scFv can form a desired structure capable of binding to the antigen. For example, the anti-HPV 68 antibody may include V H and V L V H and V L Linking peptides may be provided between the links, and the single-chain anti-HPV 68 antibody may sequentially include V from the N-terminus to the C-terminus. L , linker peptides and V H The anti-HPV 68 single-chain antibody may also sequentially include V from the N-terminus to the C-terminus. H , linker peptides and V LThe linker peptide can be any linker peptide suitable for forming scFv in the art. For example, the linker peptide can be a G4S3 linker. The selection or design of the G4S3 linker can be found in the references Michel Sadelain et al., Science Translational Medicine, 2013; Carl H. June et al., Science Translational Medicine, 2015.

[0032] The “Fab” fragment comprises the variable and constant regions of the light chain, and the variable and first constant region (CH1) of the heavy chain. An F (ab') antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near the carboxyl terminus by a hinge cysteine ​​residue between them.

[0033] In some embodiments of the present invention, the anti-HPV 68 antibody is a monoclonal antibody, which includes a constant region, which may be a natural sequence constant region (e.g., a human natural sequence constant region) or a variant of its amino acid sequence.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the various antibodies constituting this population are identical, except for possible naturally occurring mutants, which are usually present in very small quantities. Monoclonal antibodies are highly specific, targeting a single epitope on an antigen. Furthermore, unlike polyclonal antibody formulations which contain different antibodies targeting different determinant regions (epitaxes), each monoclonal antibody targets a single determinant region on an antigen. In addition to their specificity, one advantage of monoclonal antibodies is that they can now be synthesized without contamination from other antibodies. The modifier "monoclonal" indicates the nature of antibodies derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring any specific method to produce the antibody.

[0035] The monoclonal antibodies described in this article explicitly include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence derived from a particular species or belonging to a particular antibody type or subtype, and the remaining portion is identical or homologous to a corresponding sequence derived from another particular species or belonging to another particular antibody type or subtype, as well as fragments of these antibodies, provided they exhibit the desired biological activity.

[0036] In some embodiments of the present invention, the anti-HPV 68 antibody is obtained by screening a hybridoma cell line, and the nucleotide sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively, and the amino acid sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID No. 9 and SEQ ID No. 10, respectively.

[0037] In some embodiments of the present invention, the heavy chain variable region and the light chain variable region may further include a framework region, which may be located between complementarity-determining regions or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0038] Another aspect of the present invention provides an isolated polynucleotide encoding the heavy chain variable region and / or light chain variable region or full-length amino acid of the anti-HPV 68 antibody.

[0039] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0040] The present invention also provides an antibody expression system, wherein the expression system contains the aforementioned nucleic acid construct or genome in which exogenous polynucleotides are integrated.

[0041] The present invention also provides a method for preparing the anti-HPV 68 antibody, comprising the following steps: culturing the expression system of the antibody under suitable conditions for expressing the anti-HPV 68 antibody, thereby expressing the anti-HPV 68 antibody, and purifying and separating the anti-HPV 68 antibody.

[0042] Another aspect of the present invention provides a kit for detecting HPV 68 antigen, comprising the anti-HPV 68 antibody or its immunoconjugate.

[0043] The kit typically uses HPV 68 antigen as a target for detection. The kit may also include a label for anti-HPV 68 antibodies, which can be used to label anti-HPV 68 antibodies. The types of labels that can be used include, but are not limited to, one or more combinations of fluorescent labels, radioactive labels, enzyme-linked immunosorbent assay (ELISA) labels, and chemiluminescent labels. Depending on the detection principle of the kit, the kit may also contain one or more reagents required for the detection. Furthermore, the kit may, as needed, include: containers, controls (negative or positive controls), buffers, and auxiliary agents, which can be selected by those skilled in the art according to specific circumstances.

[0044] In one embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0045] In one embodiment, the kit is a double-antibody sandwich ELISA kit. In another embodiment, the coating antibody in the double-antibody sandwich ELISA kit is a rabbit monoclonal antibody, and the primary antibody is a mouse monoclonal antibody.

[0046] Another aspect of the present invention provides the use of the anti-HPV 68 antibody and the kit in drug screening, drug quality control, or the preparation of diagnostic or therapeutic drugs.

[0047] The drug may be a drug that targets the HPV 68 antigen, binds to or acts on the HPV 68 antigen, thereby treating and / or preventing indications.

[0048] In some embodiments of the present invention, the drugs used in drug screening and quality control may be tumor therapeutic drugs, tumor diagnostic drugs, or tumor preventive drugs. The tumor diagnostic, therapeutic, or preventive drugs may be drugs that target the HPV 68 antigen on the functional surface of tumor cells, binding to or acting on the HPV 68 antigen to diagnose, treat, and / or prevent tumors. The tumor may be HPV68-positive tumors such as cervical cancer or precancerous cervical lesions.

[0049] In some embodiments of the present invention, the tumor prevention drug is a vaccine.

[0050] The drug screening was conducted in vitro.

[0051] Drug quality control, for example, is the in vitro efficacy assay of the drug.

[0052] The present invention also provides the use of the anti-HPV 68 antibody or the kit in detecting HPV 68 antigen levels.

[0053] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0054] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0055] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0056] Example 1: Detection of anti-HPV 68 antibodies by Western blot The experimental steps are as follows: Prepare a 10% gel. Take 300 μl of HPV 68L1 stock solution and add 60 μl of 6× loading buffer, mix well, boil in a 100℃ water bath for 5 minutes, centrifuge at 10,000 rpm for 5 minutes, load the sample, stacking gel voltage 100V, separating gel voltage 120V. SDS-PAGE wet transfer conditions: 100V, 90 min; blocking overnight with 5% milk powder; cell supernatant containing different monoclonal antibodies numbered 1-18 as primary antibody, diluted 1:20, incubated at room temperature for 20 h. Goat anti-mouse HRP (BIO-RAD batch number L1706516) diluted 1:1000 as secondary antibody, incubated at room temperature for 3 hours; color development: DAB color development, and stop with water.

[0057] like Figure 2 As shown, the results indicate that all 26 monoclonal antibodies that recognize HPV68L1 contain spatial epitopes.

[0058] Example 2: Identification of cross-specificity between antibody subtypes There are over 200 HPV subtypes. Because the production of multivalent HPV vaccines requires identification tests for each subtype, cross-reactions must be avoided during antigen content testing to ensure the accuracy of each subtype's antigen content test results. This experiment identified 17 HPV subtypes, confirming that this monoclonal antibody was positive for HPV 68, while being negative for the other 16 subtypes.

[0059] Experimental steps: Coating: Rabbit anti-HPV 6L1, 11L1, 16L1, 18L1, 31L1, 33L1, 45L1, 52L1, 58L1, 35L1, 39L1, 51L1, 53L1, 56L1, 66L1, and 68L1 were purified by diluting with carbonate coating buffer according to the dilution factor. 100 μl / well was added to the microplate and incubated overnight at 2–8°C.

[0060] Blocking: Wash the plate 5 times with 300 μl / well washing buffer, add 200 μl blocking buffer (5% skim milk powder-PBS) to each well, and block at 37°C for 2 hours.

[0061] Sample loading: Wash the plate 5 times with 300 μl / well washing buffer; dilute the stock solution of each subtype to 40 μg / ml; add 100 μl / well; incubate at 37℃ for 1 hour.

[0062] Add primary antibody: Wash the plate 5 times with 300 μl / well washing buffer. Dilute the cell supernatant 1:100 with diluent, add 100 μl / well to each microplate, and incubate at 37°C for 1 hour.

[0063] Add enzyme-labeled antibody: Wash the plate 5 times with 300 μl / well washing buffer. Dilute HRP-goat anti-mouse lgG 5000 times with dilution buffer, add 100 μl / well of enzyme-labeled antibody, and incubate at 37°C for 1 hour.

[0064] Color development: Wash the plate 5 times with 300 μl / well washing solution, add 100 μl / well freshly prepared color development solution, and incubate at 37°C in the dark for 10 min.

[0065] Termination reading: Add 50 μl of stop solution to each well of the plate, gently vortex to mix, and then read the values ​​using a microplate reader at a wavelength of 450 nm and a reference wavelength of 620 nm. Results are as follows: Figure 3 , Figure 4 and Figure 5 As shown.

[0066] The results showed that, excluding the cell supernatants of antibodies numbered 1, 5, 7, 8, 11, 12, 13, 19, 20, 23, and 26 which showed cross-identification with the other 16 subtypes, the other 15 monoclonal antibodies did not show cross-identification with the other 16 subtypes.

[0067] Example 3: Identification of Antibody Subtypes Coating: Dilute HPV68L1 stock solution with phosphate coating buffer at a rate of 5 μg / well, add 100 μl / well to the microplate, and incubate overnight at 2–8°C.

[0068] Blocking: Wash the plate 5 times with 300 μl / well washing buffer, add 200 μl blocking buffer (5% skim milk powder-PBS) to each well, and block at 37°C for 2 hours.

[0069] Sample loading: Wash the plate 5 times with 300 μl / well of washing buffer. Number the HPV68L1 cell supernatant as follows: 11, 15, 7, 6, 12, 13, 3, 16, 18, 4, 9, 17, 14, 2; dilute 1:10 and 1:100 respectively. Add 100 μl / well; incubate at 37°C for 1 hour.

[0070] Add enzyme-labeled antibody: Wash the plate 5 times with 300 μl / well of washing buffer. Dilute HRP-goat anti-mouse lgG 5000 times with dilution buffer, and add 100 μl / well of enzyme-labeled antibody; dilute Goat anti-mouse-lgG1-HRP, Goat anti-mouse-lgG2a-HRP, and Goat anti-mouse-lgG2b-HRP 2000 times with dilution buffer, and add 100 μl / well of enzyme-labeled antibody. Incubate at 37°C for 1 hour.

[0071] Color development: Wash the plate 5 times with 300 μl / well washing solution, add 100 μl / well freshly prepared color development solution, and incubate at 37°C in the dark for 10 min.

[0072] Termination reading: Add 50 μl of the stop solution to the plate, shake slightly to mix, and then read the value using an ELISA reader at a wavelength of 450 nm and a reference wavelength of 620 nm.

[0073] The results showed that among the 26 monoclonal antibodies against HPV 68L1, antibodies numbered 2, 3, 6, 15, 20, 22, 24, and 25 were more predominantly IgG1-based. The other 18 antibodies were more predominantly IgG2a-based. Example 4: Detection of antibody neutralizing activity The neutralizing antibody titers of each monoclonal antibody strain were detected using a pseudovirus neutralization method.

[0074] Cell seeding: 293FT cells were seeded at a density of 1.5 × 10⁻⁶ cells / cm². 4 / well was pre-coated at a density of 100 μl per well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cells adhered. The cell supernatant containing each antibody strain was serially diluted with complete culture medium to the appropriate dilution factor.

[0075] Neutralization: Add 60 μl of pseudovirus diluent to 60 μl of serum diluent, mix well, and incubate in a constant temperature incubator at 25±2℃ for 60±2 minutes.

[0076] Sample addition: Accurately aspirate 100 μl of serum-pseudovirus mixture and slowly and carefully add it to a 96-well pre-coated cell plate.

[0077] Culture: After incubation at 37℃ in a 5% CO2 incubator for 68–76 hours, observe the results using an inverted fluorescence microscope. The highest dilution factor of 50% of the fluorescence count of the neutralizing pseudovirus control is the neutralizing antibody titer.

[0078] like Figure 1 and Figure 6As shown, the results indicate that among the 16 monoclonal antibodies against HPV 68L1, the order of neutralization titers is as follows: No. 16 > 3 = 6 = 18 = 24 = 25 > 2 = 4 = 5 = 9 = 14 = 22 > 10 > 15 > 17 = 21.

[0079] Example 5: Screening of monoclonal antibodies for in vitro relative potency assay of HPV 68 Coating: Dilute the HPV68L1 rabbit antibody appropriately with coating buffer at a certain dilution factor, add 100 μl / well to the microplate, and incubate overnight at 4°C or for 2 hours at 37±2°C.

[0080] Blocking: Wash the plate 6 times with 300 μl / well washing solution, add 200 μl blocking solution to each well, and block at 37 ± 2℃ for 2 hours.

[0081] Sample and reference sample preparation and loading: Wash the plate 6 times with 300 μl / well washing buffer. Pre-dilute the recombinant human papillomavirus 68L1 stock solution and reference sample with diluent to an appropriate factor, then increase the dilution by 2-fold or appropriate factor to 9-11 dilutions. Add 100 μl / well to the ELISA plate, and perform double-duplicate testing for each dilution. Incubate at 37±2℃ for 1 hour.

[0082] Add primary antibody: Wash the plate 6 times with 300 μl / well washing buffer. Assay for recombinant human papillomavirus 6L1 stock solution: Dilute HPV68 monoclonal antibody to the appropriate dilution factor with diluent, add 100 μl / well to the ELISA plate, and incubate at 37±2 ℃ for 1 hour.

[0083] Add enzyme-labeled secondary antibody: Wash the plate 6 times with 300 μl / well washing buffer. Dilute horseradish peroxidase-labeled goat anti-mouse IgG (H+L) with appropriate dilution buffer, add 100 μl / well to the ELISA plate, and incubate at 37±2℃ for 1 hour.

[0084] Color development: Wash the plate 6 times with 300 μl / well washing solution, add 100 μl / well freshly prepared color development solution, and develop at 37±2℃ for 5 to 20 minutes.

[0085] Termination reading: Add 50 μl of the stop solution to the plate, shake slightly to mix, and then read the value using an ELISA reader. The measurement wavelength is 450 nm, and the reference wavelength is 620 nm.

[0086] Data Analysis: SoftMax Pro software was used for analysis. A four-parameter fitting was selected, with concentration as the X-axis and absorbance values ​​as the Y-axis. The fitting equation was Y=(AD) / (1+(X / C)). ∧ B)+D, C value is the EC of the test sample. 50 .

[0087] like Figures 7-10 As shown, Figure 10In the diagram, 68-1, 68-2, and 68-3 represent three different ELISA plate numbers. Each ELISA plate can be used to mount a maximum of six monoclonal antibodies. The results show that among the 26 monoclonal antibodies against HPV68L1, monoclonal antibodies numbered 9, 4, 10, 25, 24, and 6 are EC... 50 The value is low, making it suitable for in vitro relative potency assays of antigens.

[0088] Based on all the above experiments, the monoclonal antibody numbered 6 was selected for use in the in vitro relative potency assay kit.

[0089] Example 6: Sequencing of HPV 68 monoclonal antibody number 6 The HPV 68 monoclonal antibody, designated 68H6, was used for mRNA extraction after cell culture. The variable region sequence was determined (sent to a third-party testing company), and the obtained sequences were translated into the following amino acid sequence of the protein: CDRH1 sequence: Gly Phe Ser Leu Ser Thr Ser Gly Met Gly (SEQ ID NO: 1) CDRH2 sequence: Ile Tyr Trp Asp Asp Asp Lys (SEQ ID NO:2) CDRH3 sequence: Ala His Gly Ser Ser Ser Tyr Tyr Ala Met Asp Tyr (SEQ ID NO: 3) CDRL1 sequence: Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr (SEQ ID NO: 4) CDRL2 sequence: Tyr Ala Ser (SEQ ID NO:5) CDRL3 sequence: Gln His Ser Trp Glu Ile Pro Tyr Thr (SEQ ID NO: 6) 68H6 VH: CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCACGGTAGTAGCTCTTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCACAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCACGGTAGTAGCTCTTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:7) 68H6 VL: GACATTGTGCTGACACAGTCTCCTACTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTAGGTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCGACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAACACAGTTGGGAGATTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAAGACATTGTGCTGACACAGTCTCCTACTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTAGGTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTATGCATCCGACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAACACAGTTGGGAGATTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA (SEQ ID NO:8) Amino acid sequence of 68H6 VH: QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCAHGSSSYYAMDYWGQGTSVTVSSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCAHGSSSYYAMDYWGQGTSVTVSS (SEQ ID NO:9) The amino acid sequence of 68H6 VL: DIVLTQSPTSLAVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIKYASDLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKDI VLTQSPTSLAVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIKYASDLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK (SEQ ID NO:10) The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An anti-HPV 68 antibody, said anti-HPV 68 antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The CDR of the heavy chain variable region of the anti-HPV 68 antibody includes the amino acid sequences CDR-H1 as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2, and CDR-H3 as shown in SEQ ID No. 3; The CDR of the light chain variable region of the anti-HPV 68 antibody includes CDR-L1 with an amino acid sequence as shown in SEQ ID No. 4, CDR-L2 with an amino acid sequence as shown in SEQ ID No. 5, and CDR-L3 with an amino acid sequence as shown in SEQ ID No.

6.

2. The anti-HPV 68 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-HPV 68 antibody includes the amino acid sequence shown in SEQ ID No. 9; And / or, The amino acid sequence of the light chain variable region of the anti-HPV 68 antibody includes the amino acid sequence shown in SEQ ID No.

10.

3. The anti-HPV 68 antibody according to claim 1, characterized in that, The anti-HPV 68 antibody is a single-chain antibody and / or a monoclonal antibody.

4. An isolated polynucleotide, characterized in that, Encoding the heavy chain variable region and / or light chain variable region or full-length amino acid of the anti-HPV 68 antibody as described in any one of claims 1-3.

5. A nucleic acid construct, characterized in that, It contains the isolated polynucleotide as described in claim 4.

6. An antibody expression system, characterized in that, The expression system contains the nucleic acid construct as described in claim 5 or the genome in which exogenous polynucleotides as described in claim 4 are integrated.

7. The method for preparing the anti-HPV 68 antibody according to any one of claims 1-3, characterized in that, The method includes the following steps: culturing the antibody expression system as described in claim 6 under suitable conditions for expressing the anti-HPV 68 antibody, thereby expressing the anti-HPV 68 antibody, and isolating the anti-HPV 68 antibody.

8. A kit for detecting HPV 68 antigen, characterized in that, It contains the anti-HPV 68 antibody as described in any one of claims 1-3.

9. The reagent kit as described in claim 8, characterized in that, The kit is an enzyme-linked immunosorbent assay (ELISA) kit.

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