Antibodies against human papillomavirus E1 protein or their kits and related applications

Abstract

This invention discloses an antibody against human papillomavirus E1 protein or a kit thereof and related applications, relating to the field of immunoassay. The antibody comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:15. It can specifically bind to HPV16 and / or 18 E1 protein and has advantages such as high detection sensitivity, strong specificity, high accuracy and good repeatability.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and more specifically, to antibodies against human papillomavirus E1 protein or kits thereof and related applications. Background Technology

[0002] Human papillomavirus (HPV) is a pathogen that primarily infects the squamous epithelium of the human epidermis and mucous membranes, causing proliferative lesions. It is mainly transmitted through sexual contact or close contact. Currently, more than 200 HPV subtypes have been identified, classified into low-risk and high-risk types based on the severity of the disease they cause. High-risk types mainly include HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68, which can cause genital warts, external genital cancer, cervical cancer, and high-grade cervical intraepithelial neoplasia. HPV16 and 18 infection, in particular, are the most important pathogenic factors in the development of cervical cancer. Cervical cancer is one of the most common malignant tumors and a major public health problem affecting women's health worldwide. Its incidence and mortality rates rank fourth among female cancers globally, after lung cancer, breast cancer, and colorectal cancer. According to statistics from the World Health Organization, there are nearly 600,000 new cases of cervical cancer worldwide each year, with about 300,000 deaths. In countries with more developed societies and economies, the incidence and mortality rates of cervical cancer are lower.

[0003] Human papillomavirus (HPV) is a non-enveloped, double-stranded, closed-circular small DNA virus, composed of a viral protein coat and a core DNA. The early transcriptional region of the HPV genome contains several proteins, including E1 through E8. E1 plays a crucial role in the initiation of viral DNA replication, E2 is involved in the transactivation of viral DNA transcription, E4 can disrupt the keratin intermediate filament network, and E6 and E7 are the main oncogenes, continuously expressed throughout the carcinogenesis process and participating in the malignant transformation of cells. E6 and E7 are important causes of cervical cancer; therefore, they are widely recognized as ideal target antigens for therapeutic HPV vaccines.

[0004] Currently available preventative HPV vaccines are all based on virus-like particles (VLPs). VLPs are formed from single viral proteins and are neither infectious nor carcinogenic. However, no research has shown that VLP vaccines have a therapeutic effect. Therefore, for subjects already infected with HPV before vaccination, it cannot alter the infection and progression of the virus. Furthermore, patients infected with HPV currently lack treatment options. Therefore, developing therapeutic HPV vaccines is an ideal strategy for controlling existing HPV infections and treating HPV-related cancers and precancerous lesions, and it also aligns with clinical needs and market trends.

[0005] Quality control of the main active ingredients in HPV vaccines is crucial during the research and development and production process. Currently, there is a lack of high-performance antibodies against human papillomavirus E1 on the market, which limits the accuracy and reliability of HPV E1 antigen detection.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an antibody against human papillomavirus E1 protein or a kit thereof and related applications.

[0008] This invention is implemented as follows:

[0009] In a first aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof against HPV type 16 and / or type 18 E1 protein, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 14 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 15.

[0010] Secondly, embodiments of the present invention provide an antibody conjugate comprising: the antibody described above or its antigen-binding fragment.

[0011] Thirdly, embodiments of the present invention provide a reagent or kit comprising: the antibody described above or its antigen-binding fragment or the antibody conjugate described above.

[0012] Fourthly, embodiments of the present invention provide the use of the antibodies or their antigen-binding fragments or antibody conjugates as described above in the preparation of products for detecting HPV type 16 and / or 18 E1 protein.

[0013] The present invention has the following beneficial effects:

[0014] The antibodies or antigen-binding fragments provided in this invention can specifically bind to HPV type 16 and / or type 18 E1 proteins, and have advantages such as high detection sensitivity, good specificity, high accuracy and good repeatability, providing a way for the effective detection of HPV type 16 and / or type 18 E1 proteins. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1Here is a flowchart of the sandwich ELISA experiment.

[0017] Figure 2 Standard curves for detecting HPV18 E1 antigen using E1-4 and E1-8 monoclonal antibodies. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0020] Definition of noun

[0021] The term “antibody” as used in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to HPV E1 antigen or fragments thereof.

[0022] The term "antigen-binding fragment" as used herein refers to a portion of a complete antibody that specifically binds to the antigen to which the complete antibody is bound. Those skilled in the art will readily understand from the contents of this disclosure that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds, or by recombinant genetic techniques or by automated peptide synthesizers (such as Applied BioSystems' automated peptide synthesizers).

[0023] In this article, the term "CDR" stands for "complementarity-determining region," which refers to the highly variable region of the heavy and light chains of an immunoglobulin, specifically the region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0024] The term "framework region" in this article, synonymous with "backbone region" or "FR region," refers to the region of the antibody's heavy chain variable region excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0025] The term "identity" percentage used herein refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage can be determined using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine the appropriate parameters for the aligned sequences.

[0026] The antibodies against HPV16 and / or 18 E1 proteins or their antigen-binding fragments provided in this invention can be applied to quality control during the production process of HPV16 and / or 18 E1 proteins. They can efficiently and accurately quantify the content of HPV16 and / or 18 E1 protein antigens in products such as vaccines, thereby monitoring the quality of vaccines.

[0027] On one hand, embodiments of the present invention provide an antibody or antigen-binding fragment thereof against HPV type 16 and / or type 18 E1 protein, comprising:

[0028] The amino acid sequences are HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:15.

[0029] On the other hand, embodiments of the present invention provide an antibody or antigen-binding fragment thereof against HPV type 16 and / or type 18 E1 protein, comprising:

[0030] The amino acid sequences are HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:21 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:22.

[0031] The antibodies or antigen-binding fragments provided in the embodiments of the present invention have high affinity and high binding activity for HPV type 16 and / or type 18 E1 proteins.

[0032] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one or a combination of systems from Kabat, Chothia, IMGT, AbM, or Contact.

[0033] In some embodiments, the HPV16 and / or 18 E1 protein includes at least one of the HPV16 E1 protein, the HPV18 E1 protein, and the HPV16-18 E1 fusion protein.

[0034] In some embodiments, the amino acid sequence of the HPV16 E1 protein is shown in SEQ ID NO:27.

[0035] In some embodiments, the amino acid sequence of the HPV18 E1 protein is shown in SEQ ID NO:23.

[0036] In some embodiments, the amino acid sequence of the HPV16-18 E1 fusion protein is as shown in SEQ ID NO:28.

[0037] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1~3 and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, LVS and SEQ ID NO:5.

[0038] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, 2 and 17, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, LVS and SEQ ID NO:5.

[0039] In some embodiments, the antibody or its antigen-binding fragment further includes frame regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from (a) or (b):

[0040] (a) The amino acid sequences of HFR1, HFR2, HFR3 and HFR4 are at least 80% identical to the sequences shown in SEQ ID NO: 6-9; the amino acid sequences of LFR1, LFR2, LFR3 and LFR4 are at least 80% identical to the sequences shown in SEQ ID NO: 10-13.

[0041] (b) The amino acid sequences of HFR1, HFR2, HFR3 and HFR4 are at least 80% identical to the sequences shown in SEQ ID NO:6, 18, 19 and 9, respectively; the amino acid sequences of LFR1, LFR2, LFR3 and LFR4 are at least 80% identical to the sequences shown in SEQ ID NO:10, 11, 20 and 13, respectively.

[0042] In some embodiments, at least 80% can be any one or a range between any two of 80%, 82%, 84%, 86%, 88%, 90%, 2%, 94%, 96%, 98%, and 100%.

[0043] In some embodiments, the antibody or its antigen-binding fragment further includes a constant region.

[0044] In some embodiments, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0045] In some embodiments, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0046] In some embodiments, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions.

[0047] In some embodiments, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0048] In some embodiments, the antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0049] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand from the description of this invention that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. The antigen-binding fragments of the aforementioned antibodies can also be obtained using recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0050] On the other hand, embodiments of the present invention also provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0051] In some embodiments, the antibody-drug conjugate further includes a label, purification tag, and / or solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0052] A marker is a substance that possesses properties that can be directly observed with the naked eye or detected by instruments, such as luminescence, color development, or radioactivity. These properties enable qualitative or quantitative detection of the corresponding target analyte. In practical applications, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the scope of protection of this invention.

[0053] In some embodiments, the markers include fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0054] In some embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy...). 5. Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0055] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0056] In some embodiments, the radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and18 F.

[0057] In some embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0058] In some embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; the nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0059] In some embodiments, the solid support includes, but is not limited to, microspheres, plates, and membranes.

[0060] In some embodiments, the solid support includes any one or more of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0061] On the other hand, embodiments of the present invention also provide a reagent or kit comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or the antibody conjugate as described in any of the foregoing embodiments.

[0062] In some embodiments, the antibodies or antigen-binding fragments thereof described in any of the foregoing embodiments can be used as detection antibodies and / or capture antibodies (coated antibodies). For example, antibodies or antigen-binding fragments thereof containing HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14 and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:15 can be used as capture antibodies (or detection antibodies), and antibodies or antigen-binding fragments thereof containing HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:21 and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:22 can be used as detection antibodies (or capture antibodies). For example, antibodies or antigen-binding fragments thereof containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1~3 and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, LVS, and SEQ ID NO:5 can be used as capture antibodies (or detection antibodies), and antibodies or antigen-binding fragments thereof containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, 2, and 17 and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, LVS, and SEQ ID NO:5 can be used as detection antibodies (or capture antibodies).

[0063] In some embodiments, the kit includes a detection kit.

[0064] In some embodiments, the detection includes an immune detection.

[0065] In some embodiments, the immunoassay includes enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (IFA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), colloidal gold immunochromatography, and other non-enzyme-linked antibody binding assays or methods.

[0066] In some embodiments, the kit may further include reagents required for immunoassay. For example, an ELISA kit may also include any one or more of a diluent, an elution buffer, and a chromogenic solution.

[0067] Furthermore, embodiments of the present invention also provide the application of antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, or antibody conjugates as described in any of the foregoing embodiments, in the preparation of products for detecting HPV18 E1 protein.

[0068] In some embodiments, the product includes any one or more of reagents, kits, test strips, and chips.

[0069] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0070] Example 1

[0071] This embodiment provides two monoclonal antibodies against HPV E1 protein, named E1-4 and E1-8. Sequencing revealed the amino acid sequences of the heavy chain variable region and light chain variable region of E1-4 as shown in SEQ ID NO:14 and 15, and the amino acid sequences of the heavy chain variable region and light chain variable region of E1-8 as shown in SEQ ID NO:21 and 22. It should be noted that both E1-4 and E1-8 are IgG type antibodies, and the Fc is a mouse-derived Fc.

[0072] The preparation methods for E1-4 and E1-8 specifically include the following steps.

[0073] Mouse immunization: Female BALB / c mice were selected and subcutaneously immunized with complete Freund's adjuvant (CFA) emulsified with HPV E1 protein (the amino acid sequence of which is shown in SEQ ID NO:27). Two weeks later, the mice were immunized with incomplete Freund's adjuvant (IFA) emulsified with the same protein. The immunization was repeated multiple times after a two-week interval until the serum titer was qualified.

[0074] (2) Cell fusion: Mouse spleen cells with qualified immunogenicity were fused with mouse myeloma cells SP2 / 0 at a ratio of 5:1 with 50% PEG, suspended in 1% HAT selective medium and cultured for 7-10 days to observe the growth of cloned cells.

[0075] (3) Screening of hybridoma cell lines: Cell lines that react with all of the above antigens were screened by indirect ELISA (coated with HPV16 E1 antigen (SEQ ID NO:27), HPV18 E1 antigen (SEQ ID NO:23) and HPV16-18 E1 fusion protein (SEQ ID NO:28)). Then, the cells were repeatedly cloned to obtain stable hybridoma cells.

[0076] (4) Monoclonal antibody preparation: Hybridoma cells that can stably secrete anti-HPV E1 protein are injected into the peritoneal cavity of BALB / c mice treated with liquid paraffin to produce ascites in vivo. The ascites is then purified to obtain monoclonal antibodies (E1-4 and E1-8).

[0077] Example 2

[0078] This embodiment provides a sandwich ELISA kit for detecting HPV E1 protein and its detection method. The kit includes a polystyrene microplate, TMB chromogenic solution, PBST elution solution, coating antibody (E1-4), and detection antibody (E1-8).

[0079] The principle of sandwich ELISA detection method: Specific antibodies are diluted to a certain concentration and immobilized on the surface of a polystyrene microplate via physical adsorption. The sample to be tested is added, and it specifically binds to the antibody coated on the solid-phase support. Then, enzyme-labeled secondary antibody and chromogenic solution are added, and finally, a stop solution is added to terminate the reaction. Quantitative analysis is performed by measuring the absorbance at a specific wavelength. The absorbance value of the test sample is positively correlated with its antigen concentration. The concentration of the antigen in the test sample is calculated based on a standard curve. See the sandwich ELISA experimental flowchart below. Figure 1 .

[0080] Determination of linear range and working antibody concentration:

[0081] (1) The mouse monoclonal antibody (E1-4) was diluted to 4 μg / ml and coated under conventional coating conditions (coating at 2-8℃ for 16-24 hours, coating solution formula: NaHCO3 3.068 g, Na2CO3 1.435 g plus purified water to 1L).

[0082] (2) The next day, wash away the unbound coated antibody with 1×PBST, and then add blocking solution for blocking (block at 25℃ for 1 hour) to obtain the antibody-coated ELISA plate.

[0083] (3) When using, add the series of diluted standards, test sample and diluted horseradish peroxidase-labeled E1-8 monoclonal antibody in sequence. After reacting at 25°C, add the TMB colorimetric solution of the horseradish peroxidase colorimetric system. Finally, stop with 1 M phosphate and read the specific absorbance value. The labeling method for peroxidase-labeled E1-8 monoclonal antibody was as follows: (a) Weigh 4.2 mg HRP and dissolve it in 420 μl of ultrapure water; (b) Weigh 9.7 mg NaIO4 and dissolve it in 755 μl of ultrapure water; (c) Add 420 μl of solution (b) to solution (a) and react at 4°C in the dark for 30 min; (d) After the reaction, add 3.78 μl of ethylene glycol and react at room temperature in the dark for 30 min; (e) Add the solution obtained from reaction (d) and the antibody to be labeled to a dialysis bag at a 1:1 mass ratio and dialyze with 1×CBS buffer; (f) After dialysis, transfer the liquid in the dialysis bag to a beaker; then add 45 μl of NaBH4 (5 mg / ml) and react at 4°C in the dark for 3 h; (g) After the reaction, add saturated ammonium sulfate in the total volume of the first 6 steps and react at 4°C in the dark for 30 min; (h) After the reaction, remove the liquid and centrifuge at 11000 rpm at 4°C for 15 minutes. min; (i) Redissolve the precipitate with PBS, then add an equal volume of glycerol, mix well, and store;

[0084] (4) The results of the experiment showed that the optimal working dilution ratio of E1-8 enzyme-labeled antibody was 1:400; in the range of 8000~62.50 ng / ml, the absorbance value was highly linearly correlated with the detection concentration (ng / ml) (R2>0.98).

[0085] Example 3

[0086] The specificity of the sandwich ELISA detection method in Example 2 was verified.

[0087] Experimental design: Take stock solutions of 715, 716, 717 and 721, and dilute them 10 times. Then mix the diluted 715, 716, 717 and 721 (reference standard) separately. The sample addition process is shown in Table 1.

[0088] Table 1. Sample dilution

[0089]

[0090] Note: Protein 715 is the HPV16 E6-E7 fusion protein, amino acid sequence as shown in SEQ ID NO: 24; protein 716 is the HPV18 E6-E7 fusion protein, amino acid sequence as shown in SEQ ID NO: 25; protein 717 is the HPV16-18 E2 fusion protein, amino acid sequence as shown in SEQ ID NO: 26; protein 721 is the HPV18 E1 antigen, amino acid sequence as shown in SEQ ID NO: 23. Acceptance criteria: Recovery rate of each sample 80%~120%; the presence of other antigens does not interfere with the detection of the test sample antigen.

[0091] Recovery rate (%) = 721 mixed protein measured value / 721 protein measured value × 100%.

[0092] The results are shown in the table below.

[0093] Table 2. Specificity Validation Results

[0094]

[0095] Specificity verification conclusion:

[0096] Even with proteins 715, 716, and 717 present, the content of protein 721 can still be accurately detected (with a recovery rate within the acceptable range of 80%-120%), indicating that the detection method provided in this embodiment of the invention has good specificity. Specificity verification is successful.

[0097] Example 4

[0098] The precision (reproducibility) of the sandwich ELISA detection method provided in Example 2 was verified.

[0099] Experimental design: A batch of 721 protein (amino acid sequence as shown in SEQ ID NO:23) was selected and diluted with diluent to three concentrations: high, medium and low, namely 8000 ng / ml, 4000 ng / ml and 2000 ng / ml, respectively. Then, it was serially diluted 2-fold in slabs for detection.

[0100] During testing, each concentration was measured three times. The CV value of the three repeated measurements was calculated.

[0101] Acceptance criteria: The test sample must have at least 3 dilutions within the linear range of the standard curve; the CV of the three retests of the same batch of test samples must be ≤20%.

[0102] The results of the precision (repeatability) verification are shown in the table below.

[0103] Table 3. Precision Validation Results

[0104]

[0105] Precision (repeatability) conclusion: The CV between the three retest values ​​for each concentration was less than 20% (1.5%, 6.3%, and 1.8%, respectively). Repeatability verification passed.

[0106] Example 5

[0107] The precision (intermediate precision) of the sandwich ELISA detection method provided in Example 2 was verified.

[0108] Experimental design: On another day, three experimenters will repeat the "reproducibility" experiment and calculate the differences between daytime and individual participants.

[0109] Acceptance criteria: For the same batch of test samples from different dates and different personnel, the CV value should be ≤20%.

[0110] The results of the precision (intermediate precision) verification are shown in the table below.

[0111] Table 4. Intermediate precision verification results

[0112]

[0113] Precision (intermediate precision) conclusion: The CV of the 721 antigen detection results on different dates and by different experimenters was less than 20% (4.2%, 2.2%, and 2.5%, respectively), and the intermediate precision verification was qualified.

[0114] Example 6

[0115] The accuracy of the sandwich ELISA detection method provided in Example 2 was verified.

[0116] Experimental design: One batch of 721 stock solution (i.e. 721 protein, amino acid sequence as shown in SEQ ID NO:23) was selected, diluted with diluent according to Table 5-6, and then spiked. The spiking process is shown in Table 7. C1, C2, C3, M1, M2, M3, c1, c2, and c3 were measured and the recovery rate was calculated.

[0117] Recovery rate (%) = (Spiked sample measured value - Standard sample measured value) / Standard sample measured value × 100%.

[0118] Acceptance criteria: Recovery rate between 80% and 120%.

[0119] Table 5. Dilution process of 721 stock solution

[0120]

[0121] Table 6. Dilution process of 721 reference standard

[0122]

[0123] Table 7. Accuracy Calibration Process

[0124]

[0125] The accuracy verification results are shown in the table below.

[0126] Table 8. Accuracy Verification Results

[0127]

[0128] Note: Measured value after spike = Measured value of spiked sample - Measured value of the original sample

[0129] Accuracy verification conclusion: The recoveries of spiked samples at all concentrations were between 80% and 120% (93%, 95%, and 98%, respectively), and the accuracy verification was qualified.

[0130] Example 7

[0131] The sandwich ELISA detection method provided in Example 2 was validated using standard curves (linearity and range).

[0132] Experimental design: Summarize the standard curve data in precision, accuracy, and specificity verification to obtain the linearity of the standard curve and the optimal detection range.

[0133] Acceptance criteria: The correlation coefficients of the standard curves (four-parameter fitted curves) are all not less than 0.98, and the R-values ​​of each standard curve are [not specified]. 2 The CV is ≤10%; the optimal detection range of the standard curve is consistent.

[0134] The verification results of the standard curve (linearity and range) are shown in the table below.

[0135] Table 9. Validation results of the standard curve (linearity and range)

[0136]

[0137] Standard curve (linearity and range) verification conclusion: Based on the previous verifications, the standard curve has good linearity, and R0... 2 All are greater than 0.99, R 2 The CV% was less than 10%, meeting the validation criteria; the linear range of the standard curve was consistent, ranging from 8000 to 62.50 ng / ml. The standard curve is shown below. Figure 2 .

[0138] Example 8

[0139] This embodiment provides a kit comprising a polystyrene microplate on which E1-8 monoclonal antibody (coated antibody) is immobilized. The kit also includes enzyme-labeled detection antibody (E1-4), TMB chromogenic solution and PBST elution solution.

[0140] (1) According to the conventional coating conditions, the E1-8 antibody was diluted at a certain ratio for coating (coating at 2-8℃ for 16-24 hours, coating solution formula: NaHCO3 3.068 g, Na2CO3 1.435 g, plus purified water to make up to 1L).

[0141] (2) The next day, wash away the unbound coated antibody with 1×PBST, and then add blocking solution for blocking (block at 25℃ for 1 hour) to obtain the antibody-coated ELISA plate.

[0142] (3) When using, add the series of diluted standards, test sample and diluted horseradish peroxidase-labeled (E1-4) monoclonal antibody in sequence. After reacting at 25°C, add the TMB colorimetric solution of the horseradish peroxidase colorimetric system. Finally, stop with 1 M phosphate and read the specific absorbance value.

[0143] The sequence information involved in this application is shown in the table below.

[0144] Table 10 Sequence

[0145]

[0146] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against HPV type 16 and / or 18 E1 protein, characterized in that, It includes: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

15. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1~3 and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, LVS and SEQ ID NO:

5.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that, The antibody or its antigen-binding fragment further includes frame regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein the amino acid sequences of HFR1, HFR2, HFR3, and HFR4 are at least 80% identical to the sequences shown in SEQ ID NO: 6-9; and the amino acid sequences of LFR1, LFR2, LFR3, and LFR4 are at least 80% identical to the sequences shown in SEQ ID NO: 10-13.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that, The antibody or its antigen-binding fragment further includes a constant region; the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; the constant region includes a heavy chain constant region and / or a light chain constant region; the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

6. An antibody conjugate, characterized in that, It consists of the following components: the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5; A label, purification tag, and / or solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

7. A reagent or kit, characterized in that, It includes: The antibody or its antigen-binding fragment according to any one of claims 1 to 5, or the antibody conjugate according to claim 6.

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or the antibody conjugate as described in claim 6, in the preparation of products for detecting HPV type 16 and / or HPV type 18 E1 protein.

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