Anti-human papilloma virus E1 protein antibody or kit and related application thereof

Abstract

The invention discloses an antibody for resisting human papilloma virus E1 protein or a kit and related application thereof, and relates to the field of immunodetection, the antibody comprises HCDR1, HCDR2 and HCDR3 in a heavy chain variable region with the amino acid sequence shown as SEQ ID NO: 14 and LCDR1, LCDR2 and LCDR3 in a light chain variable region with the amino acid sequence shown as SEQ ID NO: 15, the antibody can be specifically combined with HPV16 type and / or 18 type E1 protein, and the HPV16 type and / or 18 type E1 protein can be specifically combined with the HPV16 type and / or 18 type E1 protein. The kit has the advantages of high detection sensitivity, strong specificity, high accuracy, good repeatability and the like.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay, and in particular to an antibody against human papillomavirus E1 protein or a kit thereof and related applications. Background Art

[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 primarily transmitted through sexual intercourse or close contact. Over 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 primarily include HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68, which can cause genital warts, genital cancer, cervical cancer, and high-grade cervical intraepithelial neoplasia. Infection with HPV types 16 and 18 is particularly important as a risk factor for the development of cervical cancer. Cervical cancer is one of the most common malignancies and a major public health concern affecting women worldwide. Its morbidity and mortality rate rank fourth among female cancers worldwide, 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 and about 300,000 deaths worldwide each year. However, in socioeconomically developed countries, the incidence and mortality rates of cervical cancer are lower.

[0003] Human papillomavirus (HPV) is a small, non-enveloped, double-stranded, closed-circular DNA virus composed of a viral protein coat and a core DNA core. The early transcription region of the HPV genome contains multiple proteins, including E1, E2, and E8. E1 plays a key role in the initiation of viral DNA replication, E2 is involved in transactivation of viral DNA transcription, and E4 disrupts the keratin intermediate filament network. E6 and E7 are major oncogenic proteins, expressed continuously throughout the carcinogenesis process and involved in the malignant transformation of cells. E6 and E7 are important contributors to cervical cancer and are therefore considered ideal target antigens for therapeutic HPV vaccines.

[0004] Currently available preventive HPV vaccines are based on virus-like particles (VLPs). VLPs are composed of a single viral protein and are non-infectious and non-carcinogenic. However, no studies have demonstrated the therapeutic effects of VLP vaccines. Therefore, they are unable to alter viral infection and progression in subjects already infected with HPV prior to vaccination. HPV-infected patients currently lack treatment options. Therefore, developing a therapeutic HPV vaccine is an ideal strategy for controlling existing HPV infections and treating HPV-related cancers and precancerous lesions. It also closely addresses clinical needs and aligns with market trends.

[0005] Quality control of the HPV vaccine's main active ingredients is crucial during the development and production of HPV vaccines. Currently, there is a lack of high-performance anti-human papillomavirus E1 antibodies on the market, which limits the accuracy and reliability of HPV E1 antigen testing.

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

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

[0008] The present invention is achieved in that: In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof against HPV16 and / or 18 E1 protein, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 14 and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in the amino acid sequence of SEQ ID NO: 15.

[0009] In a second aspect, an embodiment of the present invention provides an antibody conjugate comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0010] In a third aspect, an embodiment of the present invention provides a reagent or kit comprising: the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0011] In a fourth aspect, embodiments of the present invention provide use of the above-mentioned antibody or antigen-binding fragment thereof or antibody conjugate in the preparation of a product for detecting HPV type 16 and / or type 18 E1 protein.

[0012] The present invention has the following beneficial effects: The antibodies or antigen-binding fragments thereof provided in the embodiments of the present invention can specifically bind to HPV16 and / or 18 E1 proteins, and have the advantages of high detection sensitivity, good specificity, high accuracy and good repeatability, providing a way to effectively detect HPV16 and / or 18 E1 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 This is the flow chart of the sandwich ELISA experiment; Figure 2 The standard curve for E1-4 and E1-8 monoclonal antibodies to detect HPV18 E1 antigen. DETAILED DESCRIPTION

[0015] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0016] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0017] Definition of noun The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, so long as they exhibit the desired biological activity, such as specific binding to HPV E1 antigen or fragments thereof.

[0018] The term "antigen-binding fragment" as used herein refers to a portion of an intact antibody that specifically binds to the antigen bound by the intact antibody. Those skilled in the art will readily appreciate, based on the disclosure herein, that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds. They can also be synthesized using recombinant genetic techniques or automated peptide synthesizers (e.g., Applied BioSystems automated peptide synthesizers).

[0019] The term "CDR" as used herein is the same as "complementarity determining region" and refers to the highly variable regions of the heavy and light chains of immunoglobulins, comprising 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 its recognized antigen or epitope.

[0020] The term "framework region," as used herein, is synonymous with "framework region" or "FR" region and refers to the regions of the heavy chain variable region of an antibody excluding the CDR regions. The heavy chain framework region can be further subdivided into contiguous regions separated by CDRs (FR1, FR2, FR3, and FR4). The heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, comprising the HFR1, HFR2, HFR3, and HFR4 framework regions. The heavy chain variable region is formed by the following numbered CDRs and FRs (arranged from amino-terminus to carboxyl-terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0021] As used herein, the term "percent identity" refers to the degree to which amino acids in two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Comparison of amino acid sequence identity percentages can be performed using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, software well known in the art. Appropriate parameters for aligning sequences can be determined by those skilled in the art.

[0022] The antibodies or antigen-binding fragments thereof against HPV16 and / or 18 E1 proteins provided in the embodiments of the present invention can be used for quality control in the production process of HPV16 and / or 18 E1 proteins. They can efficiently and accurately quantify the HPV16 and / or 18 E1 protein antigen content in products such as vaccines, thereby monitoring the quality of the vaccines.

[0023] In one aspect, an embodiment of the present invention provides an antibody or antigen-binding fragment thereof against HPV type 16 and / or type 18 E1 protein, comprising: The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 14, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 15.

[0024] On the other hand, an embodiment of the present invention provides an antibody or antigen-binding fragment thereof against HPV type 16 and / or type 18 E1 protein, comprising: The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 21, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 22.

[0025] The antibodies or antigen-binding fragments thereof 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.

[0026] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems, or a combination of multiple systems.

[0027] In some embodiments, the HPV16 and / or 18 E1 proteins include at least one of the HPV16 E1 protein, the HPV18 E1 protein, and the HPV16-18 E1 fusion protein.

[0028] In some embodiments, the amino acid sequence of the HPV16 type E1 protein is shown in SEQ ID NO:27.

[0029] In some embodiments, the amino acid sequence of the HPV18 type E1 protein is shown in SEQ ID NO:23.

[0030] In some embodiments, the amino acid sequence of the HPV16-18 E1 fusion protein is as SEQ ID NO: 28.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 4, LVS and SEQ ID NO: 5.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 16, 2 and 17, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4, LVS and SEQ ID NO: 5, respectively.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof further comprises framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from (a) or (b): (a) the amino acid sequences of HFR1, HFR2, HFR3 and HFR4 are at least 80% identical to the sequences shown in SEQ ID NOs: 6 to 9, respectively; the amino acid sequences of LFR1, LFR2, LFR3 and LFR4 are at least 80% identical to the sequences shown in SEQ ID NOs: 10 to 13, respectively; (b) the amino acid sequences of HFR1, HFR2, HFR3 and HFR4 are at least 80% identical to the sequences shown in SEQ ID NOs: 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 NOs: 10, 11, 20 and 13, respectively.

[0034] In some embodiments, at least 80% can be any one of 80%, 82%, 84%, 86%, 88%, 90%, 2%, 94%, 96%, 98% and 100%, or a range between any two of them.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0036] In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

[0037] In some embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0038] 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.

[0039] In some embodiments, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0040] In some embodiments, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0041] The antigen-binding fragments of the aforementioned antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that functional fragments of the aforementioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds. Antigen-binding fragments of the aforementioned antibodies can also be synthesized using recombinant genetic techniques known to those skilled in the art or using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0042] On the other hand, an embodiment of the present invention further provides an antibody conjugate, comprising: the antibody or antigen-binding fragment thereof described in any of the aforementioned embodiments.

[0043] In some embodiments, the antibody conjugate further comprises a label, a purification tag and / or a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0044] A marker is a substance that possesses properties that can be observed directly by the naked eye or detected or detected by an instrument, such as luminescence, color development, or radioactivity. These properties enable qualitative or quantitative detection of the corresponding target. In actual use, those skilled in the art can select an appropriate marker based on the detection conditions or actual needs. Regardless of the marker used, it is within the scope of protection of the present invention.

[0045] In some embodiments, the labels include fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle labels.

[0046] In some embodiments, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.57, Cy3.58, Cy3.59, Cy3.60, Cy3.70, Cy3.71, Cy3.72, Cy3.73, Cy3.89, Cy3.90, Cy3.91, Cy3.92, Cy3.93, Cy3.94, Cy3.95, Cy3.96, Cy3.97, Cy3.98, Cy3.99, Cy3.91, 5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0047] 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 deoxygenase.

[0048] In some embodiments, the radioisotopes include but are not limited to 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 Such as 62 Cu, 64 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and18 F.

[0049] In some embodiments, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanine and its derivatives, and peroxalate and its derivatives.

[0050] In some embodiments, the nanoparticle markers include but are not limited to nanoparticles and colloids; nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0051] In some embodiments, the solid support includes, but is not limited to, microspheres, plates, and membranes.

[0052] In some embodiments, the solid phase carrier comprises any one or more of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0053] On the other hand, an embodiment of the present invention further provides a reagent or kit, which includes: the antibody or antigen-binding fragment thereof described in any of the foregoing embodiments or the antibody conjugate described in any of the foregoing embodiments.

[0054] In some embodiments, the antibodies or antigen-binding fragments thereof described in any of the preceding embodiments can be used as detection antibodies and / or capture antibodies (coating antibodies). For example, an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region set forth in SEQ ID NO:14 and the amino acid sequence of LCDR1, LCDR2, and LCDR3 in the light chain variable region set forth in SEQ ID NO:15 can be used as a capture antibody (or detection antibody), and an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region set forth in SEQ ID NO:21 and the amino acid sequence of LCDR1, LCDR2, and LCDR3 in the light chain variable region set forth in SEQ ID NO:22 can be used as a detection antibody (or capture antibody). For another example, an antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, LVS and SEQ ID NO: 5, can be used as a capture antibody (or detection antibody); an antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 16, 2 and 17, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 4, LVS and SEQ ID NO: 5, can be used as a detection antibody (or capture antibody).

[0055] In some embodiments, the kit comprises a detection kit.

[0056] In some embodiments, the detecting comprises immunodetection.

[0057] In some embodiments, the immunoassay includes enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (IFA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), colloidal gold immunochromatography, and other non-enzyme-linked antibody binding assays or methods.

[0058] In some embodiments, the kit further includes reagents required for immunoassay. For example, an ELISA detection kit may further include any one or more of a diluent, an eluent, and a developer.

[0059] In addition, an embodiment of the present invention further provides use of the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, or the antibody conjugate as described in any of the foregoing embodiments, in the preparation of a product for detecting HPV18 type E1 protein.

[0060] In some embodiments, the product includes any one or more of a reagent, a kit, a test strip, and a chip.

[0061] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0062] Example 1 This example provides two monoclonal antibodies against the HPV E1 protein, designated E1-4 and E1-8. Sequencing revealed that the amino acid sequences of the heavy and light chain variable regions of E1-4 are shown in SEQ ID NOs: 14 and 15, and the amino acid sequences of the heavy and light chain variable regions of E1-8 are shown in SEQ ID NOs: 21 and 22. It should be noted that both E1-4 and E1-8 are IgG antibodies, and their Fc fragments are murine.

[0063] The preparation method of E1-4 and E1-8 specifically includes the following steps.

[0064] Mouse Immunization: Female BALB / c mice were subcutaneously immunized with HPV E1 protein (amino acid sequence shown in SEQ ID NO:27) emulsified in complete Freund's adjuvant (CFA). Two weeks later, they were immunized with the same protein emulsified in incomplete Freund's adjuvant (IFA). Immunizations were repeated multiple times at two-week intervals until the serum titer reached a satisfactory level. (2) Cell fusion: Take the spleen cells of mice with qualified immune titers and mouse myeloma cells SP2 / 0, fuse them with 50% PEG at a ratio of 5:1, suspend them in 1% HAT selection medium and culture them. Observe the growth of cloned cells for 7-10 days; (3) Hybridoma cell line screening: Cell lines that react to HPV16 E1 antigen (SEQ ID NO: 27), HPV18 E1 antigen (SEQ ID NO: 23), and HPV16-18 E1 fusion protein (SEQ ID NO: 28) were screened by indirect ELISA (using HPV16 E1 antigen (SEQ ID NO: 27), HPV18 E1 antigen (SEQ ID NO: 23), and HPV16-18 E1 fusion protein (SEQ ID NO: 28) for coating) and then cloned repeatedly to obtain stable hybridoma cells; (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 collected for purification to obtain monoclonal antibodies (E1-4 and E1-8).

[0065] Example 2 This embodiment provides a sandwich ELISA kit for detecting HPV E1 protein and a detection method thereof. The kit includes a polystyrene microplate, TMB color development solution, PBST eluent, a coating antibody (E1-4), and a detection antibody (E1-8).

[0066] The principle of the sandwich ELISA detection method is: the specific antibody is diluted to a certain concentration and then fixed on the surface of the polystyrene microplate by physical adsorption. The sample to be tested is added and specifically binds to the antibody coated on the solid phase carrier. Then, the enzyme-labeled secondary antibody and color development solution are added. Finally, the stop solution is added to terminate the reaction. Quantitative analysis is performed by measuring the absorbance value 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 the standard curve. The flow chart of the sandwich ELISA experiment is shown in Figure 1 .

[0067] Determination of linear range and antibody working concentration: (1) According to conventional coating conditions, the mouse monoclonal antibody (E1-4) was diluted to 4 μg / ml for coating (coating at 2-8°C for 16-24 hours, coating solution formula: NaHCO3 3.068 g, Na2CO3 1.435 g, add purified water to 1 L); (2) The next day, wash off the unbound coated antibody with 1×PBST, then add blocking solution for blocking (blocking at 25°C for 1 hour), thus obtaining the antibody-coated ELISA plate; (3) When using, add serially diluted standard, test sample and diluted horseradish peroxidase-labeled E1-8 monoclonal antibody in sequence, react at 25°C, add TMB colorimetric solution of horseradish peroxidase colorimetric system, and finally terminate with 1 M phosphoric acid and read the specific absorbance value. The labeling method of the E1-8 monoclonal antibody labeled with root peroxidase is as follows: (a) 4.2 mg HRP was weighed and dissolved in 420 μl ultrapure water; (b) 9.7 mg NaIO4 was weighed and dissolved in 755 μl ultrapure water; (c) 420 μl of solution (b) was added to (a) and reacted in the dark at 4°C for 30 min; (d) after the reaction, 3.78 μl ethylene glycol was added and reacted in the dark at room temperature for 30 min; (e) the solution obtained in the reaction (d) and the antibody to be labeled were added to a dialysis bag in a mass ratio of 1:1 and dialyzed in 1× CBS buffer; (f) after the dialysis was completed, the liquid in the dialysis bag was transferred to a beaker; then 45 μl NaBH4 (5 mg / ml) was added and reacted in the dark at 4°C for 3 h; (g) after the reaction was completed, saturated ammonium sulfate equivalent to the total volume of the previous six reactions was added and reacted in the dark at 4°C for 30 min; (h) after the reaction was completed, the liquid was removed and centrifuged at 11,000 rpm at 4°C for 15 min. min; (i) Redissolve the precipitate in PBS, then add an equal volume of glycerol, mix well, and store; (4) After testing, the results 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).

[0068] Example 3 The specificity of the sandwich ELISA detection method of Example 2 was verified.

[0069] Experimental design: Take the stock solutions of 715, 716, 717 and 721, dilute them 10 times, and then mix the diluted 715, 716, 717 with the diluted 721 (reference product). The sampling process is shown in Table 1.

[0070] Table 1. Sample dilution

[0071] Note: Protein 715 is the HPV16 E6-E7 fusion protein, with the amino acid sequence shown in SEQ ID NO: 24; protein 716 is the HPV18 E6-E7 fusion protein, with the amino acid sequence shown in SEQ ID NO: 25; protein 717 is the HPV16-18 E2 fusion protein, with the amino acid sequence shown in SEQ ID NO: 26; protein 721 is the HPV18 E1 antigen, with the amino acid sequence shown in SEQ ID NO: 23. Acceptance criteria: Sample recovery of 80%-120%; the presence of other antigens should not interfere with the detection of the test antigen.

[0072] Recovery rate (%) = 721 mixed protein measured value / 721 protein measured value × 100%.

[0073] See the table below for the results.

[0074] Table 2. Specificity verification results

[0075] Specificity verification conclusion: In the presence of proteins 715, 716, and 717, the 721 protein content can still be accurately detected (with a recovery rate within the acceptable range of 80%-120%), indicating that the detection method provided by this embodiment of the present invention has good specificity. The specificity verification is qualified.

[0076] Example 4 The sandwich ELISA detection method provided in Example 2 was verified for precision (repeatability).

[0077] Experimental Design: A batch of 721 proteins (amino acid sequence shown in SEQ ID NO:23) was selected and diluted with diluent to high, medium, and low concentrations of 8000 ng / ml, 4000 ng / ml, and 2000 ng / ml, respectively. Eight 2-fold serial dilutions were then performed within the plate for testing.

[0078] During the test, each concentration was measured three times, and the CV value of the three repeated tests was calculated.

[0079] Acceptance criteria: The test sample must have at least three dilutions within the linear range of the standard curve; the CV of three replicates of the same batch of test sample must be ≤20%.

[0080] The precision (repeatability) verification results are shown in the following table.

[0081] Table 3. Precision verification results

[0082] Precision (Repeatability) Conclusion: The CV between the three repeated test values ​​for each concentration was less than 20% (1.5%, 6.3%, and 1.8%, respectively). Repeatability verification passed.

[0083] Example 5 The sandwich ELISA detection method provided in Example 2 was verified for precision (intermediate precision).

[0084] Experimental design: Three experimenters repeated the "repeatability" experiment on another day and calculated the day-to-day and individual differences.

[0085] Acceptance criteria: CV of test values ​​of the same batch of test products obtained by different personnel on different dates is ≤ 20%.

[0086] The precision (intermediate precision) verification results are shown in the following table.

[0087] Table 4. Intermediate precision verification results

[0088] Precision (intermediate precision) conclusion: The CV of the 721 antigen test results on different dates and by different laboratory technicians was less than 20% (4.2%, 2.2%, and 2.5%, respectively), and the intermediate precision verification was qualified.

[0089] Example 6 The sandwich ELISA detection method provided in Example 2 was verified for accuracy.

[0090] Experimental design: One batch of 721 stock solution (i.e., 721 protein, amino acid sequence shown in SEQ ID NO: 23) was selected and diluted with diluent according to Tables 5 and 6. The solution was then spiked (see Table 7 for the spiked process). C1, C2, C3, M1, M2, M3, c1, c2, and c3 were measured, and the recovery was calculated.

[0091] Recovery rate (%) = (measured value of spiked sample - measured value of sample) / measured value of standard product × 100%.

[0092] Acceptance criteria: Recovery rate between 80% and 120%.

[0093] Table 5. Dilution process of 721 stock solution

[0094] Table 6. Dilution process of 721 reference product

[0095] Table 7. Accuracy Spiking Procedure

[0096] The accuracy verification results are shown in the following table.

[0097] Table 8. Accuracy verification results

[0098] Note: Measured value of spiked sample = Measured value of spiked sample - Measured value of sample Accuracy verification conclusion: The recovery rates of spiked samples at each concentration were between 80% and 120% (93%, 95% and 98% respectively), and the accuracy verification was qualified.

[0099] Example 7 The sandwich ELISA detection method provided in Example 2 was validated by standard curve (linearity and range).

[0100] Experimental design: Summarize the standard curve data from precision, accuracy, and specificity verification to obtain the linearity and optimal detection range of the standard curve.

[0101] Acceptance criteria: The correlation coefficient of the standard curve (four-parameter fitting curve) is not less than 0.98, and the R 2 The CV was ≤10%; the optimal detection range of the standard curve was consistent.

[0102] The standard curve (linearity and range) verification results are shown in the following table.

[0103] Table 9 Standard curve (linearity and range) verification results

[0104] Verification conclusion of standard curve (linearity and range): Based on the previous verifications, the standard curve has good linearity and R 2 All greater than 0.99, R 2 The CV% is less than 10%, which meets the validation criteria; the linear range of the standard curve is consistent, which is 8000~62.50ng / ml. Figure 2 .

[0105] Example 8 This embodiment provides a kit comprising a polystyrene microplate with E1-8 monoclonal antibody (coating antibody) immobilized on the surface of the plate. The kit also comprises: an enzyme-labeled detection antibody (E1-4), TMB color development solution, and PBST eluent.

[0106] (1) According to conventional coating conditions, the E1-8 antibody was diluted in a certain ratio for coating (coating at 2-8°C for 16-24 hours, coating solution formula: NaHCO3 3.068 g, Na2CO3 1.435 g, add purified water to 1 L).

[0107] (2) The next day, wash off the unbound coated antibody with 1×PBST, then add blocking solution for blocking (blocking at 25°C for 1 hour), thus obtaining the antibody-coated ELISA plate; (3) When using, add serially diluted standard, test sample and diluted horseradish peroxidase-labeled (E1-4) monoclonal antibody in sequence, react at 25°C, add TMB colorimetric solution of horseradish peroxidase colorimetric system, and finally terminate with 1 M phosphoric acid and read the specific absorbance value.

[0108] The sequence information involved in this application is shown in the following table.

[0109] Table 10 Sequence

[0110] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against HPV16 and / or 18 E1 protein, characterized in that: It comprises: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 14, and LCDR1, LCDR2 and LCDR3 in the light chain variable region with the amino acid sequence shown in SEQ ID NO:

15.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems, or a combination of multiple systems.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 4, LVS and SEQ ID NO:

5.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof further comprises framework 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 NOs: 6 to 9, respectively; and the amino acid sequences of LFR1, LFR2, LFR3 and LFR4 are at least 80% identical to the sequences shown in SEQ ID NOs: 10 to 13, respectively.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof further includes a constant region; the species origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; the constant region includes a heavy chain constant region and / or a light chain constant region; the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD heavy chain constant regions or a combination of multiple constant regions; the light chain constant region is selected from κ-type or λ-type light chain constant regions.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

7. An antibody conjugate, characterized in that It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The antibody conjugate according to claim 7, characterized in that The antibody conjugate further comprises a label, a purification tag and / or a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

9. A reagent or kit, characterized in that It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the antibody conjugate according to claim 7 or 8.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the antibody conjugate according to claim 7 or 8, in the preparation of a product for detecting HPV type 16 and / or type 18 E1 protein.

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