Monoclonal antibodies for multiplex human papillomavirus detection

Abstract

The present invention relates to monoclonal antibodies for detecting multiple types of human papillomavirus. Specifically, the present invention first provides an isolated polypeptide selected from: (1) SEQ ID NO: 1; (2) a fragment of SEQ ID NO: 1 having at least 12 to 21 amino acid residues; (3) a sequence having a cysteine ​​residue at the N-terminus and / or C-terminus of the sequence shown in (1) and (2); and (4) a fusion protein formed by the sequences (1) to (3) and a carrier protein used in antibody preparation or detection. The present invention also provides antibodies and antibody combinations that specifically bind to the polypeptide. The antibody combination of the present invention can detect multiple types of HPV.

Description

Technical Field

[0001] This invention relates to monoclonal antibodies for the detection of multiple types of human papillomavirus. Background Technology

[0002] Human papillomavirus (HPV) was first discovered in 1933, and the first case of genital HPV was identified in 1978. Currently, based on the different genes expressed by HPV DNA sequences, more than 200 HPV subtypes have been identified. Among them, gene clones of 85 HPV types have been identified, and partial genotypes of another 120 types have been identified. In addition, more than 30 types have been isolated from genital tract tissues.

[0003] HPV is classified into cutaneous and mucosal types based on the site of infection; and into high-risk (HR) and low-risk (LR) types based on its pathogenicity. High-risk types mainly include HPV16, 18, 33, 31, 58, and 52, which are associated with the development of cervical cancer; low-risk types mainly include HPV6, 11, 42, and 44, which are associated with genital warts.

[0004] HPV is now clearly a significant contributing factor to cervical cancer. Fully utilizing epidemiological survey results is crucial for developing preventative HPV screening strategies and guiding the development of screening kits. Currently, molecular biological testing dominates HPV screening as a key method for reducing cervical cancer incidence and is the only HPV testing method with clinical approval. The current research and development direction for HPV testing remains focused on genotyping, particularly distinguishing between a wider range of types. However, current testing and research still face several challenges, such as: the coverage of genotyping types; whether "low-risk" HPV infection can be ignored; the issues of alternating and multiple infections with different types; the susceptibility of individuals to HPV infection; and the relationship between the body's immune status and HPV infection. A comprehensive understanding of these issues will undoubtedly be essential for adjusting the development strategy of preventative HPV screening kits.

[0005] The primary purpose of HPV genotyping is to differentiate between HR-HPV and LR-HPV infections, thereby identifying high-risk groups for cervical cancer. Recent epidemiological surveys indicate that the potential for LR-HPV to cause tumors should be taken seriously. de Sanjose S et al. collected paraffin-embedded specimens of invasive cervical cancer from 38 countries for HPV DNA analysis. Of 10,575 invasive cancer specimens, 8,977 (85%) tested positive for HPV DNA, identifying 30 types. The most common types were HPV16, 18, 31, 33, 35, 45, 52, and 58, totaling 8,196 cases (91%, 95% CI: 90–92). Sixteen specimens showed only a single LR-HPV infection: 9 cases of HPV6, 2 cases of HPV11, 3 cases of HPV42, and 1 case each of HPV44 and HPV74, accounting for 0.18% of all cervical cancer cases. Correnti M et al. performed HPV genotyping on 28 types in cervical cancer specimens from 150 Venezuelan women. Of the 148 DNA-positive specimens, 135 were HR-HPV infections (91.2%), and one was identified as LR-HPV11 infection, accounting for 0.7%. Similarly, Alemany L et al. collected 816 cervical squamous cell carcinoma specimens from Spain between 1940 and 2007 and performed HPV genotyping on 25 types. Seven cases showed single LR-HPV infection, including three cases of HPV30, two cases of HPV6, and two cases of HPV26, accounting for 0.4%, 0.2%, and 0.2% of the positive detections, respectively. These results suggest that single LR-HPV infection may be the sole etiological factor in the development of cervical cancer.

[0006] The following two studies showed a higher detection rate of LR-HPV in cervical cancer. Alsbeih G et al. collected 100 paraffin-embedded cervical cancer specimens from Saudi Arabia and performed HPV genotyping using the Linear Array kit technique. The results showed that 89 cases were HPV positive, and 11 different types were identified. Two of these cases were single LR-HPV infections, HPV6 and HPV64, accounting for 2%. Alibegashvili T et al. collected invasive cancer specimens from 91 Georgian women for genotyping. GP5+ / GP6+ PCR amplification was first used, and positive products were then used for HPV genotyping using reverse linear hybridization, identifying 44 HPV types. The results showed a 100% HPV detection rate, with two cases being purely LR-HPV positive, HPV30 and HPV42, accounting for 2.2%. In summary, multiple studies have found that low-risk HPV can be the only detectable infection factor in cervical cancer specimens, therefore the possibility of low-risk HPV causing cancer cannot be ruled out.

[0007] Numerous epidemiological studies have demonstrated that persistent HPV infection reflects a population's or individual's susceptibility to HPV and is closely related to the individual's physical condition. When an individual's physical condition declines, such as due to immunosuppression or a compromised immune system, even a small amount of LR-HPV infection can lead to malignant transformation of cervical tissue. The distribution of cervical HPV types in women infected with human immunodeficiency virus (HIV) differs from that in the general population. HIV-infected patients with cervical CIN III and carcinoma in situ are more susceptible to infection with non-16 and non-18 HPV types (55%), and co-infected HIV patients are more prone to persistent HPV infection. LR-HPV infection also exhibits cervical malignant transformation behavior.

[0008] Entiauspe LG et al. performed HPV type testing on 100 HIV women and used PCR technology to detect 9 genotypes. Among the 68 HPV DNA positive patients, LR-HPV had the highest detection frequency, about 66.7%, of which HPV6 had the highest infection rate, accounting for 63.9%. HPV16 was the second highest, accounting for 48.5%

[17] . The results of Levi JE et al. also confirmed that HIV-infected women had the highest HPV6 infection frequency, reaching 87%

[15] . All of the above data indicate that HIV women are more likely to be infected with LR-HPV, with HPV6 having the highest infection rate. Researchers believe that in patients with co-infection of HPV / HIV, HIV can increase the viral load of HPV, promote viral persistence, and thus increase the risk of malignant transformation of the cervix.

[0009] Chaturvedi et al. conducted HPV genotyping tests on 55,871 sexually active women at the National Cancer Institute of Costa Rica and found that although HPV is a sexually transmitted disease, the distribution of HPV infection types does not seem to be significantly affected by changes in the number of sexual partners. Even women with only one sexual partner throughout their lives are still relatively common to have HPV infection. That is, regardless of whether they are infected, high-risk or low-risk, or have multiple infections, individual constitution is a very important influencing factor

[19] . In Chaturvedi's study, two patients were found to be infected with eight HPV types at the same time. He believed that these two women either had a higher risk of HPV exposure or their constitution was more likely to lead to a persistent state of infection than others. Model analysis showed that the number of sexual partners of women had a much smaller impact on the increase in multiple types of infection than the influence of constitution. Researchers believe that individual constitution is the key factor in determining HPV infection. Therefore, the role of HPV-susceptible individuals in cervical lesions should be emphasized.

[0010] The Hybrid Capture 2 Assay (HC2) is an FDA-approved HPV testing kit for clinical use. It is a semi-quantitative qualitative detection method, theoretically capable of detecting at least 5000 copies of the virus. It can detect 13 high-risk (HR-HPV) types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low-risk (LR-HPV) types (HPV6, 11, 42, 43, and 44). This method is currently the most widely used HPV clinical testing method globally.

[0011] To assess the detection capability of HC2, María-Paz Cañadas et al. compared the F-HPV typing kit (F-HPV typing™, Molgentix, Barcelona, ​​Spain) with HC2, analyzing the two tests' ability to detect HPV infection. F-HPV is a multi-pathway fluorescent PCR technology that can identify 13 HR-HPV E6 and E7 regions, and its detection type is consistent with HC2. The results showed that the HPV positivity rate detected by HC2 was 24% (37 / 155), while the F-HPV typing detection rate was 33% (49 / 155), with 22 samples showing contradictory results. The calculated consistency between the two tests was only 85.8% (133 / 155, kappa = 0.65). Based on standardized results, the sensitivity and specificity of the HC2 test were 79% and 97%, respectively. This study indicates that HC2 has a relatively high rate of false negatives.

[0012] Didelot MN et al. compared the PapilloCheck test kit (Greiner Bio-one, Frickenhausen, Germany) with HC2, and the results showed a higher evaluation for HC2. The PapilloCheck kit can additionally detect the following HPV types: HR-HPV 53, 66, 73, 82; LH-HPV 6, 11, 40, 42, 43, 44 / 55, 70. This study compared 181 cervical smear specimens. The results showed that HC2 was positive in 34 cases (18.8%), while PapilloCheck was positive in 38 cases (21.0%). Analyzing only the 13 HPV types that HC2 can detect, PapilloCheck only detected 27 positive cases (14.9%), indicating a higher detection rate for HC2. Only 11 results differed between the two methods: 9 positive and 2 negative for HC2. The two methods showed a high concordance rate (Kappa = 0.85). Nine positive samples with inconsistent results were further amplified using GP and MY universal primers for L1 sequence amplification, sequenced using a DNA sequencer, and then sequenced to determine the HPV type. Seven cases were found to be non-HC2 covered types (false positives), and two were negative. The two negative samples with inconsistent results were HPV16 and 18 infections, respectively (false negatives). The evaluation results showed that the HC2 test has low specificity but high sensitivity.

[0013] Igidbashian S et al. used the HC2 method to test for HPV in 1976 cervical excision specimens. Among them, 37 cervical lesion specimens (30 CIN III, 2 cervical squamous cell carcinomas, and 5 cervical adenocarcinomas) showed negative HC2 results. Further genotyping of the 37 negative specimens using the INNO-LiPA HPV genotyping kit revealed 33 HPV positive cases, including 22 (68.7%) HR-HPV positive cases, 10 HPV negative cases, and 5 suspected HR-HPV positive cases. Among the 22 HR-HPV specimens, most were infected with HPV types 16 or 18, with 17 cases of single infection and 5 cases of multiple HPV infection. Previous reports of false negatives in HC2 testing of CIN III and cervical cancer specimens are rare, but in this study, approximately two-thirds of the HC2 test results were false negatives, and approximately one-third were for types not covered by HC2 (misdetections).

[0014] Phillips S et al. evaluated the specificity of HC2 detection, finding that the correlation between HC2 detection results and high-grade cervical lesions was only 86.1%. Lee et al., using HC2 detection and DNA sequencing, found that only 62.6% of samples that tested positive were definitively positive. Therefore, even clinically approved tests should be treated with caution, and a single test result should not be used as a basis for diagnosis; multiple tests using different methods may be more helpful for accurate diagnosis.

[0015] Schopp B et al. compared the accuracy of three commercially available testing methods: PapilloCheck (Greiner Bio-One GmbH), SPF10PCR LiPav1 (DDL, Leiden, Netherlands), and PGMY09 / 11PCR-LBA (Roche Molecular Diagnostics). These three methods can detect 24, 25, and 38 HPV types, respectively. Results are considered consistent if two genotyping tests yield the same result. In 826 cervical samples, 716 were positive for HC2 and 110 were negative. Retesting the HC2-positive samples using the three methods yielded positive rates of 87.6%, 96.4%, and 87.1%, respectively, with SPF10PCR showing the highest positive rate. The positive rates for the 110 HC2-negative samples were 34.5%, 76.4%, and 13.6%, respectively, with SPF10PCR showing the highest positive rate and PGMY09 / 11PCR showing the lowest. The detection rates of multi-type HPV infection using the three methods were 39.5%, 55.9%, and 25.8%, respectively, with a consistency of 27.8%, indicating significant differences in detection rates. Pairwise comparisons of the results from 13 HR-HPV genotyping methods revealed average Kappa values ​​of PapilloCheck / SPF10 of 0.58±0.12, SPF10 / PGMY of 0.57±0.14, and PapilloCheck / PGMY of 0.74±0.13. Only the latter method showed satisfactory consistency. A comparison of five common HPV types (16, 18, 31, 33, and 51) revealed positive detection rates of 19.4%, 26.8%, and 17.7%; 4.0%, 16.7%, and 4.7%; 14.4%, 30.0%, and 9.0%; 4.4%, 7.3%, and 3.5%; and 9.1%, 16.6%, and 6.8%, respectively, showing significant differences in detection rates. The results suggest that different commercial detection methods exhibit significant differences in the detection of different HPV types or populations due to differences in primer design. Primers designed for different segments each have their own detection advantages and also their own omissions.

[0016] Barcellos RB compared the detection efficacy of a popular PapilloCheck kit used in the European market using a microplate colorimetric hybridization assay (MCHA). The PapilloCheck kit identifies 24 types, while the MCHA assay detects the six most common HR-HPV types (HPV16, 18, 31, 33, 39, and 45). Barcellos RB used the MCHA assay to test 108 DNA samples genotyped by the PapilloCheck kit. The concordance rates for the six HR-HPV types varied significantly: 69.4% for HPV16, 79.1% for HPV45, 82.4% for HPV18, 93.6% for HPV31, 87.9% for HPV33, and a very low 17.6% for HPV39. The study shows that although the PapilloCheck kit uses specific probes, errors can still occur under more stringent testing criteria.

[0017] Basu P et al. established a genotyping method based on MassARRAY spectrometry, which consists of three steps: competitive PCR, primer amplification, and matrix-assisted time-laser desorption / ionization (MAD) separation by product mass-flight spectroscopy. This method can detect 15 HR-HPV types. Each type uses independently designed amplification primer pairs, and the genotyping probes are also type-specific sequences. Researchers compared the detection efficacy of this method with the PreTect HPV kit (detecting HR-HPV 16, 18, 31, 33, 45) and the universal primer MY09 / MY11 PCR method. The results showed that the HPV detection rates of these three methods were 94.8% (182 / 192), 83.3% (160 / 192), and 86.5% (166 / 192), respectively, demonstrating significant differences. Only 6 cases (3.1%, out of 192 cases) tested negative for HPV using all three methods; the multiple infection rates were 14.8% (27 / 182), 4.3% (7 / 160), and 0, respectively. Analysis of the consistency of the three methods showed that MassARRAY and PreTect had an 86.0% consistency (165 / 192), while PreTect and universal PCR had the lowest consistency (81.8%). The overall consistency among the three methods was only 65.1% (125 / 192). The researchers concluded that the MassARRAY spectrophotometry method was superior to the two commercially available methods. This result was consistent with expectations; compared to the PreTect HPV kit, this method covered a wider range of HPV types; compared to universal primer PCR, this method used specific primers and type-specific probes, significantly outperforming universal primers in both sensitivity and specificity. This study demonstrates that using type-specific primers for genotyping can improve both sensitivity and specificity, but this increases testing costs significantly and complicates procedures, making it unsuitable for large-scale clinical screening.

[0018] Molecular biology detection has always been known for its high sensitivity and specificity. However, the unique nature of HPV genotyping greatly limits its advantages. Primer universality versus primer specificity, and detection universality versus detection sensitivity, are inherently contradictory. The challenge lies in primer design and amplification condition setting: achieving both coverage of as many types as possible and accurate detection. Some researchers have analyzed the potential impact of amplification temperature on detection results. Micalessi et al. performed SPF10 real-time quantitative PCR amplification on 31 HPV plasmids, showing that these plasmids had their own Tm values ​​ranging from 79.7℃ to 83.4℃. Four samples (two types 16 and two types 31) identified by SPF10 conventional PCR conditions (10 μL / 49℃) were amplified and identified using real-time quantitative PCR at three temperature gradients of 49.0℃, 49.5℃, and 50.0℃, with sample volumes of 1 μL and 10 μL respectively. As a result, one HPV16-positive sample was not detected three times and was detected three times; one HPV31-positive sample was detected four times with a peak of type 44. Real-time quantitative PCR amplification tests on HPV16 and 31 full-sequence plasmids mixed at ratios of 1:1 to 1000 showed that if the correct genotyping results were obtained, the amplified Cp (crossing point, 19.5–33.56) and Tm (melting temperature, 80.7–81.5℃) were all different.

[0019] Other researchers have explored the causes of detection differences through primer analysis. Cai Yu Pin et al. evaluated the detection efficacy of four universal HPV primer pairs (GP5+ / 6+, MGP, MY09 / 11, PGMY09 / 11), performing broad-spectrum HPV amplification on 325 cervical specimens, and then identifying the amplified products for HPV genotyping. The results showed very good consistency among different primer pairs in terms of overall HPV detection rate and identification of major types, with kappa values ​​ranging from 0.751 to 0.925. However, some minor differences remained; for example, GMP and GPMY09 / 11 showed higher efficacy in detecting multiple infections. The researchers analyzed these differences and concluded that, even among universal primer pairs, selecting a primer pair essentially selects a broad range of HPV types.

[0020] Due to the existence of HPV gene variations, it is impossible to design primers that fully cover all HPV types by trying to find completely identical sequences within the HPV genome. Therefore, false negative results are unavoidable. Improving primer universality comes at the cost of sacrificing primer specificity and optimal reaction conditions for type specificity, which inevitably reduces the sensitivity and accuracy of detection, leading to missed detections and false positives. Researchers have shown through testing that even a 0.5°C adjustment in primer binding temperature can significantly affect the genotyping results. Reduced accuracy (missed detections and false positives) may be the inevitable price of molecular biology detection technologies in improving the broad spectrum of HPV detection. Therefore, the best way to simultaneously solve the problems of type coverage and detection specificity is to use one primer and one set of conditions per type.

[0021] The above studies summarize that since the discovery of HPV, HPV types have continuously evolved and increased in number, posing increasing challenges to molecular biological typing. Furthermore, epidemiological surveys show that LR-HPV has significant value in pathogenicity and identifying HPV-susceptible individuals, especially given the prevalence of alternating HR and LR infections. Therefore, it is necessary to adjust HPV screening strategies for cervical cancer prevention. Summary of the Invention

[0022] This invention provides a polypeptide selected from:

[0023] (1)SEQ ID NO:1;

[0024] (2) A fragment of SEQ ID NO:1 consisting of 12 to 21 amino acid residues;

[0025] (3) A sequence having cysteine ​​residues at the N-terminus and / or C-terminus of the sequences shown in (1) and (2); and

[0026] (4)(1)~(3) are fusion proteins formed with the carrier proteins used in antibody preparation or detection.

[0027] In some embodiments, the fragment is 15 to 20 amino acid residues long.

[0028] In some embodiments, the fragment contains at least one of the following sequences, or consists of one of the following sequences:

[0029] (a) The amino acid sequence of SEQ ID NO:1, positions 1-15;

[0030] (b) The amino acid sequence of SEQ ID NO:1, positions 6-20;

[0031] (c) The amino acid sequence of SEQ ID NO:1, positions 11-25;

[0032] (d) The amino acid sequence of SEQ ID NO:1, positions 16-30; and

[0033] (e) SEQ ID NO:1 amino acid sequence from position 6 to 25.

[0034] The coding sequence of the aforementioned polypeptide or its complementary sequence is also included within the scope of this invention.

[0035] The present invention provides an antibody that specifically binds to the polypeptide described herein.

[0036] In some embodiments, the antibody specifically binds to SEQ ID NO:1.

[0037] In some embodiments, the antibody specifically binds to any one of (a) to (e).

[0038] In some embodiments, the antibody specifically binds to the polypeptide fragments described in both (b) and (c) simultaneously.

[0039] In some embodiments, the antibody is an antibody produced by hybridoma cells with accession number CCTCC NO:C2017239.

[0040] In some embodiments, the antibody is an antibody produced by hybridoma cells with accession number CCTCC NO:C2017240.

[0041] In some embodiments, the antibody is an antibody produced by hybridoma cells with accession number CCTCC NO:C2017241.

[0042] The present invention also provides a group of antibody compositions comprising at least two antibodies, said at least two antibodies being specifically capable of binding to epitopes of SEQ ID NO:1 of 15-20 amino acid residues selected from at least two of the following sequences, wherein said 15-20 amino acid residues contains at least one of the following sequences:

[0043] (a) The amino acid sequence of SEQ ID NO:1, positions 1-15;

[0044] (b) The amino acid sequence of SEQ ID NO:1, positions 6-20;

[0045] (c) The amino acid sequence of SEQ ID NO:1, positions 11-25;

[0046] (d) The amino acid sequence of SEQ ID NO:1, positions 16-30; and

[0047] (e) SEQ ID NO:1 amino acid sequence from position 6 to 25.

[0048] In some embodiments, the antibody comprises at least two of the following antibodies:

[0049] (1) An antibody that can specifically bind to the epitope shown in SEQ ID NO:6;

[0050] (2) An antibody that can specifically bind to the epitope shown in SEQ ID NO:8;

[0051] (3) An antibody that can specifically bind to the epitope shown in SEQ ID NO:10; and

[0052] (4) An antibody that can specifically bind to the epitope shown in SEQ ID NO:13.

[0053] In some implementations, the antibody can bind specifically to one or two of the epitopes simultaneously.

[0054] In some embodiments, the antibody can specifically bind to both the epitope shown in SEQ ID NO:8 and the epitope shown in SEQ ID NO:10.

[0055] In some embodiments, the antibody includes an antibody that specifically binds to the epitope shown in SEQ ID NO:6, an antibody that specifically binds to the epitope shown in SEQ ID NO:8, and an antibody that specifically binds to the epitope shown in SEQ ID NO:10.

[0056] In some embodiments, the antibody includes an antibody that specifically binds to the epitope shown in SEQ ID NO:6 and an antibody that specifically binds to the epitope shown in SEQ ID NO:13.

[0057] In some embodiments, the antibody includes an antibody that specifically binds to the epitope shown in SEQ ID NO:8, an antibody that specifically binds to the epitope shown in SEQ ID NO:10, and an antibody that specifically binds to the epitope shown in SEQ ID NO:13.

[0058] In some embodiments, the antibody includes an antibody that specifically binds to the epitope shown in SEQ ID NO:6, an antibody that specifically binds to the epitope shown in SEQ ID NO:8, an antibody that specifically binds to the epitope shown in SEQ ID NO:10, and an antibody that specifically binds to the epitope shown in SEQ ID NO:13.

[0059] In some embodiments, the antibody comprises at least two or all three antibodies selected from hybridoma cells with accession number CCTCC NO:C2017239, hybridoma cells with accession number CCTCC NO:C2017240, and hybridoma cells with accession number CCTCC NO:C2017241.

[0060] The present invention also provides a kit containing the antibody described herein.

[0061] In some embodiments, the kit contains the antibody composition described herein.

[0062] The present invention also provides a detection plate coated with the antibody or antibody composition described herein.

[0063] The present invention also provides the use of the polypeptide described herein or its encoding sequence in the preparation of antibodies for detecting HPV, especially monoclonal antibodies, or in the preparation of test kits for detecting HPV. Attached Figure Description

[0064] Figure 1 Western blotting detection of the reactivity of monoclonal antibody group to multi-type recombinant HPV L1. Lysates of sf9 cells expressing recombinant HPV L1 types 18, 31, 52, 58, and 45 were subjected to polyacrylamide gel electrophoresis. The gel was then transferred to a nitrocellulose membrane and reacted with biotin-labeled monoclonal antibodies 4G12, 3A9, and 5A4. Results showed that the monoclonal antibody group produced a specific reaction band at the 56 kDa position in the lysates of sf9 cells expressing recombinant HPV L1. Figure 1 In the middle, the lanes from left to right correspond to HPV18, 31, 52, 58 and 45 respectively.

[0065] Figure 2 ELISA assay for the reactivity of monoclonal antibody groups to HPV-containing cell cultures. Caski and Siha cells containing HPV16 virus, HeLa and Hep G2 cells containing HPV18 virus, and HPV-negative Hacat cells (control) were tested. Recombinant HPV16 and HPV18L1 proteins were used as positive controls. 4G12 and 7C10 were used as capture antibodies to coat plastic plates, and the cell lysates were added to each well. The plates were then reacted with biotin-labeled 4G12 and 3A9 antibodies. The results showed that all wells containing HPV-containing cell lysates exhibited positive reactions.

[0066] Figure 3Western blotting analysis of the reactivity of monoclonal antibody assays to HPV-containing cell cultures. The three lanes from left to right represent: HeLa cells containing HPV18, Caski cells containing HPV16, and the positive control recombinant HPV31L1 protein. The antibody assays elicited specific bands in HPV-containing cell cultures. The upper band is the HPV L1-specific band, and the lower band is the β-Actin internal control.

[0067] Figure 4 Immunohistochemical detection of HPV-positive tissues by monoclonal antibody assay. Left image: Genital condyloma acuminata, HRP labeling, DAB staining, hematoxylin counterstaining, HPV DNA typing result: HPV6; Right image: Cervical epithelium, CIN II, DyLight 549 labeled secondary antibody, DAPI staining, HPV DNA typing result: HPV16 (×400).

[0068] Figure 5 Immunohistochemical detection of cervical exfoliated cell specimens that tested positive for HPV genotyping using monoclonal antibody assays. Top left: HPV DNA genotyping showed HPV43; Top right: HPV DNA genotyping showed HPV16 positive; Bottom left: HPV DNA genotyping showed HPV51 positive; Bottom right: HPV DNA genotyping showed HPV33, 56, and 44 (hematoxylin counterstaining, ×400).

[0069] Figure 6 Western blotting detection of recombinant HPV18L1 protein using three antibodies: A: 3A9; B: 5A4; C: 4G12; D: 3A9+5A4; E: 3A9+4G12; F: 5A4+4G12; G: 3A9+5A4+4G12. The titers of both single and mixed antibodies were ultimately adjusted to 1:2000. Detailed Implementation

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0071] This invention provides a set of antibody compositions for detecting multiple HPV types. Specifically, the antibody compositions provided by this invention contain at least two, at least three, or all four antibodies selected from (1) an antibody specifically binding to the epitope shown in SEQ ID NO:6, (2) an antibody specifically binding to the epitope shown in SEQ ID NO:8, (3) an antibody specifically binding to the epitope shown in SEQ ID NO:10, and (4) an antibody specifically binding to the epitope shown in SEQ ID NO:13. In some embodiments, the antibodies may bind specifically to one or two of the epitopes simultaneously. For example, in some embodiments of this application, some antibodies may bind specifically to both the epitope shown in SEQ ID NO:8 and the epitope shown in SEQ ID NO:10 simultaneously. Therefore, in these embodiments, the antibody compositions may contain up to three antibodies.

[0072] In some embodiments, the present invention provides an antibody that specifically binds to the epitope shown in SEQ ID NO:6, which is an antibody produced by hybridoma cells with accession number CCTCC NO:C2017239.

[0073] In some embodiments, the present invention provides antibodies that specifically bind to the epitopes shown in SEQ ID NO:8 and 10, which are antibodies produced by hybridoma cells with accession number CCTCC NO:C2017240.

[0074] In some embodiments, the present invention provides an antibody that specifically binds to the epitope shown in SEQ ID NO:13, which is an antibody produced by hybridoma cells with accession number CCTCC NO:C2017241.

[0075] In the antibody composition of the present invention, the proportions of each antibody can be equal. Alternatively, the proportions of different antibodies in the antibody composition can be set according to the results of epidemiological surveys, such as the expression levels of common HPV L1 protein types in patients. For example, for antibodies with high expression levels, the content of the corresponding antibody in the antibody composition can be relatively high; for antibodies with low expression levels, the content of the corresponding antibody in the antibody composition can be relatively low compared to other antibodies.

[0076] In this document, the term "antibody" has the generally accepted meaning in the art, referring to an antibody that, in its presence, can inhibit a specific immune response. An antibody can be the full-length sequence of an antibody or a functional fragment thereof, such as Fab, Fab', F(ab')2, Fv, scFv, and scFv-Fc. Antibodies can be polyclonal or monoclonal, preferably monoclonal.

[0077] In this article, the term "epitope" or "antigenic epitope," also known as an antigenic determinant, refers to a specific chemical group in an antigen molecule that determines the antigen's specificity and can be recognized by specific antibodies. The nature, number, and spatial configuration of antigenic epitopes determine the antigen's specificity. Based on the different amino acid sequences, antigenic epitopes can be divided into linear epitopes and spatial epitopes. A linear epitope is an epitope composed of a sequence of adjacent amino acids, while a spatial epitope is an epitope composed of several non-adjacent but spatially adjacent amino acids.

[0078] In this document, the term "specific binding" refers to the reaction between an antibody or its antigen-binding fragment and the antigen or antigenic epitope it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen. "Specific recognition" has a similar meaning.

[0079] Antibodies can be prepared using methods known in the art. For example, animals (such as mice) can be immunized with suitable antigens, and spleen cells from the immunized animals can be fused with SP2 / 0 myeloma cells to prepare hybridoma cell lines. Positive hybridoma cell lines can be screened using ELISA, and stable monoclonal cell lines can be obtained through multiple cloning processes. When preparing antigenic peptides for immunization, a cysteine ​​residue (C) can be attached to one side of the peptide, and then it can be fused with a carrier protein (such as keyhole hemocyanin, KLH), and the fusion protein can be used to immunize animals.

[0080] Therefore, in some embodiments, the antibodies described herein that specifically bind to the epitope shown in SEQ ID NO:6, the epitope shown in SEQ ID NO:8, the epitope shown in SEQ ID NO:10, and the epitope shown in SEQ ID NO:13 may be specific antibodies prepared using the polypeptide described in SEQ ID NO:6, the polypeptide described in SEQ ID NO:8, the polypeptide described in SEQ ID NO:10, or the polypeptide described in SEQ ID NO:13.

[0081] In some embodiments, the antibodies specifically binding to the epitope shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:13 described herein may be antibodies prepared using the polypeptide shown in SEQ ID NO:3 as the antigen. In these embodiments, after the antibodies are prepared, conventional methods can be used to detect the reaction site and titer to determine whether the prepared antibodies can specifically bind to the epitopes shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and / or SEQ ID NO:13.

[0082] The antibody compositions of the present invention may contain suitable solvents, including but not limited to Freund's complete adjuvant and Freund's incomplete adjuvant. However, in some embodiments, the antibodies of the present invention may be coated onto suitable plates for the detection of HPV.

[0083] Depending on the class of antibodies contained in the antibody composition, the antibody compositions of the present invention can be used to detect multiple HPV types, including but not limited to multiple types from 12 high-risk types (HPV16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59 and 68) and 13 low-risk types (HPV6, 11, 40, 42, 43, 44, 53, 54, 55, 57, 66, 67 and 73), for example at least 10 types, at least 15 types or at least 20 types. In some embodiments, the antibody composition of the present invention contains antibodies that specifically bind to the epitopes shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:13, and can recognize low-risk HPV types 6, 11, 40, 42, 43, 44, 53, 54, 55, 66, 67 and 73, and high-risk HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59 and 68.

[0084] In some embodiments, the present invention also provides a detection kit containing at least two, at least three, or all four of the antibodies described herein. The kit may also contain other reagents required for detection using immunohistochemistry (IHC) or ELISA, including but not limited to biotin-labeled or enzyme-labeled secondary antibodies, avidin-conjugated HRP, chromogenic agents, or fluorescently labeled secondary antibodies.

[0085] In some aspects, the present invention also provides isolated SEQ ID NO:1 and fragments thereof of 12 to 21 amino acid residues in length, such as fragments of 13 to 20 amino acid residues, 15 to 20 amino acid residues in length, or 15 to 18 amino acid residues in length. More specifically, the fragments of SEQ ID NO:1 include the following fragments:

[0086] (1) The amino acid sequence of SEQ ID NO:1 from position 1 to 15;

[0087] (2) The amino acid sequence of SEQ ID NO:1, positions 6-20;

[0088] (3) The amino acid sequence of SEQ ID NO:1, positions 11-25;

[0089] (4) The amino acid sequence of SEQ ID NO:1, positions 16-30; and

[0090] (5) The amino acid sequence of SEQ ID NO:1 from position 6 to 25.

[0091] The present invention also includes a polypeptide sequence having a cysteine ​​residue (C) at the N-terminus or C-terminus of SEQ ID NO:1 and its fragment, and a fusion protein of SEQ ID NO:1 and its fragment, and the polypeptide sequence having a cysteine ​​residue at either the N-terminus or C-terminus, with a carrier protein. The carrier protein may be a carrier protein commonly used in antibody preparation or detection, including but not limited to keyhole hemocyanin (KLH) and bovine serum albumin (BSA).

[0092] In some embodiments, the fragments shown in (1) to (5) above can be used as antigens to prepare antibodies. It should be understood that the length of the fragments shown can be appropriately increased (e.g., by adding 1, 2, or 3 amino acid sequences at their N and / or C ends) or shortened (e.g., by shortening 1, 2, or 3 amino acid sequences at their N and / or C ends). For example, for the fragment shown in the amino acid sequence of SEQ ID NO:1, a sequence containing 1, 2, or 3 amino acid residues at the C end can be used, i.e., amino acid residues of SEQ ID NO:1, positions 1-16, 1-17, or 1-18 can be used as antigens; as another example, for the amino acid sequence of SEQ ID NO:1, positions 6-20 can be used, such as the amino acid sequence of SEQ ID NO:1, positions 5-21 or SEQ ID NO:1, positions 7-19.

[0093] The amino acid sequences of the present invention can be synthesized using conventional methods in the art, such as solid-phase synthesis or equivalent chemical synthesis methods known in the art to prepare the polypeptides described herein.

[0094] In some respects, the present invention also includes the coding sequence of the above-described amino acid sequence.

[0095] Therefore, the present invention also includes the use of the amino acid sequence or its encoding sequence described herein in the preparation of antibodies for detecting HPV, especially monoclonal antibodies, or in the preparation of test kits for detecting HPV.

[0096] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the materials and methods used in the embodiments are common in the art.

[0097] Example

[0098] Example 1

[0099] (a) Synthesis of HPV L1 short peptide

[0100] In this embodiment, Shanghai Qiangyao Biotechnology Co., Ltd. was commissioned to synthesize the following sequence polypeptides (sequence numbers are SEQ ID NO:1-13) using conventional methods:

[0101]

[0102]

[0103] With purities of 75% or 98%, the above-mentioned short peptides containing cysteine ​​(C) are coupled with carrier proteins—keyhole hemocyanin (KLH) or bovine serum albumin (BSA) to form peptide-KLH and peptide-BSA, respectively. Peptide-KLH is an immunogenic antigen, and peptide-BSA is a detection antigen, which are used for immunization or detection, respectively.

[0104] (b) Preparation of monoclonal antibodies

[0105] The conjugates of peptides (1) and (2) with the carrier protein KLH—peptide (1)-KLH and peptide (2)-KLH—were dissolved in physiological saline, and an equal volume of adjuvant was added to prepare an emulsion. Female Balb / c mice (4-6 weeks old) were immunized with an immunization volume of 0.2 ml / mouse (see Table 1 for specific immunization protocols).

[0106] Table 1: Immunization regimens for mice

[0107]

[0108] Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells to prepare hybridoma cell lines. Positive hybridoma cell lines were screened using ELISA, and stable monoclonal cell lines were obtained through multiple cloning processes. Several antibodies with high production rates were selected from the obtained monoclonal cell lines for reaction site and titer analysis. Antibodies with high titers and well-defined reaction sites were mass-produced and purified, resulting in a total of eight monoclonal antibodies (see Table 2 for details).

[0109] Table 2: Monoclonal antibody strains targeting each reaction site

[0110]

[0111] Among them, hybridoma cell lines 3A9, 4G12 and 5A4 were deposited on October 25, 2017 at the China Center for Type Culture Collection (CCTCC, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072), with accession numbers CCTCC NO:C2017239, CCTCC NO:C2017240 and CCTCC NO:C2017241, respectively.

[0112] Example 2: Western Blot detection of the reactivity of monoclonal antibody groups to L1 of multi-type recombinant HPV

[0113] sf9 cells expressing recombinant HPV6b, 11, 16, 18, 31, 45 and 58L1 were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0114] Lysate from sf9 cells expressing recombinant HPV L1 was subjected to polyacrylamide gel electrophoresis. The gel was then transferred to a nitrocellulose membrane and reacted with biotin-labeled Ant-1, Ant-23, and Ant-4 monoclonal antibodies. The results showed that the monoclonal antibody groups produced a specific reaction band at the 56 kDa position in the lysate of sf9 cells expressing recombinant HPV L1. Figure 1 ).

[0115] Example 3: ELISA detection of the reactivity of monoclonal antibody groups to HPV virus-containing cultured cells

[0116] Caski and Siha cells containing HPV16 virus, HeLa and Hep G2 cells containing HPV18 virus, and HPV-negative Hacat cells (control) were used for detection. All cells were purchased from the China Cell Bank, Wuhan University. HRP-labeled streptavidin was a product of Abbkine, USA. Recombinant HPV16 and HPV18L1 proteins were used as positive controls.

[0117] Ant-1 and Ant-4 were used as capture antibodies to coat plastic plates. Cell lysis buffer was then added to each well, and biotin-labeled Ant-1 and Ant-4 antibodies were used to react with the buffer. The results showed that wells containing the HPV virus-containing cell lysis buffer exhibited positive reactions. Figure 2 ).

[0118] Example 4: Western Blot detection of the reactivity of monoclonal antibody groups to HPV virus-containing cultured cells

[0119] Lysates of cultured Caski cells containing HPV16 and HeLa cells containing HPV18 were subjected to polyacrylamide gel electrophoresis. The gels were then transferred to nitrocellulose membranes and reacted with biotin-labeled Ant-1, Ant-23, and Ant-4. After binding with HRP-labeled streptavidin, color development was performed. Recombinant HPV31L1 protein was used as a positive control, and immortalized keratinocytes (Hacat cells) without the HPV viral genome were used as a negative control. Results showed that the antibody group induced specific reaction bands on the electrophoretic lanes of Caski and HeLa cell lysates containing HPV virus. Figure 3 ).

[0120] Example 5: Immunohistochemical detection of different HPV-positive clinical specimens using monoclonal antibody groups

[0121] Clinical pathological examination revealed tumors or condyloma acuminata, and pathological tissues were subjected to PCR identification. Samples positive for different PCR types were tested. The results showed that the monoclonal antibody group caused all these samples to show a positive reaction. In HPV6 and 11-positive condyloma acuminata tissues, some cell nuclei showed a positive reaction, staining brown; positive nuclei were only present in the surface layer of the epithelium. The monoclonal antibody group caused some cells in HPV-induced tumor tissues to show a positive reaction; the reaction pattern varied depending on the tissue, ranging from strong to weak, uniform, or nested. Positive cells may have a clear boundary with negative cells, or show transitional changes; the cytoplasmic reaction was strong. Figure 4 The tests demonstrated that this antibody group has excellent ability to identify and detect multiple HPV types.

[0122] Example 6: Performance determination of antibody group on multi-type detection of cervical exfoliated cells infected with different types of HPV

[0123] 339 cervical exfoliated cell specimens were collected from the gynecology outpatient clinic and HPV genotyping was performed using an HPV genotyping kit (National Drug Approval Number 2011-3401505) manufactured by Shenzhen Ganglong Biotechnology Co., Ltd. This genotyping kit is approved by the China Food and Drug Administration (FDA) for clinical testing. The kit can detect 26 HPV types, including 13 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 13 low-risk types (HPV6, 11, 40, 42, 43, 44, 53, 54, 55, 57, 66, 67, and 73). 220 cervical exfoliated cell specimens tested positive for HPV, and a total of 25 types were detected. Twenty-one types were detected as single-type infections, including eight low-risk types (18 cases) and thirteen high-risk types (131 cases).

[0124] Using the IHC method, except for HPV35, the monoclonal antibody group (Ant-1, Ant-23, and Ant-4) showed a positive reaction in all 20 HPV types, indicating that this antibody group can effectively identify the L1 of these HPV types. Of the 71 multi-type HPV infection samples tested, only one was negative, while the rest were positive. The negative sample was a combination of HPV42 and HPV54, but the other two samples with this combination showed positive reactions (Table 3). Figure 5 The test results indicate that, except for type 35, the reaction with monoclonal antibodies can react with the other 24 HPV types.

[0125] Table 3: Detection results of monoclonal antibody groups on different HPV infection specimens

[0126]

[0127]

[0128] Example 7: Immunohistochemical detection of cervical exfoliated cell specimens by monoclonal antibody group after PCR-generated HPV detection.

[0129] HPV genotyping was performed on 339 specimens using an HPV genotyping kit manufactured by Shenzhen Ganglong Biotechnology Co., Ltd., with 220 specimens testing positive for HPV, resulting in a positive rate of 64.90% (220 / 339). These cervical exfoliated cell specimens were then analyzed using immunohistochemistry, employing biotin-labeled 3A9, 5A4, and 4G12 antibodies as detection antibodies. A total of 229 specimens tested positive, with a positive detection rate of 67.55% (229 / 339). Comparison between the immunohistochemical results and the HPV genotyping kit results showed good agreement between the two methods (Kappa = 0.743, p < 0.001) (see Tables 4 and 5). This indicates that the test has a high clinical HPV detection efficacy and the ability to detect multiple HPV types.

[0130] Table 4: Comparison of HPV genotyping and IHC testing results and analysis of IHC detectability

[0131]

[0132] Table 5

[0133] Classification Youden Index Positive likelihood ratio Negative likelihood ratio Positive predictive value Negative predictive value Compliance rate ASC-US 0.720 4.522 0.096 0.836 0.902 0.864 ASC-H 0.757 4.783 0.054 0.957 0.800 0.929 LSIL 0.930 ∞ 0.070 1.000 0.733 0.941 HSIL 0.939 ∞ 0.061 1.000 0.600 0.944 AGC 0.429 1.750 0.000 0.333 1.000 0.556 total 0.730 4.620 0.085 0.895 0.864 0.885

[0134] Example 8: Comparison of the effects of monoclonal antibody group detection and single monoclonal antibody detection

[0135] (a) Western blot detection of recombinant HPV L1 protein

[0136] Ant-1+Ant-23+Ant-4, Ant-1+Ant-23, Ant-1+Ant-4, Ant-23+Ant-4, Ant-1, Ant-23, and Ant-4 were used to detect different types of recombinant HPV L1 protein, respectively. The detection effects of different antibody combinations and single antibodies were compared. The results showed that the detection effect of the three antibody combinations was the best. Figure 6 ).

[0137] (b) Western blot detection of HPV L1 protein in HPV-positive cultured cells

[0138] The lysates of tumor cell lines expressing different HPV L1 protein types were detected using Ant-1+Ant-23+Ant-4, Ant-1+Ant-23, Ant-1+Ant-4, Ant-23+Ant-4, Ant-1, Ant-23, and Ant-4, respectively, and the detection effects of different antibody combinations and single antibodies were compared.

[0139] (c) Immunohistochemical detection of HPV-positive cultured cells

[0140] Tumor cell lines expressing different HPV L1 protein types were cultured on slides and fixed with 70% ethanol when the slides reached 50% confluence. The cells were then tested using biotin-labeled monoclonal antibodies Ant-1+Ant-23+Ant-4, Ant-1+Ant-23, Ant-1+Ant-4, Ant-23+Ant-4, Ant-1, Ant-23, and Ant-4, respectively. The detection effects of different antibody combinations and single antibodies were compared. sequence list <110> Three Gorges University <120> Monoclonal antibodies for the detection of multiple types of human papillomavirus <130> 177992 <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <400> 1 Cys Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe 1 5 10 15 Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys 20 25 30 <210> 2 <211> 31 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro 1 5 10 15 Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Cys 20 25 30 <210> 3 <211> 30 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro 1 5 10 15 Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys 20 25 30 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <400> 4 Cys Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <400> 5 Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Cys 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <400> 6 Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <400> 7 Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Cys 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <400> 8 Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <400> 9 Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Cys 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <400> 10 Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <400> 11 Cys Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Cys 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <400> 13 Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys 1 5 10 15

Claims

1. An antibody composition, characterized in that, The antibody composition contains antibodies produced by hybridoma cells with accession number CCTCC NO:C2017239, antibodies produced by hybridoma cells with accession number CCTCC NO:C2017240, and antibodies produced by hybridoma cells with accession number CCTCC NO:C2017241.

2. The antibody composition according to claim 1, characterized in that, The antibody composition is used to detect low-risk HPV types 6, 11, 40, 42, 43, 53, 54 and 66, and high-risk HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59 and 68.

3. A detection plate, characterized in that, The detection plate is coated with the antibody composition according to any one of claims 1-2.

4. A test kit, characterized in that, The test kit contains the antibody composition according to any one of claims 1-2.

5. The detection kit as described in claim 4, characterized in that, The test kit contains the reagents required for detection using immunohistochemistry or ELISA.

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