TW201043958A - Detection of early stages and late stages HPV infection - Google Patents

Abstract

Embodiments of the invention provide methods, polyclonal antibodies, monoclonal antibodies, assays, and kits for detecting HPV infection, including infection by various HPV genotypes, early and/or late HPV-associated or HPV-specific proteins or antibodies. Mononoclonal antibodies are used to detect oncogenic high risk and low risk HPV types in a single assay, which is not limited to assay type or format. Useful tools for specific detection of invasive cervical cancer are provided. Cervical cancer biomarkers are identified and can be used in a detection method for early stage precancerous lesions as well as late stage cancer progression.

Description

201043958 VI. INSTRUCTIONS: The application of this invention is related to the reference [〇〇〇1]. The scope of application of this patent is benefited from the US Provisional Patent Application No. 61/131,991, June 13, 2Q08, and the US Provisional Patent Application No. 6i /i92,912, submitted on September 22, 2008. Each of the above-mentioned related patent applications is hereby incorporated by reference. TECHNICAL FIELD OF THE INVENTION [0002] This case is an immunoassay test for early and late infection of human papillomavirus. [Prior Art] [0003] There seems to be a defect in the prior art. SUMMARY OF THE INVENTION [〇〇〇4] The subject of the present invention provides various immunoassays for in situ detection of HPV protein, using various monoclonal antibodies against HPV recombinant protein to infect high-risk or low-risk hpv virus type, Specific monoclonal antibodies or general pan-antibody assays. In an implementation case ’ a method used to detect HPV infection in humans, using one or more monoclonal antibodies that bind to HPV early proteins to perform immunoassays and human nuclear staining of clinical human samples. In another embodiment, a method for detecting HPV infection in a human body uses one or more antibodies to produce a plurality of purified HPV recombinant proteins, immunohistochemically testing human cells of clinical samples, and comparing nuclei and Cytoplasmic staining. Another method for detecting HPV infection in humans is to use a variety of antibodies to screen for Hpv virus proteins from Ε6, Ε7, L1 and its binding protein to perform immunoassays for clinical human samples, and compare human cells with two or more antibodies. In the case of staining, if at least one antibody test results in a positive staining, it indicates that the human body is infected with the HPV virus. 201043958 [The present invention provides an anti-HPV monoclonal antibody which can be used in an immunoassay to cytoplasmic staining of HPV late-infected human cells. In another aspect, the present invention provides an anti-HpV E7 monoclonal antibody and an anti-_Hpv E6 monoclonal antibody, which can be applied to an immunoassay for nuclear staining of human cells to express HPV infection in a disease stage. . The nuclear staining of HPV-infected human cells indicates early infection of HPV, while cytoplasmic staining indicates that lesions are increased into advanced disease stages. Anti-HPV monoclonal antibodies used in immunoassays include anti-HPV E7, anti-HPV E6 and anti-HPV L1 monoclonal antibodies, and combinations thereof. In the other aspect, the present invention provides an anti-HPVE7 monoclonal antibody and an HPVE7 protein as biomarkers for detecting proliferation of cervical cancer. [6] In addition, the present invention provides methods for performing various immunoassays, including immunohistochemistry and immunocytology assays. Individual antibodies are highly specific for HPV viral proteins and can also be used as an immunoassay for HPV detection. In one embodiment, staining of the nuclei of epithelial tissue samples with anti-HPV monoclonal antibodies revealed general hpv infection. In another embodiment, cytoplasmic staining of epithelial tissue samples with anti-HPV monoclonal antibodies can show hyperplasia of HPV-infected lesions. [〇〇〇7] In another aspect, a reagent for clinical sample immunoassay is provided, which comprises an anti- _!^ monoclonal antibody capable of staining the nucleus of HPV-infected human cells, showing HPV infection; HPV infection The cytoplasm of human cells stains to show infection in the advanced stage of HPV. In one aspect, the detection reagent for HPV infection in human body contains a monoclonal antibody against HPV, which is used in human clinical samples for immunoassay, which can stain the cells from clinical human cells and cytoplasm with human cells. Dyeing is done for comparison. [〇〇〇8] The present invention provides various immunoassays for detecting HPV protein in situ, using various monoclonal antibodies against HPV recombinant protein, infecting high-risk or low-risk HPV disease 201043958, which can be monospecific Sexual antibodies or general pan-antibodies are detected. The present invention also provides HPV immunocytochemistry (icc) assays, HPV flow cytometry assays, and ΗΡν immunohistochemistry assays (IHC) to detect the presence of HPV protein 子宫 in cervical cells or tissues. In addition, 'monoclonal antibodies are highly specific for HPV viral proteins and can be used in HPV ICC or HPV flow cell assays. [00〇9] In an embodiment, an immunocytology test method is used to detect human infections. HPV proteins from a variety of HPV virus types are included in biopsies containing a thin layer of human cells on the in situ detection test piece. A thin layer of human cells was stained with 0 using a variety of antibodies. The method comprises providing a clinical sample from a human body, preparing a sample of a human body cell which is abnormal in shape and normal, and forming a thin layer of the cell on the test piece to produce a multi-purified HPV recombinant protein by using one or more antibodies. In an immunocytochemistry test, a mixture of abnormal and normal morphological cells in a clinical specimen on a test strip, a plurality of HPV proteins from various HPV virus types can be stained in situ. [〇〇1〇] In another embodiment, a method for detecting HPV infection in a human body is provided, which includes providing a human clinical sample, and 多种*5 has a plurality of antibodies as reagents, and the sample is made into a type containing abnormality. A solution of a mixture with normal human cells. Use multiple antibodies to produce resistance

Normal and normal human cell mixture, each of which is finely quarantined, and the presence of HPV proteins of various Ηρν virus types in a mixture of liquid clinical samples and normal human cells [0011] In an embodiment, Produces one or more antibodies 'anti-multiple purified HPV heavy

A variety of human cells produce L HPV protein-producing bonds to prepare a solution, and various antibodies are reacted with Hpv 201043958 from various Ηϊ> ν virus types, reacted with antibodies labeled with S, and detected by the performance of the reagent. In another embodiment, the reagents include colorimetric reagents, fluorescent staining reagents, and other reagents as reagents for separation and characterization of human cells in a flow cell assay. Λ , [〇〇12] In addition, a reagent for performing an immunoassay is provided. The reagent comprises a pre-antibody stop solution, a post-antibody stop solution, an anti-HPV antibody as a primary antibody, an anti-mouse or anti-rabbit immunoglobulin grafted HRP or biotin, or other reagents as secondary antibodies, comprising A solution of the appropriate reagent is detected as a matrix for the secondary antibody. [0〇13] In another embodiment, a reagent is provided for detecting human HPV infection, including an anti-Ο·ΗΡν monoclonal antibody 'Energy HPV (4) egg self-generating bond Xiao, performing immunocytochemistry test of clinical samples, will be examined The body is made into a mixed solution containing abnormal and normal human cells' and is made into thin layer cells on the test piece to in situ stain a plurality of hpv proteins from various HPV virus types. [〇〇14] The present invention provides various solid surface immunoassays for detecting ΗΡν protein, using various anti-HPV antibodies, anti-Ηρν recombinant proteins, high-risk or low-risk HPV can be used by a single specialized Individual antibodies or general pan-antibodies are detected. The invention also provides HPV film spot ink test, protein wafer microarray test, HPV particle test, 〇HPV flow fast test, Hpv vertical permeable rapid test, Hpv microfluid rapid test, direct enzyme immunoassay (ΕΙΑ) and enzyme linkage Immunosorbent assay (ELISA), - detects the presence of HPV protein in a biopsy such as a cervical cell or tissue. In addition, reagents and equipment for performing these tests are also available. [〇〇15] In an embodiment, a method for detecting a plurality of HPV proteins is provided, and an anti-HPV antibody is provided to bind to a plurality of HPV proteins from various HPV virus types in a clinical specimen to provide a solid surface. The anti-HPV antibody or the viral protein expressed in the cell lysate solution; the Pro-Clinical Cell lysis solution containing a plurality of HPV proteins. The method further comprises an anti-HPV antibody and a cell lysate solution. The HPV protein of the 201043958 antibody complex is expressed.

The liquid enters the second reaction 'formation' - contains a variety of HPV proteins and anti-HPV solid test strips, and detects the complex on the test piece to confirm the clinical examination. [〇〇16] In another case, in the case of 会 施 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The solution, which provides a phlegm-resistant phloem to the anti-HPV-class antibody, is immobilized on a solid surface and reacts with the cell-resistant anti-HPV secondary antibody. Both antibodies bind to HPV proteins from a variety of scorpion venoms. The method further comprises detecting a complex of a plurality of Hpv proteins in the biological sample by detecting a complex on the solid surface by detecting a complex on the solid surface = HPV protein and anti-HPV-class, secondary antibody. Further, in accordance with an embodiment of the present invention, a one-side flow system is provided for detecting a plurality of HPV proteins from different HPV virus types in a biological sample. The lateral flow system comprises a solid primary surface to which an anti-HP V-class antibody is immobilized, and a solid primary surface containing an anti-v primary antibody at the other end 'reacts with a cell lysis solution made from a biological sample' to laterally flow through the solid surface, A complex is formed with an anti-HPV-grade antibody. In another embodiment, a vertical rapid test system is provided to detect a plurality of HPV proteins from different HPV virus types in a biological sample. The vertical rapid test system comprises a solid membrane surface immobilized with an anti-HPV-grade antibody, reacted with a cell lysis solution made of a biological sample, and an anti-HPV secondary antibody is vertically flowed through the solid surface on the surface of the membrane. The anti-HPV-grade antibody forms a complex. [0018] In one aspect, an anti-HPV-class antibody is produced against a plurality of recombinant proteins carrying various HPV viral genes. The anti-HP V-class antibody captures a plurality of HPV in a cell lysis solution on a solid surface. protein. In another aspect, 'produces an anti-HPV secondary antibody against the same first recombinant protein with the same HPV viral gene'. The anti-7 201043958 -HPV secondary antibody is capable of interacting with a variety of hpv in a cell lysis solution on a solid surface. The protein produces a bond and is detected. [Examples] 1. The HPV recombinant protein was expressed, purified and prepared, and used as an immunogen to produce antiserum and anti-HPV antibodies, and the fusion cell lines were screened to form monoclonal antibodies. [o〇19] The HPV recombinant protein can be any HPV protein and HPV protein of early or late genes, including but not limited to E2, E6, E7, L1 and L2, and can form a variety of HPV 〇 virus types. The entire polypeptide sequence of E6, E7 and L1 is not only difficult to obtain, but also unpredictable aggregation occurs when the protein is purified, resulting in protein instability and low performance, and the purified protein immunogen is low in performance. For example, many early E6 proto-oncoproteins contain many cysteines, so the correct E6 proto-oncoprotein topography requires the formation of many suitable disulfide bonds. In addition, some immunological assays use the small peptides of the early E6 and E7 proteins, which have extremely low detection specificity and sensitivity, and are not suitable as a tool for clinical diagnosis. Therefore, the recombinant protein provided by the present invention, a partial sequence or a whole sequence of the recombinant HPV pro-oncoprotein, forms a recombinant hybrid protein. 〇 [〇〇2〇]1). Selection and production of various recombinant proteins carrying the HPV16 E6 and HPV18 E6 genes. An E6 early proto-oncogene paradigm gene from HPV paradigm HPV-16 was cloned. This cloning gene has a 474 base pair (bp) DNA fragment containing 157 amino acid coding regions of the entire HPV_16 E6 gene. , obtained by polymerase chain reaction (PCR) and amplified. The DNA sequence of the isolated DNA fragment was confirmed by alignment with the gene bank database. The recombinant protein HPV-18 E6 is also available. All colonization steps are based on the method described in "Molecular Colonization", Laboratory Manual, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. - 201043958. In addition, the HPV18 E6 gene was also cloned and confirmed for DNA sequence. [0021] 2) Selection and production of various recombinant proteins carrying the HPV16 E7 and HPV18 E7 genes. An early proto-oncogenic paradigm from HPV paradigm HPV-16. Because E7 was cloned, this cloning gene has a DNA fragment of 294 base pairs (bp), including 99 of all HPV-16 E7 genes. The amino acid coding region is obtained by polymerase chain reaction (PCR) and amplified. The DNA sequence of the isolated DNA fragment was confirmed by alignment with the gene bank database. HPV-18 E7 recombinant protein was also obtained by 0. In addition, E7 DNA fragments from different HPV virus types can be selected from different clinical samples or sources. [〇〇22] 3). Selection and production of recombinant proteins carrying the HPV16 L1 and HPV18 L1 genes. An early paradigm gene from HPV paradigm HPV-16 was cloned, a 1596 base pair (b_p.) DNA fragment containing 531 amino acid coding regions of the entire HPV-16 L1 gene, via the polymerase chain The lock reaction (PCR) was obtained and amplified. The DNA sequence of the isolated DNA fragment was confirmed by alignment with the gene bank database. In addition, LI DNA fragments from different HPV models can be repopulated from different clinical samples or sources. [0023] An N-terminal recombination fragment of the HPV16 L1 protein was obtained from a His-tagged expression system. The molecular weight of the HPV-16 LI N-terminal recombinant protein is approximately 34 KD. LI C-terminal fragments can also be obtained. HPV-18 L1 recombinant protein can also be obtained as an immunogen to produce anti-serum, polyclonal antibodies and monoclonal antibodies. [0〇24] The various recombinant proteins described in this patent can be expressed in a variety of suitable systems, such as bacterial, viral, yeast, and mammalian expression systems, such as E. coli, yeast, baculovirus, and mammalian cell culture. Although the multi-peptide can be obtained by other means, in the case of the patent provided, most of the recombinant proteins are (or almost) their original 9 201043958 initial pattern, which is more likely to be infected with HPV from the HPV infection test. The antibody of the tissue is twinned. For example, GST, MBP or His-labeled HPV16-E6, HPV18E6, HPV16E7, HPV18E7, HPV16L1, and HPV18L1 recombinant egg * white, induced by IPTG, are expressed in the "like" (7) five. Inducing eggs, After the white matter is expressed, the marker-HPV recombinant protein is obtained by lysing the lysed fraction after culturing the cells, and the purified concentration is about 0.1 to 1 mg/ml or higher. The purity of the HPV recombinant protein is estimated to be greater than 90% by PAGE analysis. HPV recombinant protein can be used to detect the presence or absence of HPV antibodies in clinical samples, and can also be used as an immunogen to produce multiple antisera and monoclonal antibodies. [0025] Cell culture of various expression vectors described in this patent Contains a variety of HPV recombinant proteins, amplified to 1L, 10 L or 100 L or even higher to obtain a large number of soluble recombinant proteins for purification. Cell lysis can be dissolved after various stages, through various column, in the appropriate performance system Labeling position binding with HPV recombinant protein; marker-HPV recombinant protein is extracted from the column and concentrated to 100 ml, 10 ml or 1 ml. Purified water-soluble HPV recombinant protein further neutral pH Value The liquid or PBS buffer is concentrated and dialyzed to produce an anti-HPV protein antiserum as an immunogen. The water-soluble HPV recombinant protein purified from the dissolved fraction is folded in a similar manner to the original folding method in the living state. It is important to produce a variety of different types of monoclonal antibodies, and to obtain high-quality purified HPV recombinant proteins. Antibodies can recognize general or specific epitopes for *detecting HPV infection. Purified HPV recombinant protein via The test confirms the binding ability with HPV antibodies from clinical HPV-infected samples. The purified HPV recombinant protein can be used as an immunogen to produce anti-serum and antibodies, and identify HP V natural viral proteins in vivo. 2. Produce anti-HPV Strain against Zhao: 201043958 [0027] Purified HPVE6, E7 or L1 recombinant protein expressed in PBS after concentration and dialysis as immunogen. The immune reaction was performed according to standard procedures, and each obtained serum was analyzed by ELISA, followed by procedural increase. And bleeding. The blood with the best titer is collected, and the produced serum is subjected to immunoglobulin (Ig) via a protein A column or an affinity column. Purified IgG is used as an anti-HPV antibody for HPV immunoassay. [〇〇28] The acquisition, purification and testing of monoclonal antibodies, multiple antibodies and serum described in this patent, regardless of the cause of the disease, cell lesions , inflammatory response or development of cancer, can be used to detect HPV infection "other researchers try to develop anti-HPV monoclonal antibodies but did not achieve results, because not enough HPV protein to produce monoclonal antibodies; can not produce a high degree of specificity The monoclonal antibody is not immunogenic for the immunogen; or the antibody produced does not recognize the HPV original virus pattern of HPV infection in the early stage of the clinical specimen. Some antibodies produce anti-mutation peptides that only recognize advanced cervical cancer, but are not sure that their antibodies recognize HPV wild-type native proteins or any early HPV infection. In addition, late HPV testing is too late for disease intervention and treatment. [〇〇29] The clinical antibody application described in this patent application can be confirmed by HPV immunodetection analysis by an appropriate clinical sample, such as an ELISA test, an immunocytochemistry test, an immunohistochemistry test. The novel monoclonal antibody and antiserum obtaining method of the present invention can react with HPV viral proteins of clinical specimens having cervical cancer associated with early stage cell lesions such as cervical intraepithelial neoplasia (CIN) and advanced HPV, and generate a bond. Knot. This monoclonal antibody and antiserum provides a powerful and advantageous detection tool for the detection and screening of HPV-associated pathogens and early-stage cervical cancer development, thus providing a conduit for disease progression intervention and early treatment. 3. Development of anti-HPV monoclonal antibodies: 11 201043958 [〇 纯化 纯化 HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP Mice's immune response test == OK, with fines - 敝 性 = = ^ ^ When the mouse is optimized, ton is used to add bleeding cells. Job fusion cells, such as fusion tumor cells, and _ υ 敲 敲 合 细 细 细 : : : : : 为 为 为 敲 为 为 为 为 为 为 为 为 为 为 为 为 为 HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP As a "HPV egg as a positive screening, non-related proteins are used as negative screening for fusion tumor cells (4). For example, two or more purified HPV recombinant proteins are _ out, (four) anti-county tumor fine colonization And used to test and understand the specificity of each antibody-producing fusion cell strain obtained. __• Fusion tumor cell bank: use positive immunoassay (such as brain A, mA and other tests) to select the positive reaction of Lang and Negative anti-red fusion tumor cells, and selected to lean to the single cell, each cell grows through cell culture, when the number of cells reaches 3 "§ '10,000 cells per ml', the cells are cold; Dongbao pure in. 匚 or liquid In nitrogen, it is stored as a cell bank. 〇Gang] 3)·The production of ascites: Each-fusion tumor cell line grows in tissue culture and is sown into the mouse to produce ascites. The ascites produced and collected is used as G protein column purified immunoglobulin The immunoglobulin purified from each of the fusion tumor cell lines is i isotype and can be applied to HPV immunoassay. 4. HPV immunohistochemistry (IHC) test: 1). HPVIHC reagent and test: [0034] In an implementation case Reagents for HpV IHC assay are provided. The reagents include antigen extraction reagent, pre-antibody stop solution, post-antibody stop solution, anti-HPV-12 201043958 antibody, anti-mouse or anti-rabbit immunoglobulin grafting. On horseradish peroxidase (HRP) or biotin and other reagents, as a secondary antibody, the solution contains the appropriate reagent as the matrix for the primary antibody to be tested. [〇〇«丨 antigen extraction reagent contains low pHΗ A buffer with a value, neutral ρΗ or high value. The 刖-antibody stop solution contains some protein, bovine serum albumin (), humulus, or other reagents to stop the non-specifically bound cells of the antibody. The antibody stop solution is similar to the pre-antibody stop solution and contains a small amount of protein or serum and is cultured with the primary antibody. The solution containing the HPV antibody may be in a concentrated form or diluted as a reagent. The anti-HPV antibody is also directly labeled with HRP, biotin or other reagents and can be detected as a suitable reagent for the matrix. The solution containing the secondary antibody can be in concentrated form or diluted as a reagent. = the reagent as a matrix 'including DAB (3.3'-diaminobenzidine) as a "combination of two components" or AEC (3-Amino-9-Ethylcarbazole) matrix as a rabbit one component or two components, or other Matrix [〇〇36] - Once the cervical tissue is made and fixed, the test is performed by RTUP), the tissue on the test piece is heated with the antigen retrieval reagent, and then the test piece is cooled to At room temperature, the pre-antibody was stopped by the solution; after the termination - after a period of time, it was cultured with the HPV antibody. Remove any unbound HPV antibodies from PBS, water, or other buffered slaves; place the test strips with secondary antibodies in 疒 π 忒 ,, - mouse IgG HRP, and detect with appropriate matrix. To give an example: when gamma, such as anti-enzyme and hydrogen peroxide, is present, DAB is oxidized to produce a plucking color, and the alcohol-insoluble precipitate on the peroxide site; the color of the precipitate is pale gold and the tongue is brown. The golden brown precipitate under the microscope indicates that the HPV antibody produces a specific-sex bond with the HPV protein expressed in the f-color to deep gold on the test piece. This test can be carried out at room temperature or in a cell-binding reaction. The HPV me test can be performed by hand or the following acceleration line, providing HPV infection and HPV proto-oncoprotein in situ detection. The HPV IHC staining test is a very useful confirmation test. For the confirmation of dysplastic cells, HPV IHC staining provides further information on the status of Hpv infection or Ningyuan oncoprotein expression. In addition, different stages of cervical cell dysplasia, HPV Ε6 and Ε7 proto-oncoprotein overexpression, may indicate hyperplasia or * cervical cancer development. [〇〇37] 2). Sample selection and preparation: In order to analyze the anti-Hpv antibody provided by the present invention, whether the HPV protein of different CIN course or cancer is measured by the in situ test, the IHC test knife is used to analyze the Neck tissue. Includes high-grade squamous epithelial lesions (HSIL) containing CIN2 (in the second stage of cervical intraepithelial neoplasia with a 0-degree lesion), CIN3 (cervical intraepithelial neoplasia with stage 3 with severe lesions), and squamous cell carcinoma (Lv, the most common cancer of cervical cancer) invasive cancer and adenocarcinoma (ADC, glandular tumor). The waxy tissue block was cut into 4 μm, and the test piece was cultured at 6 (rc overnight; the dewaxed/dehydrated portion was not masked, and a standard IHC staining procedure was performed. The purified anti-HPV protein monoclonal antibody was diluted as a primary antibody. The staining step is followed by a secondary antibody solution, washing, and a suitable matrix reagent. When the fragment is formed, the test piece is immediately suspended in deionized water and counterstained with tetrabromofluorescein, and the cover slip is covered. 〇100381 3)· Tissue microarray: In order to simultaneously analyze multiple samples in a homogenization test reaction, many samples were simultaneously placed on the test piece by the tissue microarray. A total of 84 samples from CIN2, or non-invasive cancer were prepared into three tissue microarrays: one array containing 30 CIN2 individuals and surrounding normal epidermal cells; the other array containing - 30 CIN3 individuals and surrounding normal epidermal cells Finally, the array contains 12% of cervical squamous cell carcinoma and its surrounding normal epidermal cells, 12 adenocarcinoma samples and their normal epidermal-associated cells, at least 15111 from the edge of the tumor to the vaginal or cervical mucosa. In each CIN case, select a representative tissue array point for tumor formation, and another microarray point representing its normal associated tissue; in a non-invasive cancer case, select two tissues as array points, and one normal Related tissue microarray points. After taking the HPV DNA alignment test piece on 201043958, the 2 mm circular tissue array point from the relevant wax embedded tissue block was recovered. 4). HPV DNA test: HPV DNA alignment in each case, using EasyChip8 HPV dot ink method or HR-HPV wafer, confirmed by a reverse-dot ink test modified with MY11/GP6+ PCR-reference % There are 13 specific nucleotides on the nylon filter, and all the cellular DNA is used as a source of nucleotides, amplified and detected by hybridization. 5). IHC degree and data analysis: the staining results of each array point on the tissue microarray, analyzed by the anatomical pathologist of the project, the area of tumor cells or dysplastic cells, calculated by cell staining Percentage, dyed depth is divided into 〇 _ 3 levels. Normal adjacent epithelial cells, or normal tissues with 15 mm of their associated dysplastic cells and tumors, were classified into grades; all data were graded by professional pathology ' '1G% of the stained sections were cut for positive or negative Test, all data is shown in Table 1-17.

Tao] In order to prove the non-invasive uterine cancer Hpv protein, squamous cell carcinoma (SCC), the most common cervical cancer, scc cancer tissue was subjected to Hpv IHC test. Fig. 1A_1D shows the results of IHC staining of squamous cell carcinoma by mouse HPV E7 monoclonal antibody. Fig. 1A is a view showing a representative image of sputum staining of tissue microarrayed SCC tissue by anti-sleeve mouse monoclonal antibody. Figure m - illustrates a representative image of normal epithelial cells (i5 mm from tumor tissue) in the SCC case in Figure iA. Figure 1C illustrates a representative image of the shame-color of a tissue microarray of another scc sample using the same anti-thoracic E7 mouse monoclonal antibody. Figure ι〇 Figure K: An enlarged image of the cytoplasm of tumor cells stained with the same anti-Hpv E7 antibody. The results of the study showed that 'E7 proto-oncogenes can be detected in tumor cells in scc tissues, black solid arrows indicate E7 protein in tumor cells 15 201043958-specific staining, and open arrows indicate unstained Normal cells. These results show that the HPV E7 protein in the SCC cervical tissue can be detected in situ by the IHC test by the anti-HPV E7 antibody. [〇〇4〇] In another type of non-invasive cervical cancer HPV protein detection, Figure 2A-2C' illustrates IHC staining of cervical adenocarcinoma with the same mouse HPVE7 monoclonal antibody. The results showed that tumor cells in adenocarcinoma tissues were able to detect the expression of E7 proto-oncoprotein, black solid arrows indicating specific staining of E7 protein in tumor cells, and empty arrows indicating normal cells that were not stained. Figure 2A is a representative image showing IHC staining of tumor cells of an adenocarcinoma (ADC) sample using the same anti-HPV E7 antibody of Figure 1. Figure 2B Figure 2A shows a representative image of the correlation of normal ADC cells (15 mm from tumor tissue). Figure 2C is a magnified image of the cytoplasmic staining of adenocarcinoma tumor cells of Figure 2A, and the enlarged image maps indicate the location of E7 protein expression in the cytoplasm of tumor cells. These results show that IHC staining with E7 monoclonal antibody can detect tumor cells of adenocarcinoma and is similar to the SCC staining pattern.

[0041] Analysis of HPVIHC results for each invasive cancer, Table 1 illustrates 24 IHCs

Q The results of cytoplasmic (C), nuclear (N) staining of invasive cancer samples, in Figures 1A-1D and 2A-2C, the percentage of anti-HPVE7 antibody staining expressed as C or N. Additional anti-HPV antibodies, including other anti-E7 antibodies, anti-HPV E6 antibodies, such as MAbl and MAb 7, and anti-HPV L1 antibodies, were tested using the same tissue microarray. For IHC staining of various anti-HPV antibodies, cytoplasmic staining of tumor cells was performed using another anti-HPV antibody. The results of IHC grading are shown in Table 1, and the results of HPV DNA alignment are also shown in the table as correlation samples. [0042] The results in Table 1 indicate that staining of tumor cells of SCC and ADE with anti-E7 antibody revealed that both nuclei and cytoplasm were stained; however, compared with the staining of tumor cells in 16 201043958, the staining of cytoplasm was more Big. In the adjacent normal epithelial cells, the expression of HPVE7 protein was detected only in the nucleus, but the expression of HPVE7 protein was not detected in the cytoplasm. Compared with normal adjacent cells, the cell mass of the tumor cells was markedly stained. These results indicate that tumor cells in SCC and ADE tissues can detect the expression of HPV E7 protein in their cytoplasm and nucleus. The E7 protein is expressed in the cytoplasm of tumor cells, rather than normal epithelial cells or stromal cells, showing its specificity for tumors. The HPVE7 protein, which is expressed in normal adjacent epithelial cells and tumor cells, is detected by an anti-E7 antibody, and shows HPV infection exhibited by the proto-oncoprotein. Similar staining patterns using other anti-HPV antibodies are described in Table 1, and the results show that the HPV IHC assay described herein can detect HPV early genes, such as E6 and E7, which are expressed in cervical tissue tumor cells, and Late gene L1 protein. [〇〇43] Comparing the results of HPV IHC and HPV DNA alignment, anti-E7 antibody was positive for HPV virus type in all test samples. The anti-E7 monoclonal antibodies described herein detect single HPV viral infections such as HPV-16, HPV-18, HPV-33 and HPV-45, which are cancer-associated HPV viruses (high-risk HPV virus). . Single anti-E7 monoclonal antibodies can also detect HPV11, HPV-16, HPV-18, HPV-52, HPV-58, HPV-51 and HPV_59 combinations of these viral types, including high HPV11, HPV-58, HPV-51 and HPV_59 Risk, low risk and non-proto-oncogene α-HPV; however, multiple HPV virus type infections, at least one high-risk HPV. These results show that the anti-E7 antibody described in the present invention is non-specific, and thus can be used as an advantageous tool for detecting the HPV E7 protein of a general high-risk virus type common to cervical cancer. [0044] Table 1: IHC staining results (% staining) of 12 see biopsies and 12 ADC biopsies and HPV DNA alignment results (C: cytoplasm; N: nuclei; 17 201043958)

Dys: structural dysfunction or tumor cells)

Sample # HPV type Anti-E7 Another anti-E7 Anti- E6 Another anti-E6 Anti-L 1 Dys (%stained) Normal epith. (% stained) Dys (%) Dys (%) Dys. (%) Dys. ( %) CNCNCCCC SCC-1 18 85 85 0 20 12.5 10 70 55 SCC-2 16, 52 90 85 0 25 15 15 10 55 SCC-3 16 60 65 0 40 5 0 10 20 SCC-4 16 92 50 0 40 5 0 10 85 SCC-5 16, 52, 58 92 55 0 50 20 5 15 88 SCC-6 18, 52, 58 90 60 25 18 10 70 SCC-7 16, 52 92 75 0 30 30 5 10 20 SCC-8 16, 58 10 10 0 5 0 0 10 50 SCC-9 noDNA 95 60 0 40 25 8 15 8 SCC-10 18 92 65 0 60 45 25 20 65 SCC-11 16,58 0 80 5 0 0 SCC-12 33 95 90 0 0 30 1 20 55 ADE-1 16, 18 30 20 0 50 15 25 20 82 ADE-2 noDNA 62 40 0 30 35 70 35 78 ADE-3 16 20 30 0 20 35 55 60 ADE-4 16, 18 80 80 0 0 10 5 0 90 ADE-5 51,52 95 80 0 50 10 70 15 92 ADE-6 11,16,52 0 40 5 0 0 15 ADE-7 18 50 40 0 60 25 20 20 75 ADE -8 18 85 60 0 40 15 50 15 82 ADE-9 45 82 55 0 30 30 2 20 40 ADE-10 18 15 10 0 40 15 15 5 70 ADE-11 18, 59 70 0 0 50 15 8 5 65 ADE -12 18 30 〇〇[0045]Detecting non-invasion HPV protein of severely structurally poor cervical tissue (HSIL; highly squamous • epidermal lesions, stage 3). Figure 3 shows the results of IHC staining of CIN3 tissue with the same mouse HPVE7 monoclonal antibody. In CIN3 tissues, E7 proto-oncoproteins were detected; black solid arrows indicate specific staining for E7 proteins in structurally defective cells, and blank arrows indicate normal cells that were not stained. Fig. 3A is a view showing a representative image of IHC staining of the same anti-HPVE7 antibody against invasive cancer tissue, and CIN3 tissue of dysfunctional cells, as shown in Fig. 1 and Fig. 2 . Figure 3B illustrates a contiguous image of adjacent normal epithelial cell generation of CIN3 tissue in Figure 3A. These results show that the IHC assay described in this section can be used to detect the HpvE7 protein expression of CIN3 cervical tissue in situ by the mouse anti-HPV E7 antibody.

[〇〇46] analysis of HPVIHC results for each CIN3 case, Table 2 illustrates 3 (M@cin 3 sample IHC grades for fine (10), cytoplasmic (c) and nuclear (n) staining, with M, C or N Indicates the degree of staining (%) of the anti-E7 antibody. Additional anti-Hpv antibodies including anti-HPVE6 antibodies, such as MAbl*MAb7, and anti-Hpvu antibodies, were tested in the same tissue microarray. Different anti-caffe antibodies As a result of the staining, the cytoplasm of tumor cells was stained with another anti-HPV antibody, and the ihc level is shown in Table i. The HPV DNA alignment results are also shown in the table as a correlation sample. 2 shown in the experiment - using the play seven 7 sinking test, in all CIN3 samples 'the nucleus of the structurally defective cells was found to be stained' only part of the sample was found to be stained as the cytoplasm. The results showed that the granules of the fine-grained cells were stained. Partially more than the nucleus'. In the invasive cancerous tissue as described above, the scorpion 7 protein in the adjacent normal epithelial cells is only found in the nucleus' rather than in the cytoplasm; the normal cells adjacent to the poorly documented are compared, Cytoplasmic staining in the structure is not It is most clear in sex cells that proteins are expressed in the cytoplasm of structurally defective cells, rather than normal epidermal or stromal cells, indicating their specificity for HSIL. This indicates that cells f and dysplasia in CIN3 tissues Sex cells = HPVE7 protein was detected; HPV E7 was detected by anti-HPV polar A ^ ^ ^ antibody, white in positive, adjacent to the nucleus and benign cells of epidermal cells, showing HPV infection and accompanying The original oncogenic protein performance. In the case of egg heart (4) innocuous performance, the cytoplasm of the dysfunctional cells of the Detective J can specifically indicate the dysplastic cells. Table 2 shows the use of another anti-Hpv The antibody has been similar to the staining pattern, and the HPV IHC test, which is not listed here, can be used in the CIN3 dysplastic cells. The Hpv early genes such as 19 201043958 E6 and E7, and the late gene L1 protein. [0048] Comparing the results of HPV IHC and DNA alignment, the anti-E7 antibody was positive for all HPV virus types present in the test sample. For example, the anti-HPV E7 monoclonal antibody described herein can be detected. Single HPV infection, at least one of HPV-16, HPV-18, HPV-31, HPV-33, HPV-39 and HPV-58 and cancer-related (high-risk HPV virus). Single anti-HPV E7 Individual antibodies can also detect infections of more than two HPV strains, such as the common high-risk HPV-type HPV-16, HPV-18, HPV-33, HPV-39, HPV-52 and HPV-58. It is shown that the anti-HPVE7 antibody described in the present patent is non-specific and can be used as a favorable detection tool for detecting HPV E7 protein of a common high-risk HPV virus type in CI3 tissues. Table 2: Results of IHC staining (% staining and grade 0-3) and HPV DNA alignment of 30 CIN 3 samples (M··cell membrane; C··cytoplasm; N′·nuclei; Dys·· dysplastic cells )

ID# HPV type anti-E7 Anti-E6 Another anti-E7 Anti-Ll Dysplasia (%stained) Normal epithelium (%stained) Dys. (%) Dys. (%) Dys. (%) M c NM c N Cyto Cyto Cyto 31 33 0 80 80 0 0 50 70 40 80 32 16 0 80 80 60 0 0 5 33 16,58 0 0 60 34 31 0 50 70 0 0 50 0 0 10 35 16, 39 0 70 90 0 0 40 0 10 30 36 31 0 70 60 0 0 50 0 20 20 37 39 0 0 40 0 0 0 0 0 0 38 16 0 0 40 39 16 0 60 70 0 0 40 0 0 40 58 0 90 90 0 0 50 50 0 30 20 201043958 ❹ ID# HPV anti-E7 Anti-E6 Another anti-E7 Anti-Ll type Dysplasia (%stained) Normal epithelium (%stained) Dys. (%) Dys. (%) Dys. (%) 41 16 0 0 50 0 0 50 0 20 20 42 16 0 70 70 0 0 30 0 0 43 33 0 0 90 0 0 50 0 0 5 44 52 0 70 80 0 0 50 70 10 50 45 51,52 0 90 90 0 0 30 80 50 10 46 16 0 0 80 0 0 50 0 0 5 47 16 0 60 80 0 0 50 30 10 20 48 16, 58 0 0 80 0 0 50 0 0 10 49 31 0 80 60 50 70 40 40 50 16 0 0 60 0 0 30 0 20 20 51 6 0 0 20 0 52 16, 18, 33,39 0 0 20 0 0 30 0 0 0 53 51,52, 58 0 70 60 0 0 60 60 40 54 16, 45 0 0 70 0 0 50 0 20 20 55 16 0 0 75 0 0 50 0 0 0 56 33, 52 0 0 80 0 0 50 0 0 10 57 16 0 0 50 0 0 40 0 0 0 58 33 0 0 80 0 0 0 20 10 59 16 0 0 60 0 0 20 0 10 5 60 16, 52, 58 0 70 80 0 0 50 70 0 20 Ο [〇〇49] Detection of HSIL with HPV protein in moderate dysplasia (CIN2), Figure 4 illustrates mouse anti- The results of HPV E6 monoclonal antibody IHC staining of CIN2 tissue showed that it can detect the expression of early E6 proto-oncoprotein in CIN2. Figure 4A - Representative image showing immunohistochemical staining (IHC) of CIN2 tissue with anti-HPV E6 monoclonal antibody. Figure 4B illustrates a representative image of adjacent normal epithelial cells of the CIN2 sample structurally deficient tissue of Figure 4A. Figure 4C shows a representative image of IHC staining of dysfunctional tissue epithelial cells of another CIN2 sample with the same anti-HPV E6 monoclonal antibody. Figure 4D is an enlarged representative image of Figure 4C with black solid arrows indicating specific staining of E6 protein in structurally defective cells, and open arrow 21 201043958 indicating normal cells that have not been stained. A similar staining of cin2 was found in CIN3. The E6 protein was expressed in the cytoplasm of dysfunctional cells rather than other cells and stromal cells. These results show that the in situ expression of HPVE6 protein in c-leg cervical tissue can be detected by *IHC staining by mouse anti-HpvE6 monoclonal antibody. [00 is the analysis of HPV IHC results for each CIN2 case, Table 3 shows the IHC levels of 3 CIN 2 samples, cell membrane (10), cytoplasmic (c) and nuclear (N) staining, expressed as M, C or N - The degree of staining of the E7 antibody (〇/〇). Additional anti-Hpv antibodies 包括 including anti-HPV Ε6 antibodies such as MAbl and MAb7, as well as anti-HPV L1 antibodies, were tested in the same tissue microarray. The results of mc staining of different anti-ingue antibodies stained the cytoplasm of tumor cells with another anti-HPV antibody, and the results of eHPV DMA alignment shown in Table 3 are also shown in the table as correlation samples. _] As shown in Table 3, experiments with anti-E6 or anti-E7 antibodies were performed, and in all CIN2 samples, the nuclei of the structurally defective cells were stained, and only some samples were found to be stained with cytoplasm. The results showed that in the CIN2 sample. In the middle, the nucleus of the structurally defective cells is stained more than the cytoplasm, as in the aforementioned scc, adc and c-leg samples 〇 + 'HPVE7 eggs adjacent to normal epithelial cells are found only in the cell, not in, cytoplasm It was found that the staining of .CIN2 cytoplasm with anti-antibodies was most clearly seen in dysplastic cells compared to adjacent normal cells. The expression of other proteins was in cells of structurally defective cells, rather than normal epidermis. Cells or stromal cells show their specificity for glandular L. These results show that E6 protein can be detected in the nucleus of tissue, cytoplasmic and structurally defective cells. Performance; a case in which the high expression of Hpv E6 protein was detected in the cytoplasm of dysfunctional cells 'can explain the proliferation of dysplastic cells. Table 3 shows that similar anti-HPV antibodies can be used to obtain similar staining patterns. Show what is described here

The HPV IHC 22 201043958 test 'detects the performance of HPV early genes such as E6 and E7 in CIN2 dysfunctional cells. And late gene L1 protein

❹ [〇〇52] Comparing the results of HPV IHC and DNA alignment, all anti-E7 antibody pairs were positive for HPV virus type. For example, a single antibody of the sample described herein can detect a single HPV infection, at least one of hpv-m, ΗΡν^8 HPV-52 and ΗΡν_58 associated with cancer (high-risk Ηρν virus type), viral type, and HPV6 And HPV 53 non-high-risk Hpv virus type 1, single-ΗΡΈ7 monoclonal antibody can also produce two or more infections of Ηρν virus type, such as $ see 冋 risk or low risk HPV virus type HPV6, HPV-16, HPV-18 , HPV-31, HPV-39, HPV-44, HPV-52, HPV-58, HPV-66, HPV-68, and combinations thereof. These results show that the anti-Ηρν Ε7 antibody described in the present patent is non-viral-specific and can detect HPV Ε7 protein from common high-risk and low-risk HPV virus types in CI Ν2 tissues. The cause of dysplastic cells may be derived from the expression of the original oncogenic protein, rather than the genotype of the Ηρν virus type; this explains that the infection is still at risk for the HPV virus type, and when the proto-oncoprotein is not detected in the cytoplasm, The color produces a recovery. Therefore, the Hpv ihC test described in this patent not only provides additional information for clinical detection of HPV infection, but also detects signs of structurally undesirable cell proliferation. Table 3: Results of 30 groups of CIN 2 samples (: staining (% staining and grade 〇3) results and HPV DNA alignment results (μ: cell membrane; c: cytoplasm; N: nucleus; Dys: dysplastic cells) ID # HPV type ---Anti-E7 Anti-E6 another anti-E7 Anti-Ll— Dysplasia (% stained) Normal epithelium (%stained) Dys. (%) Dys· (%) Dys. (%) MC n MCN Cyto Cyto Cyto 1 6 0 0 0 30 70 40 80 2 31 〇0 90 0 40 0 23 201043958 Ο

ID # HPV type Anti-E7 Anti-E6 another anti-E7 Anti-Ll Dysplasia Normal epithelium Dys. Dys. (% stained) (%stained) (%) (%) (%) 3 52 0 25 50 0 0 70 0 20 20 4 16 0 0 40 0 0 30 0 5 0 5 58 0 0 50 0 0 10 ❶ 0 〇6 52 0 80 70 0 0 50 0 5 〇7 53 0 0 80 0 0 30 0 10 10 8 52 0 50 90 0 0 20 60 10 20 9 31 0 8Θ 80 0 0 50 70 20 40 10 16 0 50 80 0 0 50 60 20 10 11 no DNA 0 0 50 0 0 70 0 0 10 12 33 0 60 60 0 0 50 0 10 30 13 no DNA 0 70 80 0 0 70 0 20 10 14 52 0 0 70 0 0 70 0 30 20 15 no DNA 0 0 70 0 0 50 0 20 ξ 16 52 0 0 10 0 0 30 0 0 5 17 52 0 0 60 0 0 80 0 0 IS 16 0 50 60 0 0 30 50 "10 20 19 16 0 50 70 0 10 20 52, 44 0 SO 80 0 0 40 0 30 21 16 0 0 50 0 0 50 0 20 V 10 JZ 16, 18, 6 0 0 40 0 0 0 0 10 n 〇_Λ 16,31 0 0 30 0 0 60 0 0 6 0 0 80 0 0 50 0 — 10 c 16 0 0 10 0 0 60 0 〇2, υ 77 58 0 0 40 0 0 40 0 10 U e 9R 16, 39, 52 0 0 70 〇6 0 0 50 0 0 70 0 10 30 16 0 0 70 0 0 5 0 10 5 66, 68 , 0 0 30 0 0 60 0 10 2U 0 ] compared with positive hanging cells Cytoplasmic staining was found only in the cell in significant structural failure can thus be based on a helmet undesirable proliferation of cells in the test structure. In order to further compare the different CIN @ _ 随 々 或 or cancer cell dysfunctional cells, buy the color, analyze the data in Table 1_3 * ^ to obtain the test results. In each case, dyeing 24 201043958 is more than 10%, which is judged as positive, and less than 1% is negative. As shown in the data in Table 4, the positive rate of the trial increased with the severity of CIN, from 38%, 52% to 100% in the case of CIN2, CIN3, SCC or ADE. Positive predictive value (PPV) and negative predictive value (NPV) are also shown in the table, showing the percentage of cytoplasmic staining as the specificity of the test. Table 4: Tissue section samples of various developing cervical cancer and tissue lesions, results of IHC staining with anti-E7 antibody

Anti-E7 dysplasia or Tumor Cyto. Stain Normal Cytoplasmic stain IHC positive rate Specificity CIN2 Positive 11 0 38% 100% 100% PPV Negative 18 28 61% NPV CIN3 Positive 14 0 52% 100% 100% PPV Negative 13 28 68% NPV SCC Positive 11 0 100% 100% 100% PPV Negative 0 11 100% NPV ADC positive 10 0 100% 100% 100% PPV negative 0 10 100% NPV

[〇〇54] The expression of HPV E7 proto-oncoprotein can be detected by another anti-E7 monoclonal antibody and tested in the same tissue microarray. The results are presented in Table 5, indicating that another E7 antibody is used. The test analysis of CIN2, CIN3, SCC and adenocarcinoma, with 72%, 48 ° /. , 67°/. , 83% positive ratio. Positive predictive value (ppV) and negative • Predicted value (NPV) are also indicated in the table, indicating that the specificity of the test with this antibody is not good. Table 5: Tissue section samples of various developing cervical cancer and tissue lesions, results of IHC staining with another anti-E7 antibody

Another dysplasia Normal IHC Specificity 25 201043958

anti-E7 or Tumor Cyto. stain Cytoplasmic stain positive rate — CIN2 Positive 21 16 72% 57% PPV negative 8 9 36% 53% NPV CIN3 Positive 13 4 48% 76% PPV negative 14 25 86% 64% NPV see Positive S 5 67% 62% PPV negative 4 6 55% 60% NPV ADC Positive 10 6 83% 63% PPV negative 2 6 50% 75% NPV

[Cry to confirm that HPVE6 protein can be tested by anti-Εό monoclonal antibody and tested in the same tissue microarray, the results are presented in Table 6-7, indicating the analysis of the test with another two anti-Ε6 antibodies. As shown by the data, dysfunctional cells from CIN2, CIN3 and non-invasive cancer cells, the expression of E6 protein in the cytoplasm can be detected by the anti-E6 antibody described in this patent. In the anti-E7 antibody ΙΗ (: the positive rate increased with the severity of CIN in the test, the same increase trend in the anti-E6 antibody test. The two anti-E6 antibodies showed the same positive with the severity of CIN in the test. The ratio increased, while the towel-anti-E6 antibody performed better than the other anti-E6 antibody in the test towel; MAbl may recognize the antigenic epitope different from MAb7, which resulted in different test results. However, as shown in the table As shown, both monoclonal antibodies have a high positive predictive value (PPV) and a negative predictive value (Npv). All IHc tests with anti-E7 resistance have a higher positive rate than tests using anti-E6 antibodies. The monthly b is due to the earlier expression of E7 protein than E6 protein, so it can be used as a biomarker for early detection of cervical cancer.

Sample, 才 / U Table 6 · Tissue sections of various developing cervical cancer and tissue lesions _E6 antibody for IHC staining results

Anti-E6 dysplasia Normal IHC or Tumor Cytoplasmic positive Specificity Cyto. stain rate 26 201043958

Stain CIN2 Positive 5 1 17% 83% PPV Negative 25 29 97% 54% NPV CIN3 Positive 17 7 57% 71% PPV Negative 13 23 77% 64% NPV SCC Positive 7 1 64% 88% PPV Negative 4 10 91% 71 % NPV ADC Positive 9 0 75% 100% PPV Negative 3 12 100% 80% NPV total Positive 38 9 46% 0 81% PPV Negative 45 74 0 89% 62% NPV ^ Table 7: Cervical cancer and various developing cervical cancers Tissue section samples of tissue lesions, results of IHC staining with another anti-E6 antibody

Another anti-E6 dysplasia or Tumor Cyto. Stain Normal Cytoplasmic stain IHC positive rate Specificity CIN2 positive 5 0 17% 100% PPV negative 24 28 100% 54% NPV CIN3 positive 8 0 30% 100% PPV negative 19 29 100% 60% NPV SCC positive 11 0 92% 100% PPV negative 1 11 100% 92% npv ADC positive 9 0 75% 100% PPV negative 3 12 100% 80% NPV total positive 33 0 41% 0 100% PPV negative 47 80 0 100 % 63% NPV

[0056] The IHC assay was performed using the same tissue microarray using an anti-L1 antibody to detect the performance of the L1 viral protein at different CIN stages. The cytoplasm was stained by the L1 antibody to obtain different IHC grade results, and the positive ratio of all samples was analyzed. The correlation between HPV early and late protein in situ manifestations at different CIN stages was studied. Table 8 illustrates the positive ratio of E7 and L1 protein expression 27 201043958 in the cytoplasm of IHC test, CIN2, CIN3 and invasive cancer. According to the hpvihc test of the present invention, the correlation between the different (10) stages: non-invasive cancer tissue E7*L1 expression was observed, and the results showed that Η? appeared to be "early staged cancer" & [good biomarkers for speculation]. Table 8 shows that the cin sample showed an L1 positive result in the excitation test, in which αΝ2 was about 6〇% (1 of 9 samples), • (:1^3 was about 58% (11 out of 19 samples) Positive in the test. Non-k cancer samples were positive for L1 cytoplasm, 11 in scc (1) samples, and adenocarcinoma (1 in 10 samples) were 100%, positive for E7 cytoplasm The development of cancer is 1%% related to the performance of E7. The results of the study showed that 〇CIN2/3 samples were positive for both L1 and E7 cytoplasm, and had a higher risk of dysplastic cell development than L1 positive but E7 negative. [〇〇57] Table 8 also shows the correlation between the positive expression of E7 and E6 in the cytoplasm of the sample. As shown by the data, the positive expression of E7 cytoplasm, the positive ratio of E6 cytoplasm, CIN2 is about 45% (11 5 out of 5), CIN3 is 57% (8 out of 14), SCC is 100% (11 out of 11), ADE is 90% (9 out of 1) ). These results show that E6 is expressed after E7 in the early development of structurally defective cells, but is common in the late stage of cervical cancer. 〇28 201043958 [0058] Table 8: Cervical cancer and tissue lesions in various developments Tissue section samples, IHC staining with anti-E7 antibody, and comparison with anti-L1 and anti-E6

Cl m CIN3 SCC ADC | Total No. LI Ll Ll Ll (-) Ll Ll Ll Ll of samples (+) (-) (+) (+) (-) (+) (Ο E7 positive 9 2 11 3 11 0 10 — 0 E7 negative 6 8 8 5 0 0 0 0 No. of E6 E6 E6 E6 E6 E6 E6 E6 | samples (+) (-) (+) (-) (+) (-) (+) (-) E7 positive 5 6 8 6 11 ° 9 1 E7 negative 0 17 0 13 0 〇° 0

[0059] In order to allow samples of various disease levels to be tested under homogeneous conditions in a single reaction, various samples of CIN2, CIN3 and non-invasive cancer were collected on the same test piece using a tissue microarray. Two sets of tissue microarrays were prepared: one array of CIN2, CIN3, SCC and its adjacent normal epithelial cells each containing 10 sets of samples; the other array was CIN2, CIN3, ADE and 10 sets of samples adjacent to normal epithelial cells. To further confirm the specificity of the HPV IHC test for HPV-associated dysplastic cells or cervical cancer, a tissue microarray containing more than 90 samples from various normal human tissues was used as a negative control in the HPV IHC trial. group. The same HPVIHC assay with anti-Ε6 and anti-E7 antibodies can be applied to these tissue microarrays to obtain the aforementioned percentage of IHC staining; those with a nuclear or cytoplasmic staining rate of 10% or more are classified as positive. [〇〇6〇] IHC data for all tissue microarray experiments are presented in Tables 9-14. Table 9 shows the results of IHC staining of mouse anti-HPV Ε7 antibody in the development of various cervical cancer and tissue lesions. The study data show that the different stages of the disease 29 201043958 cervical tissue, from CIN2, CIN3 and invasive cancer tissues such as squamous cell carcinoma (SCC) or adenocarcinoma (AD) samples, can be used to test the hpv E7 by increasing the positive ratio In situ expression of the protein. The positive ratios of CIN2 and CIN3 in the samples were 72% and 90%, respectively. In cancer tissues (squamous cell carcinoma and adenocarcinoma), IHC-staining with anti-HPV E7 antibody was positive for 100% of the samples, indicating that 1% of the cancers exhibited HPV proto-oncogene proteins. These results show that the HPVE7 antibody described in the present invention performs the IHC test' as an advantageous tool for diagnosing cervical cancer from tissues of different disease courses. 0 [0061] Table 9: Tissue section samples of various developing cervical cancer and tissue lesions, IHC staining with anti-HPV E7 monoclonal antibody IHC CIN2 CIN3 SCC ADC total Anti-HPV E7 positive 34 43 22 23 122 Anti -HPV E7 negative 13 5 0 0 18 Positive rate 72% 90% 100% 100% 87%

[〇〇62] The data of Table 9 was further analyzed to obtain the results of the test. Table 1 〇 Description The results of IHC staining in Table 'are taken as a sample of αΝ2 negative reaction in normal human tissues. The experimental data showed that IHC staining with anti-HPV E7 antibody, the sensitivity to CIN2+ was 87%, and the specificity was 92%. These results show that the test can be effectively applied to detect HPV protein' for cervical lesions cm2. Confirmation. CIN2+ CIN negative HPVE7 positive 122 7 95% PPV HPVE7 negative 18 85 83% NPV Sensitivity 87% Specificity 92% Table 10: Immunohistochemical staining of CIN2+ lesions with mouse anti-HPV E7 monoclonal antibody' and negative samples with CIN Results of the comparison 30 201043958 [〇〇63] For further analysis of the data, Table 11 illustrates the results of IHC staining in Tables 9 and 10 'showing the anti-HPV E7 antibody described in the present invention, for CIN3+ (including CIN3 and invasiveness) Cancer) The mc test has a sensitivity of 95%, a specificity of 92%, a positive predictive value of 93%, and a negative predictive value of 95%. These results show that ‘ this test can detect HPV protein in clinical applications for confirmation of different stages of cervical lesions. Table Π Immunohistochemical staining of CIN3+ lesions with mouse anti-HPV E7 monoclonal antibody and comparison with CIN negative samples CIN3+ CIN negative HPVE7 positive 88 7 93% PPV HPVE7 negative 5 85 94% NPV sensitivity 95 % Specificity 92% [0064] In order to confirm whether the EHC protein can be detected by the IHC test using the anti-E6 antibody described in the present invention, the same tissue microarray was used, and the IHC test was performed with the anti-E6 antibody. Tables 12-14 illustrate the results of another HPV IHC assay with an anti-HPV E6 antibody, as shown by the data, comparing IHC results for anti-E6 and anti-E7 antibodies. Table 12: Tissue section samples of various developing cervical cancer and tissue lesions, IHC staining with anti-HPV E6 monoclonal antibody IHC CIN2 CIN3 see AD total HPVE6 positive 32 35 23 17 107 HPVE6 negative 17 15 1 7 40 sensitivity 65% 70% 96% 71% 73% [〇〇65] Table 12 shows the results of IHC staining with anti-HPV E6 antibody in tissue sections of various developing cervical cancer and tissue lesions. The study data showed that the HPV E6 protein was detected by the IHC test of the mouse anti-HPV E6 monoclonal antibody described in the present invention. HPV E6 protein from cervical tissue at different stages of the disease can be detected in situ. As shown by the data, in situ expression of HPV E6 protein can be detected by an increased positive ratio from CIN2, CIN3 and invasive cancer tissue 31 201043958, such as squamous cell carcinoma (SCC) or adenocarcinoma (AD) samples. As shown by the data, HPV E 6 protein was positive in cin2, CIN3 and squamous cell carcinoma samples of cancer tissues, and increased with the ratio • The positive ratios of CIN2 and CIN3 in the samples were 65% and 7〇, respectively. %. In cancer 'tissue' 96% of SCC samples were positive for ihC staining with anti-HPV E6 antibody, while AD samples were only 71% positive, indicating that HPV E6 proto-oncogenes performed better than AD in SCC. The IHC test with the HpvE6 antibody described in the present invention is provided as a diagnostic tool for confirming cervical cancer tissues of different disease courses. Table 13: Immunohistochemical staining of CIN2+ lesions with mouse anti-HPV E6 monoclonal antibody, and comparison with CIN negative samples CIN2+ CIN negative HPVE6 positive 107 12 90% PPV HPVE6 negative 40 80 67% NPV Sensitivity 73% Specificity 87% CIN3+ CIN negative HPVE6 positive 75 7 91% PPV HPVE6 negative 23 85 79% NPV sensitivity 77% Specificity 92% [〇〇66] For further analysis of the data, Table 13 shows the IHC staining in Tables 11 and 12. As a result, it was shown that the anti-HPV E6 antibody described in the present invention was subjected to the HIC test for CIN2+ with a sensitivity of 73% and a specificity of 87%. These results show that the test can detect HPV protein in clinical applications for confirmation of different course of cervical lesions. Table 14: Immunohistochemical staining of CIN3+ lesions with mouse anti-HPV E7 monoclonal antibody, and comparison with CIN negative samples 32 201043958 [〇〇67] For further analysis of data, Table i4 illustrates Table u As a result of the staining, the anti-HpVE6 antibody described in the present invention is used. The brain is sensitive to muscle tests. It has a 77/d sex rate of 92%, a positive predictive value of 91%, and a negative predictive value of 79^. These results are not obvious. The test can be used in clinical applications to extract |J HPV protein, to - Confirmation of the course of the disease. 5. HPV ICC test [嶋] Sample preparation: Traditional Pap smear samples and liquid cytology samples, these two types of samples can be applied to the Icc test. The cervical cells are directly divided into two parts by the smear of the smear on the test piece, as cytological smear staining, and the other part is subjected to IHC staining with the HpV antibody described in the present invention, according to the result of smear staining. The sample is divided into 0-17 levels, the level 丨_3 is normal, and the level 4 or above is abnormal. Abnormal cells, including squamous epithelial cells at different stages of the disease, may develop defective cells or lesions; for example, LSIL: low-grade squamous epithelial lesions, HSIL: highly squamous epithelial lesions, CIN 1: cervical intraepithelial neoplasia Mild cell variability, CIN2: Cervical intraepithelial neoplasia with proliferative lesions, αΝ3: Cervical intraepithelial neoplasia with dysplastic hyperplasia 'Invasive cancer includes squamous cell carcinoma (scc), adenocarcinoma O ( AD) and others. Undefined variability cells, such as ASCUS: Atypical squamous cells (ASCUS) of unknown importance and abnormal or atypical cells in smear, still have no clear conclusions ‘the difference has not been determined. Variant cells that have been defined, HPV ICC staining can provide additional information on Hpv infection or proto-oncoprotein expression, so 'Hpv ICC staining can be more effective in detecting LSIL or HSIL abnormalities compared to Pap smear staining Cells, or detect other undefined abnormal cells, such as ASCUS or AGUS. [〇〇69〗 ICC method as an example of an immunoassay, the cells of the Pap smear are directly applied to the test piece' or collected in a liquid solution, centrifuged and purified and immunostained 33 201043958: ^_ICC step ° collected in The Pap smear in the liquid solution is subjected to cytocentrifugation or thin layer technique to make a single layer of cells according to the operation of the smear cells, and the test 4 layers of cells are fixed to the anti- _HPV antibody for HPV ICC steps, breaking the color The cells were observed under a microscope.

1 ^ Implement a case towel to provide reagents for ICC testing. The reagent comprises: a second, a stop antibody stop solution, an anti-HPV antibody as a grade antibody, a rabbit immunoglobulin graft coffee or biotin, or other four reagents as a secondary antibody, and a solution containing a suitable reagent. The substrate as a secondary antibody is detected. [0071] The anti-HPV antibody can be directly labeled with HRp, biotin or other reagents to be detected when the appropriate reagent is used as a substrate. The pre-antibody stop solution contains a portion of the protein, BSA, serum or other reagents to terminate the non-specific binding of the cells to the antibody. The post-antibody stop solution is similar in composition to the pre-antibody stop solution and contains a small amount of protein or serum and is cultured with the primary antibody. The solution containing the ΗΡν antibody may be in a concentrated form or diluted as a reagent. Anti-HPV antibodies are also directly labeled with HRP, biotin or other reagents and can be detected as appropriate reagents for the matrix. The solution containing the secondary antibody may be in a concentrated form or diluted as a reagent. The solution contains a suitable reagent as a matrix, including DAB (3.3'-(^111丨11(^61^出116) as a component or two components, or a 3-Amino-9-Ethylcarbazole matrix) As a component or two components, or other matrix. [〇〇72] When the human cells in the Pap smear are isolated and fixed on a test piece into a thin layer or a sheet, the stop solution is first co-cultured with the HPV antibody. The antibody-stop solution is used to terminate the test piece for a period of time, and the ICC test is performed. The test piece is then washed 3 to 5 times with PBS, H20 or other solution to remove the unbound HPV antibody; the test piece and the secondary antibody are as Anti-mouse IgGHRP was cultured and detected with a suitable substrate. Test 34 201043958 - Example 'When peroxide and hydrogen peroxide are present, DAB is oxidized to produce a color and move' and alcohol on the active site of the enzyme Insoluble Shen Dian; Shen Dian color according to the enzyme content is pale gold brown to deep golden brown, under the microscope, the golden plucking color sinks the temple, indicating that the antibody and the foot protein expressed in the cell produce a specific bond. This test can be Accelerate at room temperature or high temperature In the case of a knot reaction, the HPVICC test can be performed by hand or by an ICC robot, providing a powerful tool for in situ detection of HP infection and HPV proto-oncoprotein in the epithelial cells of the Pap smear. [〇〇73] To further confirm HPVICC The test can be applied to different stages of dysplastic sputum cells, and the lesions of different early, middle or late stages include, but are not limited to, early igniting soil LSIL, CIN1 $ ASCUS, metaphase lesion CIN2, CIN3 or HSIL, and Late lesions scc and ADE. Samples of different disease stages were prepared using different solution solutions. The ICC test described in this patent was performed to confirm that the test can be applied to samples of different sources, different disease stages and different liquid solutions. 〇〇74] HPV ICC test can be used to confirm abnormal cells that cannot be confirmed by standard cytology smear staining, such as ASCUS (atypical squamous cells, abnormal or atypical cells in smear, inconclusive and uncertain The difference), or AGUS (not a definite atypical gland), the HPV ICC test is used to test the ASCUS sample. As shown in Figure 5A, the ICC test It is indicated that some of the smear-stained cells were diagnosed as ASCUS Pap smear cells, and ICC staining was positive with anti-E6 monoclonal antibody. Figure 5B shows the results of ICC test of the same sample as Figure 5A, indicating that some Pap smear cells (diagnosed as ASCUS by smear staining) 'Ic staining with anti-E7 monoclonal antibody was positive. As shown in Figures 5 and 5B, high N/c (nuclear/cytoplasmic) values were not Normal cells (marked by black arrows) were positively stained, and normal cells (large and irregular, with small nuclei) stained negative, indicated by white arrows. Figures 5A and 5B illustrate a Pap smear ASCUS sample that detects HPV E6 and HPV E7 proteins in abnormal cells. This result shows that ASCUS samples contain 35 201043958 HPV-infected cells with E6 and E7 originals. The performance of oncogenic proteins, so that mouse anti-HPV E6 and anti-HPV E7 monoclonal antibodies can be detected in situ by ICC test. [0075] The HPV ICC assay detects HSIL cells. Figure 6A shows that cervical cells diagnosed as CIN2 by smear staining are prepared in another liquid solution, and the ICC test is positive staining using the anti-E7 monoclonal antibody. As shown in Figure 6A, CIN2, HSIL abnormal cells have high N/C (nuclear/cytoplasmic) values (indicated by black arrows) with positive nuclear and cytoplasmic staining. These results show that the ICC test of mouse-HPV E7 in liquid solutions from different sources can detect HPV E7 protein expressed in situ in abnormal cells of tumors at different stages. [〇〇76] In another example, Fig. 6B illustrates a CIN2 sample of another cervical cell, and after preparation with another liquid solution, the ICC staining test by the anti-E6 monoclonal antibody was positive. If shown in Figure 6B, CIN2, HSIL abnormal cells have high N/C (nuclear/cytoplasmic) values (indicated by black arrows) and their nuclei and cytoplasm are stained positive. These results show that the ICC test of mouse-HPV E6 in liquid solutions from different sources can detect HPV E6 protein expressed in situ in abnormal cells of tumors at different stages. [〇〇77] The ICC test described in the present invention can confirm the mid- to late-stage CIN cells, and the CIN3 Pap smear sample prepared with different/trocol solutions can also perform the ice test. Figure 7A illustrates Pap smear cells (diagnosed as smear staining for CIN3) and positive for ICC staining with anti-E6 monoclonal antibody. Figures 7B-7E illustrate the analysis of another CIN3 sample with the same anti-E6 mouse monoclonal antibody; as shown, 'hsil abnormal cells N/C (nuclear/cytoplasmic) values are high (indicated by black arrows), so with other The cells are linked together and the staining results are positive. These results show that the in situ performance of HPV E6 protein in medium/late stage tumor abnormal cells can be extracted by mouse anti-HPV monoclonal antibody in the liquid solution from different sources; from 36 201043958 CIN3 liquid The in situ performance of the abnormal cells in the solution can be detected by the ice test using mouse anti-HPV monoclonal antibody. • Figure 7F illustrates the results of icc staining with the anti-HpvE7 mouse monoclonal antibody, the same αΝ3 sample as in Figure 7B.胄7G indicates another ICC staining result, and the same anti-HPVE7 mouse monoclonal antibody was used as in Fig. 7F; as shown in the figure, the N/C (nuclear/cytoplasmic) value of HSIL abnormal cells in CIN3 was high (indicated by black arrows), Therefore, it is linked with other cells and the staining result is positive. These results show that in situ metastasis of HPVE7 protein in mid-stage tumor abnormal cells can be detected by ice test using mouse anti-HPV monoclonal antibody in different liquid solutions of sputum; The in situ expression of Hpv E7 protein in normal cells can be measured by ICC test using mouse anti-HPV monoclonal antibody. [〇〇79] In order to confirm that pl6 also reached a transient state of expression in advanced tumors, ICC staining was performed on the same CIN3 sample with anti-pl6 old mouse monoclonal antibody. Figure 7G illustrates the results of Icc staining of anti-pi6 mouse monoclonal antibodies in the same CIN3 sample as in Figures 7B-7F. As shown, the N/C (nucleus/cytoplasmic) values of HSIL abnormal cells in CIN3 are still The black arrow does not list ', so it is linked with other cells, and the staining result is positive for Q. These results indicate that the in situ expression of pl6 protein in mid- to late-stage tumor abnormal cells can be detected by ICC test using mouse anti-HPV monoclonal antibody in liquid solutions from different sources; from CIN3 liquid solution The in situ manifestations of HPV E6, HPV E7 and pl6 in abnormal cells can be detected by the ICC test of mouse monoclonal antibodies. [0080] The ICC test described in this section can be used as another example for the detection of cervical cancer cells in a liquid liquid for testing. Different cancerous tumors in a Pap smear sample are prepared in different solutions for ICC test analysis. 8 indicates that cervical cancer cells (diagnosed as adenocarcinoma by smear staining)-E6 monoclonal antibody positive for ICC staining 37 201043958 should be; among them, have high N/C (nuclear/cytoplasmic) values (with black arrow The abnormal cells expressed by the cells were positive for staining, and the HSIL abnormal cells linked to other cells also showed a positive staining reaction. These results show that the expression of HPV E6 protein in the adenocarcinoma of the adenocarcinoma in the solution can be tested by ICC in the anti-HPV E6 mouse monoclonal antibody. [〇〇81] The ICC test described in this section can also be applied to detect another type of cervical cancer in another liquid solution. Figure 9A illustrates another type of cervical cancer cell gee (diagnosed as smear by smear staining) "Cellular cell carcinoma" is positive for the acetaminophen with anti-E6 monoclonal antibody. Figure 9B shows the results of ICC staining of the same SCC sample with anti-HPV E7 mouse monoclonal antibody. Figure 9C shows the results of ICC staining with anti-pl6 mouse monoclonal antibodies in the same SCC samples. As shown in the figure, the nuclear and cytoplasmic staining of HSIL SCC cells is positive. 'Indicating the in situ expression of HPVE6 and HPVE7 proteins in different viral cervical cancer cells in liquid solution. Anti-HpvE6 or E7 mouse can be used. The antibody is tested by ICC test. In order to confirm that P16 also reached a transient state of expression in advanced tumors, ICC staining was performed on the same scc sample with anti-pl6 mouse monoclonal antibody. Figure 9C shows the same SCC sample as in Figures 9A-9B, the results of ICC staining with anti- ◎ -P16 mouse monoclonal antibodies, showing that pl6 protein expression in advanced tumors can be detected; indicating liquid cancer from different viral cervical cancers In situ expression of HPV E6, HPV E7 and pl6 proteins in abnormal cells in solution can be detected by ICC test using mouse monoclonal antibodies. [0082] In order to confirm the ICC staining results described in this section, it is indicated that the HPV protein and the HPV antibody expressed in situ in the Pap smear cells are specifically bonded, and the normal cervical cells obtained in the liquid solution are also subjected to ICC test analysis. . Figure 10A illustrates Pap smear cells prepared in a liquid solution (diagnosed as normal by smear staining), negative staining for WC test with anti-E6 monoclonal antibody; ICC for the same sample with anti-E7 monoclonal antibody The test results are illustrated in Figure 10B. The results of the study showed that the HPV E6 or E7 protein expressed in normal cells of 38 201043958 was subjected to ICC test with anti-HPV E6 or E7 monoclonal antibodies, all of which showed negative staining results; indicating that the icc test described in the present invention is a specific one. Staining method, using HPV-specific antibody to detect Hcv protein 0 [0083] ICC staining results in the cell layer, Figure i iΑ illustrates the anti-HPV Ε6 mouse monoclonal antibody, the preparation of Pap smear cells in liquid solution The cytoplasm is stained. Fig. 7B shows the results of nuclear staining with anti-HpvE7 mouse monoclonal antibody in the same sample as Fig. 11A. Figure 11C is a representative image showing the cytoplasmic staining of another sample with anti-HPV E7 mouse monoclonal antibody. As shown in the figure, abnormal cells have high N/C (nuclear/cytoplasmic) values (indicated by black arrows), and the staining results are positive; while normal cells (large 'cell type is irregular and the nucleus is small) staining results Negative reaction, indicated by white arrows. These results demonstrate that the anti-HPVE6 and E7 mouse monoclonal antibodies described in the present invention can detect the expression of HPVE6 and E7 proteins in cells or nuclei. [〇〇84] HPV E6 and E7 proto-oncoproteins can be expressed in certain LSIL (low-grade squamous intraepithelial lesions) or CIN 1 (cervical intraepithelial neoplasia moderate cytopathic), Figure O 12A_12C shows resistance _HPV E6 mouse monoclonal antibody, the result of ICC staining for liquid solution of clinical diagnosis of CIN1 sample. Fig. 12B-12C shows the results of ICC staining using the same anti-HPVE6 mouse monoclonal antibody as in Fig. 12A. Fig. 12D is a view showing the results of ICC staining of the CIN1 sample identical to Fig. 12A with anti-HpvE7 mouse monoclonal antibody. Fig. 12E shows the results of icc staining using the same anti-HPV E7 mouse monoclonal antibody as in Fig. 12D. As shown, the abnormal LSIL and CIN1 cells are linked to each other, or have a high N/c (nucleus/cytoplasmic) value (indicated by black arrows), and their nuclear and cytoplasmic staining results are positive. These results indicate that the in situ expression of Ηρ“ν邡^ E7 protein can be detected in early tumor samples prepared in liquid solution by using the anti-HPV E6 or E7 antibody for ICC assay. 39 201043958. To confirm early tumors Whether the pl6 protein is expressed or not, ICC staining is performed on the CIN1 sample with anti-pl6 mouse monoclonal antibody. Fig. 12F shows the results of ICC test of the same CIN1 sample as the anti-pl6 mouse monoclonal antibody, which is the same as that of Fig. 12A-12E. The pl6 protein could not be detected in the tumor. All the results indicated that the in situ expression of HPVE6. and HPV E7 protein could be used to detect the abnormal cells of the early tumor with the anti-HPV E6 or E7 mouse monoclonal antibody described in the present invention. Detection, while anti-_pl6 mouse monoclonal antibody for ICC test, can not detect the performance of pi6 protein.

[〇〇85] The icc test not only detects HPV infection, but also detects Hpv pro-carcinogenic prion protein in situ. Therefore, a single ICC assay, or a combination of various specific or general anti-Hpv antibodies, can be an advantageous tool for in situ detection of HP V relative to standard HPV DNA assays or smear assays. Table 15: Liquid solution of various Pap smear samples by anti-HPVE6 mouse monoclonal antibody

Ap negative, using an anti-HPV E6 antibody 25 6 4 6 〇1 total 29 9 8 17 17 Positive rate 14% 33% 38% 65% 100% D 80% Q Results of the ICC test Table 15 shows the results of a test of liquid samples of various Pap smear samples by anti-HPVE6 mouse monoclonal antibody. The results showed that the monoclonal antibody of the marginal _HpvE6 mouse was subjected to ICC test on the single-cell fixed on the test piece, and the performance of the HPVE6 protein could be in situ extracted and the sample could be a purely diseased stage of the Pap smear cancer solution. sample. The smear staining of the phase (10) sample was compared with the ICC staining results. As shown in Figure 15, the performance of HPVE6 protein was positive with the Pap smear, and the Ascus, asc-h, CIN1 and CIN2/3 samples showed a positive trend. . 201043958

Pap smear normal CIN2+ ICC positive, using an anti-HPV E6 antibody 4 21 84% PPV ICC negative, using an anti-HPV E6 antibody 25 1 96% NPV Sensitivity 95% specificity 86% * [〇〇87] Diagnosed by smear For the CIN2/3 sample, 100% was positive, and the sample that was diagnosed as normal staining, ICC staining test with the same anti-HPV E6 antibody, 14% was positive. In the ASCUS or ASC-H samples, approximately 33% to 50% of the anti-HPV E6 antibodies were used in the same sample as the CIN1 and CIN2/3 samples in Table 15, indicating positive carcinogenicity in these ASCUS or ASC-H samples. The Ο ^ expression of the protein will further form the proliferation of cancer. Samples stained with ASCUS but negative for ICC staining (anti-HPV E6 antibody) may have a lower risk of developing a proliferative lesion. Table 16: Results of ICC staining of CIN2+ Pap smear liquid solution samples by anti-HPVE6 mouse monoclonal antibody [Table 88] Table 16 shows the ICC staining results from Table 15. As shown by the data, the ICC staining test was carried out using the anti-HPV E6 antibody described in the present invention, and the sensitivity to CIN2+ was 95%, and the specificity was 83%. The results of the study show that the test is beneficial for the detection of HPV protein, and can be used for screening cervical cancer in the general public smear staining. • Table 16: Results of ICC staining of various Pap smear liquid solutions with anti-HPV E7 mouse monoclonal antibody

Pap smear normal ASCUS ASC-H CIN1 CIN2/3 see ICC positive, using an anti-HPV E7 antibody 3 4 3 11 16 4 ICC negative, using an anti-HPV E7 antibody 25 6 5 6 1 1 total 28 10 8 17 17 5 positive rate 11% 40% 38% 65% 94% 80% 41 201043958 [0089] Another ICC test detects HPV samples, and Table 17 and Table 18 illustrate the results of ICC staining with anti-ΗΡν Ε7 antibody. As shown by the data, the results of the ICC test for anti-HPV Ε7 antibody are different from those for anti-HPV E6. Table 17 shows the performance of HPV E7 protein, with normal Pap smear, ASCUS, ASC-H, CIN1 and CIN2/3 samples. , showing a positive trend of increasing. The sample diagnosed as smear CIN2/3 was 94% positive, and only 11% of the smear samples were positive for the ICC test with the same anti-HPVE7 antibody. In the ASCUS or ASC-H samples, the same anti-HPVE7 antibody was used in the CIN1, CIN2/3 samples of Table 17, and 40% showed a positive staining reaction, indicating that the expression of the original oncogene protein of these samples may cause further cancer proliferation. Smear Diagnostics® ASCUS and ICC staining (anti-HPVE7 antibody) negative samples have a lower risk of developing lesions. [〇〇9〇] Table 18 illustrates the ICC staining results from Table 17. As shown by the data, the ICC staining test was carried out using the anti-HPV E7 antibody described in the present invention, and the sensitivity to CIN2+ was 91%, and the specificity was 89%. The results of the study show that the test is beneficial for the detection of HPV protein, and can be used for screening cervical cancer in the general public smear staining. Table 18: Results of ICC staining of CIN2+ Pap smear liquid solution samples with anti-HPV E7 mouse monoclonal antibody 〇

Pap Smear Normal CIN2+ ICC positive, using an anti-HPV E7 antibody 3 20 87% PPV ICC positive, using an anti-HPV E7 antibody 25 2 93% NPV Sensitivity 91% specificity 89% 42 201043958

Pap smear normal ICC HPV positive ICC HPV negative high-grade HPV DNA positive 0 6 high-grade HPV DNA negative 0 26 Table 19: Results of ICC staining of normal smears with various anti-HPV antibodies, and with HP V Comparison of DNA Tests [〇〇91] In order to test whether the HPV icC test described in the present invention can be used as a tool for early screening of cervical cancer, a normal sample of smear is compared with HPV DNA test and HPV DNA test. As shown in the data in Table 19, the anti-HPV antibody was tested, and all sputum smears were negative in normal samples (32 out of 32 samples). Of the 32 samples, 16 were stained with anti-HPV E7 antibody. The results of 4 samples stained with anti-HPV L1 antibody showed that the ICC staining test described in the present invention has a very good specificity, compared with the HPV DNA test, only 19% (6 out of 32 samples) The normal smear samples were positive in the HPV DNA test. In this experiment, the HPV DNA test was performed with the only FDA-approved hc2. HPV DNA was positive, smear staining was normal, and HPV ICC test was negative, may be false negative, or did not express HPV proto-oncogene protein. The experimental results show that the HpviCc test 提供 provides a higher positive predictive value than the HpV DNA test. Therefore, ICC provides a good basis for clinical screening of cervical cancer. Flow Cytometry • [0092] Another example of HPV protein immunoassay detection is the Ηρν flow cytometry test. Pap smear cells were collected into a liquid solution and centrifuged according to standard procedures. [Substantially followed by immunostaining similar to the ICC staining procedure. The cells are kept in the ridge without being immobilized on the test piece, analyzed by flow cytometry, and the cells in the solution are fixed, terminated and cultured with an anti-Hpv antibody, followed by appropriate secondary antibody and matrix 3 The reagent was subjected to flow cytometry reading. The best of the flow cytometry test 43 201043958 points, the number of samples can be tested at a high flow rate, without the need for professional cytologists to analyze the test piece. [〇〇93丨 In an embodiment] provides reagents for ICC flow cytometry experiments. The reagent may comprise a pre-antibody stop solution, a post-antibody stop solution, an anti-HPV antibody as a primary antibody, an anti-mouse or anti-rabbit immunoglobulin grafted fluorophore or biotin, or other as a secondary antibody. The reagent, the solution containing the appropriate reagent is used as a matrix for the secondary antibody, and is detected by a flow cytometer. 〇 〇 [〇〇94] Indirect calibration requires a two-stage culture step, first using a graded antibody followed by a compatible secondary antibody, which can be accompanied by a fluorescent dye PE C 5 , etc.). Anti-ΗΡν antibody village directly marks the topping substance, *element or other reagents'. It is made of silk with appropriate reagents. The pre-antibody stop solution contains certain proteins, BSA, serum or other cells that attempt to terminate non-specific binding to the antibody. The post-antibody stop solution is similar to the pre-antibody stop solution and contains a small amount of protein or serum and is cultured with the -grade antibody. A solution containing a HPV antibody is either in a concentrated form or diluted as a reagent. A solution containing a secondary antibody may be in a concentrated form or diluted as a reagent. It is called [secret] HPV Ε6, Ε7 or L1 protein can be carried out by specific antibodies and secondary antibodies labeled with fluorescent dyes (grafted with FITC, PE, Cy5, etc.) (4). Cells are analyzed by flow cytometry and can be sorted according to their size or other parameters and can be stained or not stained. The degree of staining of the cell population of the smaller cells was compared with the larger Z cell population and served as a control group for normal cells in the experiment. The number of cells in which the assay provides per-cell specific staining to enable staining can be known by calculation, and the degree of coloration can be quantified by analysis. This test can be used in a large number of analyses, without the use of mirrors, and without the need for cytologists to perform staining. The computer software of the cytometer can accurately analyze the data without cytology. Specialized in 44,43,958,000, it can be effectively used in clinical, as a screening or auxiliary tool for detecting HPV-related proteins. [〇〇96] In another example, after the cells are stained, they are suspended in a 4% dark environment. Under the condition and analyzed by a cytometer as soon as possible. If the analysis time is more than one hour, the cells need to be fixed. Keeping the cells for several days stabilizes light dispersion and deactivates most of the biohazard factors. Different tests require different reagents to fix and find optimized parameters. Format 1· 〇_〇1〇/0 to 1〇/〇paraformaldehyde 1〇_15 minutes, each sample is ΙΟΟμΙ, format 2. Acetone or decyl alcohol: 1„11 ice acetone, Q mixed in each sample After homogenization, let stand at -20oC for 5-10 minutes, centrifuge and wash twice with pbs 1% BSA. [〇〇97] Fix the cells first to confirm the stability of short half-life antigens or antibodies, and perform intracellular staining. The position of the labeled protein in the original cells can be maintained. The antigen in the cell is subjected to a cell permeabilization step before the staining, and the antibody is prepared in a permeabilization buffer to ensure that the cells can maintain permeation when the cells are When the group begins to permeate, the cell light dispersion data on the flow cytometer changes after permeabilization. The antigens in the cell organelles and particles 'have different fixation and permeability methods depending on the antigen type, Q to maintain The antigen is determined to be usable. [〇〇98] Cell fixation is an important factor affecting the quality of the results of the staining test. The method is as follows: (1) using formaldehyde and surfactant: in 0. 01 〇 / 〇 formaldehyde Fix it. 10-15 minutes ( Stabilizing the protein), breaking the cell membrane with a surfactant. Surfactant: Triton or ΝΡ-40 (0.1-1% in PBS), can partially dissolve the nuclear membrane, and is suitable for nuclear antigen staining. Loss of cell membrane and cytoplasm can result in reduced light dispersion and reduced non-specific fluorescence. Tween 2〇, Saponin, Digitonin and Leucoperm are mild cell membrane solubilizers, configured at 0.5% pBC, providing a large enough pore size Enables antibody to penetrate without lysis of cells 45 201043958 Plasma membrane, suitable for cytoplasmic or cell surface antigens, also for lytic nuclear antigens. (2) Use formic acid (〇.〇1%) and fermentation, and Surfactant: Add lm ice lyase to each sample, mix well, let stand for 5_1 〇 minutes at _2 〇〇c, centrifuge and wash twice with PBS 1% BSA. Fix and permeate with acetone: Add 1m ice acetone to each of the 'mixtures, mix evenly, and let stand for 5-10 minutes at -20 °C. After centrifugation, wash with PBS 1% BSA two: owe. 7. Detect solid state with primary antibody-HPV antibody Surface Hpv protein υ. Direct test : A variety of HPV proteins were coated on microplates, and a variety of anti-ϋ-HPV anti-cancer assays were used to detect HPV Ε6, Ε7 or L1 protein in a direct mA test. Different samples of cervical cells can be obtained from liquid cell solutions, in transport media (for HPV DNA testing) or in cell lysates. For the sputum test, samples are prepared, centrifuged, purified and lysed to produce cell lysates for analysis. Analyze the protein in the cell lysate and lay it in each well of the microplate, and bind the protein in the microplate with HPV monoclonal antibody and extract it. A secondary antibody having HRP (in the case of mouse or rabbit anti-igG) was used for binding, and a TMB matrix was added as a stop solution, and the absorption value of 〇D450 was detected by an ELISA enzyme immunoassay. 1001001 collects cervical cells from different stages of tumor progression in a liquid solution to obtain their cell lysates for detection of HPV DNA and proteins. In the HPV DNA 1 assay, the 'pick-down PCR step; direct EIA assay for HPV protein detection' direct cell lysate on the microplate for bonding, followed by grafting HRP specificity The secondary antibody is reacted. The OD450 absorbance value of the primary HPV antibody added or not added was read by a micropore analyzer. The microplates were coated with various proteins in the cell lysate, and the OD value of 46 201043958 was measured without a sample of the primary HPV antibody, indicating that the cell lysate and the secondary antibody were non-specifically bound. The Hpv protein and the anti-HPV antibody produce a specific bond (10) value, which is the same: the 〇d value is subtracted from the OD value of the secondary antibody non-specific bond, and the net 〇D value is Ηρν protein and the primary antibody. - The 〇D value of the HPV antibody-specific bond. The sample with negative pCR was 'self-side's net 0D value, and the average value was used as the benchmark for the test. In the off-test, the net OD value of the sample was expressed as positive when the net OD value of the PCR negative sample was more than twice the average 〇D value. Then it is negative.

Liquid based cervical scrapes HPV DNA ~ by PCR Direct EIA by poly anti-E7 Sample No. Dx or pap smear results 1 ASCUS pos pos 2 ASC-H pos pos 3 ASCUS pos pos 4 ASCUS jm___ pos 5 ASCUS neg nee 6 CIN1 pos pos 7 CINl pos pos 8 CIN1 pos pos 9 CINl pos neg 10 CIN1/ASCUS neg pos 11 CINl neg neg 12 CIN2 pos neg 13 CIN2 pos neg 14 CIN2 neg neg 15 CIN2 neg neg 16 CIN3 pos pos 17 CIN3 pos pos 18 CIN3 pos pos 19 CIN3 neg neg 20 CIN3 neg nee 21 CIN3 neg nee 22 SCC pos 1 pos 23 SCC pos pos 24 AD neg neg 〇〇[〇〇1〇1] Table 20: hpVDNA and protein detection clinical in liquid Pap smear samples The samples were diagnosed as normal cells by histology or smear staining, and ASCUS, CIN1, CIN2, CIN3, SCC or adenocarcinomas were directly lysed on microplate 47 201043958. The clinical diagnosis or smear results were compared, HPV DNA was detected by PCR, and HPV protein was detected by EIA. The results are shown in Table 2〇. The data showed that the agreement between HPV DNA and HPV protein was 79% (19 out of 24 samples) with anti-E7 antibody, and the non-conformity rate was 21〇/0 (24 samples included 5), 3 of which were PCR positive and EIA negative (samples N0.9/CINI, No.l2/CIN2 and N0.13/CIN2), indicating HPV infection but no E7 proto-oncoprotein performance, or No performance was detected. In samples positive for PCR and negative for EIA, samples N〇. 4 (ASCUS) and No. 1〇 (CINi/ASCUS) may be pCR-negative, or old proto-oncoproteins may be accompanied by loss of HPV DNA. [〇〇1〇2] PCR is positive, eia is negative, may be a false positive or positive for HPV DNA test, but HPV proto-oncogene protein does not show, these results show that HPV EIA test can be used as Another basis for clinical screening of cervical cancer. Detection of HPV proto-oncoproteins is important for tracking the occurrence of structurally undesirable HSI1, a good biomarker for early detection or screening of cervical cancer and other HPV-associated cancers. Both the pcr and eia tests were negative, but were diagnosed as ASCUS or CIN. The HPV DNA and proto-oncoprotein could not be detected, which may be related to the preparation of the patient's Pap smear sample, or the smear staining result was false. Positive, however, there are still many samples to be tested. 2). Ink test: The cell lysate is spun on the membrane, and one or more anti-Hpv anti-ships are used to detect the HPV protein 1001031 in the biological sample. The test is rapid and does not require instrument interpretation. Ink method is a viable way to provide a visual result of the colorimetric agent to detect HPV protein in cell lysates on the membrane. The cervical cells from different stages of the tumor are collected into a liquid solution, and the cell lysate is prepared and transferred to the membrane. The membrane is air-dried to terminate the solution to terminate the breakthrough point, and the anti-HPV antibody is added to the reaction point. Next, a secondary antibody that binds to 48 201043958 anti-HPV antibody is added. Wash the break point between each step to avoid non-specifically bound antibodies attached to the membrane, and finally add TMB coloring reagent. The cell lysate and the anti-HPV antibody produce a bond, which is blue in the test. Positive reaction. The HPV recombinant protein also collapsed on the membrane as either a positive control group or a negative control group. [〇〇1〇4] Fig. 13A shows the results of the dot-ink method for detecting HPV L1 protein by anti-HPV L1 mouse monoclonal antibody. The first behavior is the liquid cell lysate of different SCC Pap smear, the second behavior is HPV16 L1 recombinant protein, from left to right concentrations of 20, 2, 0.2 and 0 ug/ml, respectively, with A, B, C, D Marked. The results of the second line of ink 〇 showed that the HPV16 L1 recombinant protein at a concentration above 0.2 ug/mH, or the purified low-level recombinant protein, was highly positive for the anti-HPV L1 mouse antibody antibody used in Figure 13A. The experimental data showed that the HPVL1 protein from HPV 16 L1 recombinant protein and cell lysate was detected by a spot ink test using the same anti-HPV L1 mouse monoclonal antibody as in Figure 13A. [00105] Figure 13B illustrates the results of another spot ink test using the same anti-HPV L1 mouse monoclonal antibody as in Figure 13A. The first and second rows of the rupture point are liquid cell lysates from different SCC Pap smear, and the third row of repulsive points are HPV16 Q E6, HPV18 E6, HPV16 E7, HPV18 E7, HPV16 L1 recombinant protein Marked from left to right as A, B, C, D, E. As shown by the results, the HPV recombinant protein spot 3E was positively tested with anti-HPV L1 antibody, and the reaction with the third row of HPV16 E7 and HPV16E6 did not react, or HPV18E7 and HPV18E6 reacted weakly. Point to compare. The clinical samples 2C and 2E were highly reactive with the HPV16 L1 recombinant protein with a weakly reactive or non-specific bond as background values. The results showed that the same anti-HPV L1 mouse as in Figure 13A was used. The monoclonal antibody was subjected to a spot ink test to detect HPVL1 protein from HPV 16 L1 recombinant protein and cell lysate. 49 201043958 [00106] The HPV Ε6 protein was detected by the dot ink method 丨PTPV ρ, and the results of the dot ink analysis by the anti-HPV Ε6 antibody are shown in Fig. 14 as shown in the figure. The first material is from the same SCC cervix. Smear the cell lysate solution (same as the first row of Figure i), the second behavior ϋΡΥ16Ε6 recombinant protein 'from left to right concentration is plus, 2, 〇2 and oug/mi, =A 'B' C' D does not mark. The second line of the break point results showed that the anti-HPV E6 used in Figure 3 was subjected to a spot ink test with a single antibody, and the concentration was 2 ug/mi positive for HPV E6 recombinant protein 'q.2 and after purification Recombinant protein reaction I·weakness was not used in the same anti-supplementary mouse monoclonal antibody as in Figure 14A. Ink test T-stimulation from HPV 16 & recombinant protein and cell lysate Η" E6 []® 13B The same dot-ink method is also applied to the detection of Hpv% protein. Figure 14B shows that the same anti-Ηρν11 antibody was used to detect HpvE6 by the same anti-Ηρν11 antibody in Figure 14A. In the third line, the recombinant protein spot 3A was inhibited, and the anti-Ηρν 16Ε6 antibody was tested positively, and compared with the third line Ηρνι8Ε6 reaction 14 weak point of collapse or other non-reactive stagnation point. Explain that 'k-HPVl6E6 mouse monoclonal antibody reacts specifically with the coffee, but does not react with Hpv 〇L1 or HPV Ε7 protein. Use the weak reaction point as the background value or the 4 test HPV18 Ε6 Sex bond, relative to other moderately reactive points, sample 2 C and 2Ε are the repulsive points of reactive resilience, and the sample 2D has no detectable "crash point. These experimental data show that 7〇% (7 of 1 samples) of the 'bed sample contains HPVE6 protein can be tested by spot ink method using anti-HPV16E6 mouse monoclonal antibody.

1〇〇1〇8] To illustrate the detection of HPV E7 protein by the dot blot method, the same break point in Figure 13B and Figure 14B was used to detect Hpv E7 protein in anti-HPVE7 mouse monoclonal antibody, as shown in Figure 15. Show. The results showed that in the third line of the map, the Ηρνι8Ε7 recombinant protein rupture point 3D was positive with the anti-HPVE7 mouse monoclonal antibody, and the other HPV 201043958 recombinant protein did not detect the fulcrum point, or with HPV16 L1. There is a very reactive transition point 3E, as shown in the third row of Figure 15. These results indicate that anti-HPVE7 mouse monoclonal antibodies are specifically reactive with HPVE7 and do not cross-react with HPVL1 or HPVE7 proteins. With the weak reaction point as the background value or the cross-linking of HPV 18 E7 in the test, the samples 2C and 2E are highly reactive points with respect to other non-reactive points. These experimental data show that samples 2C and 2E contain HPV18E7 protein, which can be detected by dot blot assay using anti-HPV18E7 mouse monoclonal antibody. 0 3). Antibody microarray··The antibody is cleaved on the protein wafer to detect the HPV protein and the endogenous protein in the cell lysate labeled with the biometric sample [00109], for example, in a protein wafer As the surface of the protein slope/bonding, it may be a surface-treated glass or film that can be covalently or non-covalently bonded to a capture reagent or protein. Capture reagents, such as recombinant proteins, antigens, antibodies, or other proteins, are embedded in appropriate pinholes on the tumbling machine and placed in appropriate buffers to facilitate binding to surface-coated proteins or antibodies. As also described on the surface of the microplate, the capture protein or antibody is strongly bonded to the Q surface after chemical treatment on the surface of the protein wafer, and the surface is retained to allow the capture protein to interact with the target protein, antibody or antigen and to be specific. Bonding, after multiple cleanings to remove non-specific bonds, is detected by grafting the Cy3 or Cy5 detection system. The fluorescence intensity of the image of the break point is measured by a microarray scanner to determine whether or not a specific reaction is generated. [00110] In one example, antibody microarrays can be used in protein wafer assays to detect HPV proteins or other cellular proteins. The cells, samples or cultured cells to be tested are collected, centrifuged, washed, and lysed to produce cell lysates as analytes. Quantify the protein content in the cell lysate and calibrate biotin, Cy3, Cy-5 or other coloring agents to detect the binding of the protein labeled on the surface of the antibody at the point of reversal 51 201043958. The surface of the protein wafer, depending on the analytical and quantitative techniques, can be a film or glass surface. [00111] Table 21 illustrates the results of protein wafer assays for detecting the expression of different HPV proteins and host cell proteins. The antibody microarray first reverses the antibody, which is resistant to HPV and various cellular proteins and is used to detect HPV protein and host cell protein expression in clinical Pap smear samples. Ten groups of Pap smear samples (labeled S1-S10 in Table 2) were diagnosed with keratin squamous cell carcinoma (grade 2 or 3), stored in a liquid solution and lysed to produce protein lysate, and labeled appropriately A marker (such as biotin) is used to facilitate detection (eg, avidin-Cy3). Fluorescence intensity indicates that the protein in the sample binds to the antibody, including but not limited to HPV-16E7, HPV-16L1, p63, p53, p21WAF1, pl6INK4a, phosphorylated Rb and unphosphorylated Rb, as shown in Table 21. Fluorescence intensity indicates that specific proteins are specifically linked to antibodies on the microarray, showing varying degrees of HPV infection, as compared and analyzed in Figures 6-12. [00112] Table 21: Fluorescence intensity (after background value subtraction) of each array spot of the antibody microarray, indicating the protein expressed in the cell lysate of cervical cancer patients,

Ab spotted SI S2 S3 S4 S5 S6 S7 S8 S9 S10 HPV16 E7 849 1407 422 355 443 403 316 337 383 267 HPV 16 1309 236 1477 418 620 1206 251 205 700 3407 p63 398 128 205 51 167 146 215 230 427 174 p53 325 102 86 161 119 83 226 242 465 335 P21WAF1 594 100 130 92 167 54 177 178 493 250 pl6INK4a 164 549 97 107 116 87 72 128 87 174 Retinoblastoma 753 170 140 185 109 70 219 247 448 317 Rb (phosph) 491 236 269 143 238 245 156 224 310 171 Heterospecific antibody-specific binding [00113] Comparing the performance of 10 groups of tested SCC samples, measuring the sample-specific protein fluorescence intensity mean and standard deviation, can show its protein performance, such as Figure 16 shows. The results showed that in the cell lysates of 10 groups of Pap smear samples, 52 201043958 various Η PV proteins and endogenous proteins can be detected by antibody microarray experiments, as shown in Figure 16, HPV16 and HPV16E7 Compared with other cellular proteins, it is over-expressed. [〇〇114] Comparing the differences in HPV16 in different samples, Figure 17 illustrates the fluorescence intensity values of the HPVL1 protein detected from the cell lysate of the Pap smear sample in Figure 16. As a result, the HPV16 antibody (anti-HPVL1 antibody) binds to the Hpv protein, particularly the L1 viral protein expressed in cell lysates from cervical cancer patients on the antibody microarray. The HPV16 L1 protein is mainly expressed in the samples S1, S3, % and sl〇〇, moderately in the samples S4, S5 and S9, and in the samples S2, 87 and S8, the expression is poor. The sample may be another HPHV virus. Infection is not recognized by HPV16 antibodies in this assay. 1001151 HPVE6 and E7 proteins play an important role in HPV proto-oncogenes in cervical cancer. To study the interaction between HPVE6E7 proto-oncoproteins and cellular proteins, antibody microarray assays can be used as a tool to detect Hpv proteins and cells. A protein such as ρ53 or Rb interacts with HPV proto-oncoprotein or cellular proteins P16, P21, etc., which are affected by HPV infection. P16INK4a is often used to detect cervical cancer in Russia. To detect HPV viral proteins, such as E6 or E7 proto-oncoproteins can be used as good biomarkers. Antibody microarray (protein wafer assay) analysis can simultaneously detect a variety of HPV protein and cellular proteins. Figure 18 illustrates the detection and comparison of HPVE7 and pi6 protein expression in 10 sets of SCC samples. The fluorescence intensity value (from 1 to 1 〇) of each sample indicates the performance of cell lysates in cervical cancer patients on antibody microarrays. Protein, which produces specific binding to HPV16E7 and pl6INK4a antibodies. The fluorescence intensity of each of the HPVE7 antibodies was higher than that of the pl6 antibody, indicating that the HPVE7 protein was more abundant than the pl6 protein. The results indicated that HPVE7 is a good marker for detecting uterine carcinoma. 53 201043958 [00116] Cervical cancer HPV affects p53, and Figure 19 illustrates the fluorescence intensity of HPV16 and p53 antibodies, indicating that overexpression of HPV16 inhibits p53 in clinical samples of HPV infection. Comparing the performance of HPV16 and p53, the results showed that in most samples (except S7 and S8 samples), p53 performance was much lower than that of HPV16, and S7 and S8 might be other viral infections other than HPV16. Low expression of p53 in clinical samples indicates that most of the p53 protein is degraded by HPV E6 proto-oncoprotein in the development of cervical cancer. [00117] E7, retinoblastoma (Rb) protein and phosphorylated Rb, which are affected by cervical cancer HPV, FIG. 20 illustrates the fluorescence intensity of HPV16E7 and pRb antibodies, and deactivates Rb with an anti-Rb-phosphate specific antibody. At the time, the HPV16 E7 is over-expressed. Comparing the expression of HPV16 E7 and Rb-phosphorylation, it was evident in sample S2 that overexpression of HPV16 E7 inhibited phosphorylation of Rb, indicating that deactivation of Rb by E7 (reduced reactivity with anti-Rb-phospho antibody) will lead to development Malignant transformation of cancer in cervical cancer. [00118] Analysis of protein wafer test performance of cervical cancer, and FIG. 21 illustrates performance analysis of selected HPV proteins and cellular proteins in sample S2. The results showed that HPVE7 and pl6 were overexpressed when other cellular proteins were inhibited. All the results in Figure 8, Figure 20 and Figure 21 indicate that the excessive expression of HPVE7 protein in sample 2 deactivates Rb and induces pl6INK4a. Performance, leading to a malignant transformation of the tumor and the development of cervical cancer. The high performance of HPV E7 in sample S1 may not be related to Rb, so pl6INK4a is also not highly expressed. [00119] The expression of another cellular protein p21 WAF1 in cervical cancer is related to ρ53. Figure 12 illustrates the cell lysate of Pap smear cells, and the fluorescence intensity values of the cellular p21 WAF1 and p53 protein expression are detected. As shown in Figure 22, there were significant correlations between the performance of p21WAF1 and p53 in 9 of the 10 samples (except for sample S1), 54 201043958 The results of the study showed that path inhibition via HPVE6? 21|八1?1, the tumor suppressing gene p53 is degraded, thus causing the malignant transformation of the tumor into the development of cervical cancer. [〇〇12〇] Protein wafer assays can be used as a tool to detect HPV proteins and to detect cellular proteins induced or inhibited by HPV infection in the development of malignant cancers. The results of the protein wafer test study used in the present invention indicate that the cervical cancer disease in this study has a different path of disease, and thus the development of cancer is also different. The technology is applied to other HPV-related cancers to predict the development path of malignant tumors, and can also develop relevant data analysis to identify various related proteins in the process of cancer development, so that it can develop drugs for personal use. Specific treatment. 8. Capture anti-HP-class anti-II on a surface and identify it by anti-hpv secondary anti-ship. The HPV protein 1001211 antigen sandwich test in biopsy is a protein wafer, a film or a micropore. The disk is a bottom layer that is coated with a primary antibody, such as a capture antibody or a spotted antibody, which has affinity for the antigen of interest and can produce a bond. The antigen of interest may be an HPV protein, a proto-oncoprotein, an outer sheath protein carrying an Hpv viral gene, such as an early gene or a late gene. After blocking the unbonded portion of the wafer surface, the analyzed clinical sample can be bonded to the capture antibody to form an immune complex, which is detected by secondary antibody or detection antibody binding to the antigen of interest. Therefore, primary antibodies, • secondary antibodies, capture antibody pairs, and detection antibodies interact with antigens of interest, • like sandwiches, so they are known as sandwich assays. The capture antibody or spotting antibody can be the same or a different antibody, and the binding antibody can specifically bind to an antigen of interest such as HPV viral protein, HPV proto-oncoprotein, and outer sheath protein. [〇〇122] Secondly, the 'sandwich-bonded antigen_antibody complex can be detected by a secondary antibody'. The antibody has affinity for the detection antibody and can be easily measured by a standard immune complex detection system, such as a colorimetric reagent. , chemically luminescent, fluorescent, and other substrates of different types 55 201043958. The final data reading and presentation is performed by instrument settings for appropriate absorption of light, or directly by visual comparison with the control group. The positive reaction result indicates that the antigen of interest expressed in the clinical sample is linked with the primary antibody, the capture antibody and the detection antibody; conversely, the negative reaction indicates that the primary antibody does not bond with the antigen of interest, representing There is no antigen of interest in the clinical sample. 1) Enzyme-binding immunosorbent assay: anti-HPV-class antibody is applied to the upper microplate, and HPV protein is detected by anti-HPV secondary antibody [〇〇123], indicating that the sandwich is bound to the enzyme on the microplate. In an immunosorbent assay, G is diagnosed as containing a SCC, HPV PCR positive or HPV PCR negative serum and diluted to prepare a cell lysate for the presence of HPV E6, E7 or L1 protein. The test procedure consists of coating a layer of rabbit anti-E6, E7 or L1 monoclonal antibody as primary antibody 'plus cell lysate (金清), and its associated anti-HPV secondary antibody, and then grafting another The antibody with HRP was detected, and after culturing with the substrate and the terminating reagent, the absorption value of 〇D450 was measured by a microplate analyzer. As shown in Figure 8, the results of the experiment indicated that the ELISA test used rabbit anti-HPV multi-strain antibody to detect the expression of HPV E6, E7 and L1 peptone in human serum samples diagnosed as SCc, HPV positive or HPV negative. 1001241 Figure 23 illustrates the detection of E6, E7 proto-oncoproteins and u-viral proteins from the serum of patients diagnosed as scc and hpv positive with HPV-negative as a control group. The experimental data showed that compared with the L1 protein, the main protein in the E7 protein was clear, compared with the HPV negative samples, the Ε6 and Ε7 proteins were the main proteins in the scc and Ηρν positive samples, while the HPV positive samples did not have the L1 virus. The performance of the protein, in other cases, 'U protein is not as good as E6 and E7 protein. These results indicate that depending on the degree or cycle of viral infection, L1 protein in serum may or may not be expressed, while SCC and HPV positive samples are detected in serum by E6 or E7 proto-oncogene 56 201043958 white, labeled Εό or E7 It is a good biomarker for HPV serum detection. This is the first report of E6, E7 proto-oncoprotein detected from serum and requires more serum samples for analysis. 2) Flow magnetic bead test: anti-HpV primary antibody was coated on a magnetic bead, and anti-HpV-class antibody was coated on the surface of the magnetic bead by detecting HPV protein 1001251 by anti-HPV secondary anti-purine. The HPV protein in the biopsy cell lysate is reacted to form a complex on the surface of the magnetic beads to capture the anti-HPV secondary antibody. When the anti-HPV secondary antibody is previously labeled, the complex steroid can be detected directly, or the pre-labeled antibody capable of binding to the secondary antibody can be added to detect the complex. Pre-labeled antibodies can be labeled with a detectable antigen, including but not limited to horseradish peroxidase, biotin, nanogold particles, fluorescent dyes, or combinations thereof. As an example, a complex on a solid surface of a magnetic bead can be detected by FACS (Fluorescence Activated Cell Sorting) using anti-mouse or rabbit PE as a secondary antibody. When multiple HPV proteins are captured onto the surface of the magnetic beads, multiple anti-HPV secondary antibodies labeled with different fluorescent dyes can be detected simultaneously by FACS. Therefore, the magnetic beads test with FACS is an advantageous multiple test tool for detecting one or more HPV proteins in a bio-Q sample. [〇〇126] T-type magnetic bead assay can be used to detect various HPV proteins, using various anti-HPV E6, HPV E7 or HPV L1 antibodies as coating and detection antibodies to detect HPV E6, HPV E7 and HPV L1 proteins. Figure 24-27 illustrates the results of a magnetic bead test by FACS to detect HPV 16 Ll, HPV 16E6, HPV 18 E6 and HPV 16E7 proteins. Figure 24 illustrates the sandwich magnetic beads test with FACS, using rabbit anti-ΡίΡνίόΕΙ antibody as a surviving antibody, and mouse anti-HPV10U monoclonal antibody as a detection antibody, and then using a mouse antibody grafted with PE reagent as a second. Level antibody, detecting HPV16L1 recombinant protein. As shown in the data, containing purified HPV16L1 57 201043958

The sample of recombinant protein is captured on the surface of the magnetic bead, and R has a discontinuous peak, indicating that she knows j, which can show the buffer solution from the sample containing Xu, 々, / E (the peak is located on the right side of Figure 24) As a test... left). The results of the experiment showed that the average value of the _ to the egg was about 12) and the sample containing the specific protein (the difference between the fluorescence intensity was 250 times and the value of the table was about 2958). Qing L1'^k performance analysis method, dynamic analysis Ο Ο = 7] In another example, Figure 25 illustrates the rabbit anti-HPVi 6 E6 multi-strain antibody and 'and mouse anti-Μ6 monoclonal antibody paste The mouse antibody against the two-grafted PE reagent was used as the secondary antibody, and the FACS was used for the second test. (4) HPV (4) Recombinant egg As shown in the data, the sample containing the pure recombinant protein was captured on the surface of the magnetic bead and detected by FACS. The measurement shows that there is no discontinuous peak, indicating that there is a strong glory pE (the peak is located on the right side of Figure 25) from the buffer solution containing the sample as the negative control group of the test (peak [on the left side of Figure 25]). The experimental results showed that the control group sample (with an average of about 17) and the sample containing the specific detection protein (average value of about U4) had a difference in fluorescence intensity of 7 times, indicating that the magnetic bead test was performed. Analytical methods can dynamically analyze the performance of HPVE6L1 protein in different clinical samples. T〇01281 Figure 26 illustrates the use of rabbit anti-HPV18E6 antibody as a coating antibody, and mouse anti-HPV18 E6 monoclonal antibody as a detection antibody, and then a mouse antibody grafted with PE as a secondary antibody. The sandwich magnetic beads test was performed by FACS, and the HPV16E6 recombinant protein was measured. As shown by the data, the sample containing the purified HPV18E6 heavy protein 'protein was captured on the surface of the magnetic bead and detected by FACS, showing a discontinuous peak, indicating a strong fluorescent PE (the peak is located on the right side of Figure 26). ) from the buffer solution containing the sample as the negative control group for the test (the peak is located on the left side of Figure 26). The results of the experiment showed that the control group samples with no protein expression (average 58 201043958 value of about 148) and the samples with specific detection proteins (average value of about 2294) had a difference of glory intensity of 15 times. The experimental analysis method can dynamically analyze the performance of HPV 18E6 protein in different clinical samples. [00129]® 27 indicates that rabbit anti-Hpvi6E7 antibody was used as a surviving antibody, and mouse anti-HPV16E7 monoclonal antibody was used as a detection antibody, and then a rat antibody with a grafting reagent was used as a secondary antibody. FACS performed a sandwich magnetic bead assay to detect HPV16E7 recombinant protein. As shown by the data, samples containing purified recombinant protein were captured on the surface of the magnetic beads and detected by FACs, which showed that the 连续*continuous peaks indicated a strong fluorescence pE (the peak is located on the right side of Figure 27). 3 There is a buffer/serum solution of the sample as the negative control group of the test (the peak is located on the left side of the figure). The experimental results showed that the control group samples with no protein f expression (average of about 5.5) and the samples with specific predictive proteins (average value of 673) showed a difference of 122 times in the intensity of light. The experimental analysis method can dynamically analyze the performance of HPV 16 E7 protein in different clinical samples. The performance of the 1001301 magnetic bead assay varies with the coating antibody. Figure 28-29 illustrates the different test methods for detecting HPV 16 E6 and Hpv 18 E6 and comparing them with Figure 26 and Figure 26. Figure 28 shows that rabbit anti-HPV10 E6 antibody was used as a coating antibody and rat anti-HPV16 L1 monoclonal antibody was used as a detection antibody, and then a mouse antibody of PE reagent was used as a secondary antibody to FACS. A sandwich magnetic bead assay was performed to detect HPV16E6 recombinant protein. As shown by the data, the sample containing the purified hPV16 E6 recombinant protein was captured on the surface of the magnetic bead and detected by FACS, showing no discontinuous peaks, indicating a strong fluorescent pE (the peak is located on the right side of Figure 28). ) From the buffer solution containing the sample 'as the negative control group of the test (the peak is located on the left side of Figure 28). The experimental results showed that the control group samples with no protein expression detected (average about 1223) and the samples with specific detection proteins (average about 2755) had a difference of 2 times the fluorescence intensity; comparing Figure 28 and Figure The 25 magnetic bead assay detects HPV 16 E6 59 201043958 protein results, showing that Figure 25 provides a large dynamic range of detection that can detect HPVE6 protein expression in various clinical samples. [00131] In another example, FIG. 29 illustrates a rat anti-HPV18E6 antibody as a coating antibody, and a mouse anti-HPV18 E6 monoclonal antibody as a detection antibody, and a mouse antibody grafted with a PE reagent. As a secondary antibody, a sandwich magnetic bead assay was performed by FACS to detect HPV18 E6 recombinant protein. As shown by the data, the sample containing the purified HPV18 E6 recombinant protein was captured on the surface of the magnetic beads and detected by FACS. It showed a discontinuous peak indicating a strong fluorescent PE (the peak is located on the right side of Figure 29). ) from the buffer solution containing the sample as the negative control group for the test (the peak is located on the left side of Figure 29). The results of the experiment showed that the control group samples (average value of 787) and the samples containing specific detection proteins (average approx. 3803) with no detectable protein expression were up to 5 times different in fluorescence intensity. Comparing the results of the magnetic bead assays of Figures 29 and 26 to detect HPV18 E6 protein, it is shown that Figure 26 provides a larger dynamic detection range that can detect the expression of various levels of HPVE6 protein in clinical samples. 3). Rapid fluid test to detect HPV infection: Rapid immunological tests can be performed vertically on the membrane or in parallel on the test strip. The single-step rapid immunoassay for flow measurement or diffusion can also refer to immunochromatographic assays. The simple start-up method takes about 5-15 minutes to obtain results without training or instrumental analysis. The basic principle of the test is solid nitrocellulose or a test piece containing a capture reagent, which is reacted with a smear sample. If the patient sample contains a standard reagent, the capture reagent on the nitrocellulose reacts with it to form a complex body. Transferred to the nitrocellulose by diffusion or capillary reaction. [00132] The test film or test strip can also be used for sample collection or in combination with cotton gauze, so that the designed immune response can be started, and the test result can be obtained immediately, as in the case of inserting a visitor mirror into the human body. The inner cervix can be observed immediately. Therefore, a one-step rapid immunoassay can be used as a primary screening method for HPV validation test 201043958 combination trials, such as smear cytology, immunology, and nucleic acid hybridization assays or assays. 〃 [00133] 4 direct rapid immunoassay, performed in a device with a membrane as a capture/bonding surface, with r Ο 披 coating or trans-capsule capture reagent; The device contains -, 塾' under the membrane to facilitate flow of the sample and test reagent through the test membrane. Any target contained in the sample = white, antibody or antigen, can capture the reagent to produce a specific reaction and bond, ^ over the test membrane, even if (4) multiple wash step remove the non-specific bond, captured The sample can still be maintained on the surface of the membrane. A secondary antibody grafted with HRp or other substance = any egg-antibody complex applied to the surface of the membrane and maintained laterally on the membrane, and can be visually observed by a coloring reagent. [〇明本(四) provides the single (four) fast New Zealand: = the test side r is like the pregnancy of the 2 ί, m 4 step rapid immunoassay can be used as a test, can be directly qualitative analysis - general antigen, high Risk of deleting disease ^= special antigen or related antibodies. The one-step duty-free money-free test can be used as a nursing station to diagnose the second clinic or as an aid to the smear test in the laboratory. The one-step rapid immunization test is suitable for use in a short-to-shirt, and the diluted sample is not required to be diluted. After a short reaction time, a visual result can be produced. [00] The rapid immunoassay for measuring flow is a one-step test method. (4) The capture protein or antibody is coated on the surface of the membrane, and the sample with the labeled protein or antibody is grafted with the antibody, and with the nanogold. The particles are combined and the conjugate is directly applied to the film test piece, so that the sample direction-measuring flow i« test 4' is reached until the design of the test piece (4) is reached. Capture _ 'the system of egg-immune complex, capture the egg or antibody coating at the designed position into H to the film surface Φ, no need to be cleaned or separated at the design position, 61 201043958 is visually positive The silk of the reaction is therefore called a single step. The entire test procedure took less than 15 minutes and was therefore called a one-step quick test. The 1001361 single-step rapid immunochromatographic assay is a simple, fast and easy-to-use test that can be easily used in care stations. The present invention provides a mixture of a test sample and a detection antibody which can be attached or fixed to the surface of the membrane or glass and reacted for several minutes at a suitable culture temperature (e.g., room temperature). The reaction time can be shortened according to the reaction conditions of the test and the quality of the detected antibody, so that the waiting time of the rapid immunoassay is short, and the test can be visually obtained without detecting the instrument. Ο A.) Single-step HPV flow test: Anti-HPV-grade antibody was immobilized on the test membrane and detected by anti-HPV secondary antibody grafted with nano gold particles [00137] Single-step rapid immunoassay It may be a membrane or a test rod immobilized with a capture reagent, such as a purified HPV antibody, a recombinant protein or an HPV-related antibody and protein, etc., and the target reagents captured, such as HPV-related antibodies and proteins in clinical samples. Etc. Then, the detection is performed by an immunoassay system. [00138] Figures 30A-30G illustrate the use of the antibodies described herein for a single-step flow measurement q assay to detect HPV proteins. Fig. 30A illustrates that a rabbit anti-L1 polyclonal antibody was immobilized on a membrane and grafted with nano gold particles, and a single-step flow measurement test was performed to detect HPVL1 recombinant protein. The test control group (TC) was shown on the first line, the second line (arrow) was positive, and the right arrow was negative. L1 recombinant protein • Concentration from left to right is 6, 3, 1_5, 0.75, 0.375, 0 ug/ml. Experimental data shows that the single-step HPV flow test can detect HPV L1 recombinant proteins at concentrations below 375 ng/ml. .

[00139] The rapid flow test can detect HPV L1 protein in clinical samples, and FIG. 30B shows that the same rabbit anti-L1 antibody is immobilized on the membrane as shown in FIG. 30A, and the result of detecting HPVL1 protein in serum sample 62 201043958 is detected. The test control group (TC) is shown on the first line, and the second line (arrow) is positive. The L1 protein (left) is detected in the serum sample of the see patient, and the HPV-negative serum is not visible by PCR. The strip pattern was used as the negative control group in the test (right side). Experimental data shows that the single-step HPV flow test can detect HPV L1 protein in SCC serum samples by comparing HPV-negative serum. [〇〇14〇] The rapid flow test can detect HPV E6 protein. Figure 30C shows that mouse anti-HPV E6 monoclonal antibody is immobilized on the membrane, and nano-particles are grafted for single-step zero-flow measurement. Detection of HPV E6 protein. The test control group (TC) is shown on the first line, the second line (arrow) is positive, and the right arrow has no visible banding. The E6 recombinant protein concentration was 10, 2, 0 ug/ml from left to right. Experimental data shows that the single-step HPV flow test detects HPV E6 recombinant protein at concentrations below 2 ug/ml. [〇〇141] The flow test system can be further used to detect HPV E6 protein in clinical samples. Figure 30D shows that the same mouse anti-E6 monoclonal antibody is immobilized on the membrane in Figure 30C to detect HPV E6 protein in serum samples. result. The test control group (TC) is shown on the first line of 〇, and the second line (arrow) is positive. The serum sample (first and second from the left) is positive for E6 protein detection. There was no visible banding for HPV-negative serum as a negative control group (right) in the trial. 1 Experimental data showed that the single-step HPV flow test can detect HPVE6 protein in SCC serum samples by comparing HPV-negative serum. Fig. 30E shows that another mouse anti-E6 monoclonal antibody was immobilized on a membrane, and grafted with nanogold particles, and a single-step flow test was performed to detect HPVE6 recombinant protein. The test control group (TC) is shown on the first line, the second line (arrow) is positive, and the right arrow has no visible banding. The concentration of E6 recombinant protein was from left to right 63 201043958 was 875, 438, 0 ug/ml. Experimental data shows that the single-step HPV flow test can detect HPV E6 recombinant protein at a concentration of less than 43 5 ng/ml. [〇〇143] The rapid flow test can detect HPVE7 protein. Figure 30F shows that mouse anti-HPVE7 monoclonal antibody is immobilized on the membrane, and nano-particles are grafted for single-step flow test to detect HPVE7 protein. . The test control group (TC) is shown on the first line, the second line (arrow) is a positive reaction, and the right arrow has no visible banding as a negative reaction. E7 recombinant protein concentration from left to right is 〇, 660, 66, 6.6, 0.66 ug/m. The experimental data shows that the single-step HPV flow test can detect HPVE7 recombinant protein at a concentration lower than 660 ug/ml. [〇〇1441 The flow test system can be further used to detect HPV E7 protein in clinical samples. Figure 30G shows that the same mouse anti-HPV £7 antibody was immobilized on the membrane as shown in Figure 30F. Detecting HPV E7 in serum samples. The result of the protein. The test control group (TC) is shown on the first line, indicating that the PCR reaction is HPV positive (left) serum sample Ε7 protein positive detection, PCR reaction is Ηρν negative serum no visible band pattern 'as test Negative control group (right). Experimental data show that the single-step HPV flow test can detect HPV Ε7 protein in known (four) positive Q serum samples by comparing Ηρν negative serum. [The state is called a seed or a variety of immunological tests, using antibodies and recombinant proteins purified from early or late ‘mystery, and is a credible indicator substance when HPV infection occurs. In addition, 'HPV-related malignant tumors or pre-neoplastic cell transformation can be tested and analyzed. Among them, the most important aspect of the present invention is to diagnose cervical cancer, squamous cell and adenocarcinoma, etc. Anything related to HPV infection is carcinogenic. Normal epidermal cells such as cytoplasmic holes, excessive keratinization, precancerous lesions include epithelial cell degeneration or endothelial cell lesions, high structural dysplasia, and non-invasive or malignant cancers. 64 201043958 i〇0146] Early diagnosis of HPV infection plays an important role in the successful prevention and treatment of cervical cancer. In the prevention stage of cervical cancer, it is necessary to promote a large range of HPV trials of the population, and to track the patients with Ηρν facial and precancerous lesions. Most importantly, women infected with 12-15 years old may cause the development of money to attack the cancer of the Wei. Therefore, the biomarker test for early screening of HPV infection in women of the present invention is important, and the field test can be used to prevent uterus from early detection of uterus. "Development without relying on chemotherapy or radiotherapy for cancer malignant neoplasms. [Simple description of the schema] Figure 147 shows a representative image of squamous cell carcinoma (SCC) tissue stained with anti-Ε7 monoclonal antibody by immunohistochemistry (mc) microarray. [. _ ® 1B illustrates a representative representation of the normal epithelial cells (15 mm from the tumor tissue) in Figure 1A. [〇〇149] Figure ic shows a representative image of SCC samples stained with immunohistochemistry (ihc) microarrays using the same anti-E7 monoclonal antibody. [〇_ffl 1D illustrates a representative image of the f-staining of cells in Figure 1C_ cells. [〇_Fig. 2A] Representative images of adenocarcinoma (adc) tumor cell samples stained with ffic with anti-E7 monoclonal antibody. Figure 2B is a representative diagram showing the relevant normal epithelial cells (15 mm from the tumor tissue) of the adenocarcinoma sample of Figure 2A. [001531 ffl 2C] A representative map showing the cytoplasmic staining of gj 2A adenocarcinoma cells. 65 201043958 [〇〇154] Figure 3A illustrates a representative staining map of CIN3 dysplastic cells, and according to another embodiment of the invention, an IHC assay was performed using a mouse monoclonal anti-HPVE7 antibody. [Fig. 3B] Fig. 3B is a magnified table showing the poorly constructed epithelial cells of Fig. 2A, showing nuclear staining of CIN3 dysplasia. 1001561 Figure 4A illustrates a representative staining map of CIN2 dysplastic cells, which was subjected to an IHC assay using mouse anti-HPVE6 monoclonal antibody according to an invention. [〇〇157] Figure 4B is a diagram showing a representative staining of normal epithelial cells adjacent to the poorly structured tissue of the CIN2 sample of Figure 1A, and the IHC test was performed using the same mouse anti-jjpV E6 monoclonal antibody. Fig. 4C shows the result of dysplastic epithelial cell staining of CIN3 tissue. According to another inventive example, the mouse anti-ηρυΕ6 monoclonal antibody identical to Fig. 1A was subjected to the 1HC test. [00159] Figure 4D illustrates dysplastic epithelial cell staining results of another CIN3 tissue. According to another inventive example, the mouse anti-HPVE6 monoclonal antibody identical to Figure 1C was subjected to an IHC assay. ❾ 1〇0160】Circle 5Α indicates the staining results of the clinical specimens diagnosed as atypical squamous cells (ASCUS). According to an embodiment of the invention, the ICC test was performed in a solution of mouse anti-Ηρν Ε6 monoclonal antibody. [00161] _ 5B illustrates the same clinical sample staining results as in Fig. 5A, and the ICc test was performed using mouse anti-HPVE7 monoclonal antibody according to an embodiment of the invention. [00162] Fig. 6A illustrates the results of staining of the clinical specimen for diagnosis of αΝ2, and according to an embodiment of the invention, an ICC test was carried out in a solution with mouse anti-HPV Ε7 monoclonal antibody. 66 201043958 [Fig. 6B] Fig. 6B illustrates another staining result of a clinical specimen diagnosed as CIN2, and according to another embodiment of the invention, an ICC test was carried out in a solution with mouse anti-HPV E6 monoclonal antibody. Fig. 7A illustrates the results of staining of a clinical specimen for diagnosis of CIN3, and according to another embodiment of the invention, an ICC test was carried out in a solution with mouse anti-HPV E6 monoclonal antibody. 7B illustrates another staining result also diagnosed as a CIN3 clinical specimen, which was stained with the same mouse anti-HPV E6 monoclonal antibody as in FIG. 7A. [00166] Figure 7C illustrates another ICC staining of the same clinical specimen as Figure 7B, stained with the same mouse anti-HPVE6 monoclonal antibody. [00167] Figure 7D illustrates another ICC staining of the same clinical specimen as Figure 7B, stained with the same mouse anti-HPVE6 monoclonal antibody. [00168] Figure 7E illustrates another ICC staining of the same CIN3 sample as Figure 7B, but stained with mouse anti-HPVE7 monoclonal antibody according to another inventive example. [00169] Figure 7F illustrates another ICC staining of the same CIN3 sample as Figure 7E, stained with the same mouse anti-HPVE7 monoclonal antibody. [00170] Figure 7G illustrates the same CIN3 sample staining results as Figure 7B, but with the mouse anti-pl6 monoclonal antibody for ICC assay. [Fig. 8] Fig. 8 illustrates the results of clinical diagnosis of adenocarcinoma staining. According to another embodiment of the invention, an ICC test was performed using mouse anti-HPV E6 monoclonal antibody. [00172] Figure 9A illustrates the results of a clinical diagnosis of squamous cell carcinoma (SCC) staining, according to another embodiment of the invention, ICC test with mouse anti-HPV E6 monoclonal antibody 0 67 201043958 [00173] Figure 9B illustrates and 9A identical SCC sample staining results, according to another invention example, the mouse anti-HPVE7 monoclonal antibody was used for ICC test. 9C illustrates the same SCC sample staining results as in FIG. 9A, and according to another embodiment of the invention, an ICC test was performed using a mouse anti-pl6 monoclonal antibody. 10A illustrates the results of a clinical diagnosis of a normal sample, and according to another embodiment of the invention, an ICC test was performed using a mouse anti-HPVE6 monoclonal antibody. [0076] Fig. 10B illustrates the same clinical sample staining results as in Fig. 6A. According to another embodiment of the invention, an ICC test was performed using mouse anti-HPVE7 monoclonal antibody. [00177] Figure 11A illustrates the results of cytoplasmic staining of another Pap smear sample, according to another inventive example, the ICC test was performed with mouse anti-HPV E6 monoclonal antibody. 11B illustrates the results of nuclear staining of the same sample as that of FIG. 11A, which was tested with mouse anti-HPVE7 monoclonal antibody. [00179] Figure 11C illustrates the results of cytoplasmic staining of another Pap smear sample, which was tested with an anti-HPVE6 monoclonal antibody. [00180] Figure 12A illustrates the results of a clinical diagnosis of a staining of a CIN1 sample. According to another embodiment of the invention, an ICC test was performed using a mouse anti-HPV E6 monoclonal antibody. [00181] Figure 12B illustrates another ICC staining result for the same CIN1 sample as in Figure 12A, tested with the same mouse anti-HPV E6 monoclonal antibody. [00182] Figure 12C illustrates another ICC staining result for the same CIN1 sample as in Figure 12A, tested with the same mouse anti-HPVE6 monoclonal antibody. 68 201043958 [00183] Figure 12D illustrates another ICC staining result for the same CIN1 sample as Figure 12A, but according to another inventive example, the ICC test was performed with mouse anti-HPV E7 monoclonal antibody. [00184] Figure 12E illustrates another ICC staining result for the same CIN1 sample as in Figure 12D, tested with the same mouse anti-HPVE7 monoclonal antibody. 12F illustrates the results of staining of the same CIN1 sample as in FIG. 12A, and according to another embodiment of the invention, an ICC test was performed using a mouse anti-pl6 monoclonal antibody.

[00187] Figure 13A illustrates the results of an ink dot method for detecting HPV L1 protein, which was tested with mouse anti-HPVL1 monoclonal antibody according to an embodiment of the invention. [00188] FIG. 13B illustrates another dot method for detecting HPVL1 protein, which was tested in the same mouse anti-HPV L1 antibody as in FIG. 1. [00189] Figure 14A illustrates the results of the same HPV E6 protein dot method as in Figure 13A, and was tested with mouse anti-HPV E6 monoclonal antibody according to another embodiment of the invention. 14B illustrates the results of the same method for detecting the HPV E6 protein dot method as in FIG. 13B, and according to another embodiment of the invention, the mouse anti-HPVE6 monoclonal antibody identical to that of FIG. 14A was used for the test. [00191] Figure 15 illustrates the results of the same dot method as in Figures 13A and 14A. According to another embodiment of the invention, HPV E7 protein was detected by mouse anti-HPV E7 monoclonal antibody. 69 201043958 [00192] Figure 16 illustrates the results of average fluorescence intensity of cell lysates from 10 groups of Pap smear samples, antibody microarray assays, and detection of various HPV proteins and endogenous properties according to another embodiment of the invention protein. Figure 17 is a graph showing the fluorescence intensity of each of the 10 sets of cell lysates of Figure 16 and detecting HPVL1 protein according to another embodiment of the invention. 18 is a result of antibody microarray analysis fluorescence intensity of each sample of 10 sets of cell lysates of FIG. 16. According to another embodiment of the invention, HPVE7 protein and cell pl6 INK4a protein are detected. [00195] Figure 19 is a graph showing the results of antibody microarray analysis of each of the 10 sets of cell lysates of Figure 16 for fluorescence intensity measurements. According to another embodiment of the invention, HPVL1 protein and cellular p53 protein are detected. [00196] Figure 20 is a graph showing the results of antibody microarray analysis of each of 10 cell lysates of Figure 16 for fluorescence intensity measurements. According to another embodiment of the invention, HPV E7 protein and cytosolic acid from Rb protein are detected. [〇〇197] Figure 21 is another photograph illustrating the antibody microarray analysis fluorescence intensity 样本 of sample S2. [00198] Figure 22 Figure 16 shows the results of the fluorescence microscopy of the antibody microarray for each of the 10 sets of cell lysates. According to another embodiment of the invention, the cells p21 WAF1 and p53 protein were detected.

[00199] Figure 23 illustrates the results of an ELISA test for HPV6, E7 in human serum samples that were clinically diagnosed as squamous cell carcinoma (SCC) or HPV-positive (PCR-tested) after comparison with HPV-negative serum samples. And L1 protein, according to another embodiment of the invention, rabbit anti-HPV multi-strain antibody was used as a cover and detection. 201043958 100200] Figure 24 illustrates the results of detection of HPV16U recombinant protein by Sanming immunoassay using a fluorescence activated cell sorter (FACS), using mouse anti-HPV monoclonal antibody as a surviving antibody, and % anti-visual V16 L1 Strain antibodies are used as detection antibodies. Figure 25 shows the results of a sandwich immunoassay for detecting hpV16E6 recombinant protein by fluorescence activated cell sorting (FACS), using mouse anti-shear "π monoclonal antibody as a monoclonal antibody 1 rabbit anti-Ηρνι6 E6 multi-strain Antibodies as (four) antibodies. [00202] Objective 26 illustrates the detection of HPV18E6 recombinant protein by sandwich immunoassay using Camp Light Activated Cell Sorting (FACS), using mouse anti-Ηρνΐ8Ε6 monoclonal antibody as a separate antibody' to rabbit anti-Ηρνΐ8 Ε6 The strain antibody is used as a test antibody. 1〇〇2〇3] Figure 27 illustrates the results of a sandwich immunoassay for detection of HPV16 E7 recombinant protein by fluorescence activated cell sorting (FACS) using mouse anti-Hpvl6 E7 monoclonal antibody as a coating antibody. Rabbit anti-Hpvi6 E7 polyclonal antibody was used as a detection antibody. [〇〇2〇4] Figure 28 illustrates the results of a sandwich immunoassay for detection of HPV16E6 recombinant protein by fluorescence activated cell sorting (FACS), using mouse anti-HPV16E6 monoclonal antibody as a coating antibody to rabbit anti- - HPV16 Εό multiple antibodies as detection antibodies. [〇〇2〇5] Figure 29 illustrates the results of a sandwich-type, immunoassay method for the detection of HPV18E6 recombinant protein by fluorescence activated cell sorting (FACS), using mouse anti-HPV18E6 monoclonal antibody as a coating antibody to rabbits. Anti-HPV18 E6 polyclonal antibody was used as a detection antibody. 71 201043958 [〇〇2〇61 Figure 30A illustrates the results of a single-step flow assay to detect Hpy L1 recombinant protein (from left to right: 875, 435, 0 ug/ml), and rabbit anti-Ηρν£1 antibody from membrane It is removed and grafted with gold particles (as a detection reagent). [〇〇2〇7] Figure 30B illustrates the results of a single-step flow test to detect serum samples of squamous cell carcinoma (scc) patients (left) HPV L1 recombinant protein, compared with normal serum (HPV negative), use and Figure 30B is the same method. [〇〇2〇8] Figure 30C illustrates the results of a single-step flow test to detect Hpv% recombinant protein (from left to right.1〇' 2, 〇ug/ml), and mouse anti-HPV E6 monoclonal antibody The film is removed and grafted with gold particles for detection. [〇〇2〇9]胄30D Description Single-step flow measurement to measure serum samples from squamous cell carcinoma (scc) patients (first to second from left to right) HpvLi protein results and normal Jinqing (HPV) Negative) For comparison, use the same method as in Figure 3〇c. [〇〇21〇1Fig. 30 E illustrates the results of a single-step flow test for Hpv E6 recombinant protein (from left to right: 875, 435, 0 ug/ml), and another mouse anti-HpvE6 monoclonal antibody was obtained from the membrane. Remove and graft with gold particles for detection. [〇〇211]® 30F demonstrates the results of a single-step flow assay to detect HpvE7 recombinant protein (from left to right. 0, 660, 66, 6.6, 0.66 ug/ml), mouse anti-HPV E7 monoclonal antibody It is removed from the film and grafted with gold particles for detection. Tao 2] Figure 3 〇 G illustrates the single-step flow test to test the results of HPV E7 protein in the Hpv-positive serum sample (left), and compare it with HPV-negative serum (right) using the same method as Figure 30F. [Main component symbol description] 72 201043958 Ο

73

Claims (1)

201043958 VII. Patent Application Range: 1. A method for detecting human infection with papillomavirus, comprising: collecting clinical human samples for immunological testing, using one or more monoclonal antibodies to the nucleus of human cells in clinical samples. Dyeing. 2. According to the method of the first paragraph of the patent application, when the human nuclear staining is positive, it indicates that the stage of disease development is caused by early stage of structural dysplasia, low grade squamous cell lesion (LSIL), and highly squamous cell lesion. (HSIL), cervical intraepithelial neoplasia (CIN1, CIN2, CIN3), and the composition group of the above composition were selected from the mountain. 3. According to the method of the first paragraph of the patent application, when the human cytoplasmic staining is positive, it indicates that HPV infection progresses toward the disease stage, and may progress to advanced dysplasia, high squamous cell lesion (HSIL), cervical epithelium. Endocarcinoma (CIN 2/3), invasive cervical cancer, squamous cell carcinoma (SCC), adenocarcinoma, and a group consisting of the above. 4. Develop a variety of monoclonal antibodies against the purified HPV 重组 recombinant protein according to the method of claim 1 of the patent application. 5. According to the method of claim 1 of the patent application, a plurality of monoclonal antibodies developed can be linked to at least one HPV early viral protein. 6. According to the method of claim 1, the plurality of monoclonal antibodies developed can be linked to at least one HPV late viral protein. 7. According to the method of claim 1, the plurality of monoclonal antibodies developed can be linked to at least one HPV early and late viral protein. 74 201043958 8_According to the method of claim 1, the plurality of monoclonal antibodies developed comprise an antibody capable of binding to an HPV early viral protein and having another antibody that binds to a late HPV protein. . 9. According to the method of claim 1, the various monoclonal antibodies selected from the group include anti-HPV E6 monoclonal antibody, anti-HPV E7 monoclonal antibody, anti-HPV L1 monoclonal antibody, and A group consisting of the above. 10. According to the method of claim 1, further comprising: performing immunohistochemistry 人体 test on human cells of a clinical sample by using antibodies against a plurality of anti-HPV recombinant proteins; and comparing cell nuclei and cytoplasm of human cells in clinical samples. dyeing. 11. According to the method of claim 10, the nuclear staining of human cells in clinical samples is more positive than that of cytoplasm, indicating that the HPV-infected patients screened in the ethnic group are at an early stage of the disease, such as early dysplasia. Low squamous cell lesions (LSIL), highly squamous epithelial cell lesions (HSIL), cervical intraepithelial neoplasia (CIN1, CIN2, CIN3), and a group consisting of the above. 12. According to the method of claim 10, the cytoplasmic q staining of human cells in clinical samples is more positive than that of nucleus, indicating that HPV-infected patients selected from the population are in advanced stages of the disease, such as advanced dysplasia. Hyperplasia, cervical intraepithelial neoplasia (CIN3), invasive cancer, and a group consisting of the above. 13. The method of claim 1, further comprising: performing immunohistochemistry on human cells of a clinical sample by using a plurality of antibodies capable of binding to HPV viral proteins, and selecting related proteins selected from the population For example, E6, E7, L1 protein, and the composition group consisting of the above; comparing the results of human cell staining with the two upper antibodies, the staining result is positive, indicating that the human body is infected by the HPV virus. 75 201043958 14. According to the scope of the patent in Zhongming! The method of immunohistochemistry can be used to detect HPV infection at various stages of the disease. The disease stages screened by the group include early HPV infection, advanced HPV infection, early cervical cell lesions, advanced cervical cell lesions, and low Squamous epithelial cell lesion (LSIL), highly squamous cell lesion (HSIL), atypical squamous cell (ASCUS), cervical intraepithelial neoplasia ((: leg, salt 2, _3), developing cervix Cancer, adenocarcinoma (adc), squamous cell carcinoma (SCC), and a group consisting of the above. 15. The method according to claim 1, further comprising: providing a clinical sample from the human body The clinical sample contains normal and abnormal human cells, and the sample is prepared into a thin layer of cells which are plated on the surface of the test piece, and the purified anti-HPV recombinant protein produced by various antibodies is subjected to immunocytochemical analysis. At least one of the antibodies binds to the HPV early viral protein; and stains the HPV viral proteins produced by various HPV strains in clinical samples on the test strip. 16. According to the method of claim 2, further comprising: performing a plurality of flow cytometry tests, separating each cell from a mixture of normal and abnormal human cells to detect each individual The cells are analyzed; the clinical sample liquid solution is analyzed, and the sputum type is abnormal and the normal cell mixture contains various HPV virus types to detect the performance of HPV virus proteins from various HPV virus types. 17. Detect various Hpv in the human body A method for providing a viral protein, comprising: providing an anti-Ηρν antibody capable of binding to various HPV proteins from various HPV virus types; preparing a clinical sample into a cell lysate solution containing various Hpv viral proteins; - coating a solid surface of at least one protein selected from the group consisting of an anti-HPV antibody, and various proteins expressed in a cell lysate solution; reacting the anti-HPV antibody with a cell lysate solution; On the solid surface, shape 76 201043958 into a complex composed of anti-HPV antibody and various HPV proteins; analyze the complex to detect Whether the various HPV proteins in the sample behave. 18. According to the method of claim 17 of the patent, various HPV proteins selected from the population, including HPV E6 protein, HPV E7 protein, HPV L1 protein, and the above composition Forming a group 19. Solid surface selected by the group according to the method of claim 17 including magnetic bead surface, test paper surface, rapid test strip surface, membrane surface, heavy DC system membrane surface, microfluid The surface of the system, the surface of the membrane, the surface of the protein wafer, the surface of the bismuth glass, the bottom surface of the microplate, and the constituent groups consisting of the above. 20. The method of fluorescence activated cell sorting according to the method of claim 19 (FACS) detects complexes of various magnetic bead solid surfaces. 21. According to the method of claim 19, an anti-HPV-class antibody is fixed on one end of the test paper, and an anti-HPV secondary antibody and a cell lysate solution are added to the other end of the test paper, and the solution is measured and directed to flow through the surface of the test paper. Previously, it forms a complex with anti-HPV-grade antibodies and can be detected. Ο 22. According to the method of claim 19, the anti-HPV-class antibody is immobilized on the membrane surface of the heavy DC system, and the anti-HPV secondary antibody and the cell lysate > solution are sequentially added as the solution. When passing through the heavy DC rapid test system, it forms a complex with the anti-HPV-class antibody and can be detected. 23. According to the method of claim 19, the composite flows through the microfluidic system and is detected on its surface. 24. According to the method of claim 17, the various HPV proteins in the cell lysate solution are coated on a solid surface before being reacted with the anti-HPV antibody. 77 201043958 25. According to the method of claim 24, various HPV proteins in the cell lysate solution are coated on the solid surface of the tumbling membrane. 26. According to the method of claim 24, the various HPV proteins in the cell lysate solution are coated on the solid surface of the microplate, and the anti-HPV antibody is pre-labeled with the detection reagent. The reagent includes enzyme-grafted biotin and nanogold particles, and a group consisting of the above; the performance of the detection reagent indicates that a complex composed of various HPV proteins and anti-HPV antibodies is formed on the solid surface. , indicating the presence of various HPV proteins in the sample. Ο 27. According to the method of claim 17, anti-HPV antibodies are applied to a solid surface prior to reaction with a solution of cell lysates containing various HPV proteins to detect HPV infection in the sample. 28. According to the method of claim 27, the anti-HPV antibody is coated on the solid surface of the protein wafer before being reacted with the cell lysate solution. 29. According to the method of claim 27, the anti-HPV antibody is coated on the solid surface of the microfluidic system before being reacted with the cell lysate solution. Ο 30. Detecting the complex on the solid surface according to the method of claim 17 of the patent application, further detecting the expression of the labeled protein detecting reagent containing the anti-Η PV antibody, and the various proteins in the cell lysate solution The performance, the secondary antibody and the anti-HPV antibody are linked, and the composition group consisting of the above, wherein the type of the secondary antibody varies depending on the anti-HPV antibody, and the anti-HPV antibody can also be used in the clinical sample. Multiple HPV proteins from various HPV virus types produce linkages. 31. According to the method of claim 30, the screening test method is used to analyze whether the detection reagent is performed, including the color change of the reagent as a direct qualitative analysis, 78 201043958 to an ELISA analyzer, a microarray scanner, and FACS and the like read data 'and a constituent group composed of the above. 32. According to the methods of claims 31 and 1 of the patent application, further comprising coating an antibody on a solid surface to detect cellular proteins and comparing the expression of 'cellular proteins and various HPV proteins in clinical samples . Cellular proteins are selected from the population, such as p16INK4a, E2F, Ki-67 (MIB-1), MYC protein, CDK4, cyclin Α, cyclin Β, cyclin-E, telomerase-TERC , MCM2 protein, ΤΟΡ2Α protein, heat shock protein 40 (HSP40), heat shock protein 60 (HSP60), heat 0 shock protein 70 (HSP70), CA9/MN protein, laminin 5, laminin, brn-3a, CDK N2 protein, topoisomerase 2A, microsomal maintenance protein-2, microsomal maintenance protein-4, microsomal maintenance protein-5, auxin protein, VEGF protein, p53, RB, p27 (kipl), and p21 (waf), and a constituent group composed of the above. 33. The flow measurement system can detect a plurality of HPV proteins from various HPV virus types in a clinical sample prepared as a cell lysate solution, including: covering the solid surface at one end of the first test piece with an anti-HPV antibody, The anti-HPV-class antibody can bind to a variety of HPV proteins from various HPV virus types, thereby capturing the HPV protein in the cell lysate solution Q onto the solid surface; on the other end of the second test piece After the anti-HPV secondary antibody flows through the cell lysate solution of the first test piece, it reacts with the secondary antibody to form a complex, and the secondary antibody can bond with various HPV proteins, thereby detecting clinical The performance of HPV protein in the sample. 34. — A reagent for detecting HPV infection in humans, comprising: anti-HPV monoclonal antibody for immunoassay of clinical samples, and comparing the staining degree of the sample nuclei and cytoplasm. 35. — A reagent for detecting HPV infection in humans, comprising: an anti-HPV monoclonal antibody capable of binding to an early Hpv protein, and preparing a clinical sample into a solution containing abnormal and normal cells for immunocytochemistry test. The sample solution was plated on a test piece 79 201043958 into a thin layer of cells, and various HPV proteins from various HPV virus types were stained in situ. 36. Anti-HPV antibodies, including anti-HPVE7 monoclonal antibody, anti-HPVE6 monoclonal antibody, anti-HPV L1 antibody and anti-HPV E7 monoclonal antibody, were selected from the population according to the reagent method of claim 35. Binding of the antibody, binding of the anti-HPV L1 antibody to the anti-HPVE6 monoclonal antibody, binding of the anti-HPVE6 antibody to the anti-E7 monoclonal antibody, and a composition group consisting of the above. 37. According to the reagent method of claim 35, anti-HPV monoclonal antibodies are used to detect HPV infection at various stages of the disease, and the selected stages of the disease include early HPV infection, advanced HPV infection, and early cervical cell lesions. , advanced cervical cell lesions, low-grade squamous cell lesions (LSIL), highly squamous cell lesions (HSIL), atypical squamous cells (ASCUS), cervical intraepithelial neoplasia (CIN1, CIN2, CIN3) , developing cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and a group consisting of the above.