IN SITU detection of early stages and late stages HPV infection
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.
1 . A method of detecting papillomavirus infection in a human subject, comprising:
conducting an immunological assay on a clinical sample of the human subject; and staining of a nuclear portion of a human cell from the clinical sample using one or more monoclonal antibodies capable of binding to HPV early viral proteins.
2 . The method of claim 1 , wherein positive staining of the nuclear portion correlates with the progression to a disease stage selected from the group consisting of an early dysplasia stage, low-grade squamous intracervical lesion (LSIL), high-grade squamous intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2, CIN3) and combinations thereof.
3 . The method of claim 1 , wherein positive staining of a cytoplasmic portion of the human cell indicates papillomavirus infection being progressed into a disease stage selected form the group consisting of a late dysplasia stage, cervical intraneoplasm (CIN3), invasive cervical cancer, squamous cell carcinoma (SCC), adenocarcinoma, and combinations thereof.
4 . The method of claim 1 , wherein the one or more monoclonal antibodies are generated against one or more purified recombinant papillomavirus proteins.
5 . The method of claim 1 , wherein the one or more monoclonal antibodies are also capable of binding to the HPV late viral proteins.
6 . The method of claim 1 , wherein the one or more monoclonal antibodies further comprises an antibody capable of binding to the HPV late viral proteins.
7 . A method of detecting papillomavirus infection in a human subject, comprising:
conducting an immunohistochemistry assay on humans cells from a clinical sample of the human subject using one or more antibodies generated against one or more purified recombinant papillomavirus proteins; and
comparing the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cell from the clinical sample.
8 . The method of claim 7 , wherein the one or more monoclonal antibodies are selected from the group consisting of anti-HPV E6 monoclonal antibodies, anti-HPV E7 monoclonal antibodies, anti-HPV L1 monoclonal antibodies, and combinations thereof.
9 . The method of claim 7 , wherein more positive staining being observed in the nuclear portion than the cytoplasmic portion of the human cell from the clinical sample indicates papillomavirus infection in the human subject at an early disease stage selected form the group consisting of an early dysplasia stage, low-grade squamous intracervical lesion (LSIL), high-grade squamous intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2, CIN3), and combinations thereof.
10 . The method of claim 7 wherein more positive staining being observed in cytoplasmic portion than nuclear portion of the human cell of the clinical sample indicates papillomavirus infection in the human subject at a late disease stage selected from the group consisting of a late dysplasia stage, CIN3, invasive cancer stage, and combinations thereof.
11 . The method of claim 7 , further comprising performing a hematoxylin and eosin stain on the clinical sample and comparing the results of the hematoxylin and eosin stain with the results of the one or more immunohistochemistry assays.
12 . The method of claim 7 , further comprising performing a cytological papanicolaou smear assay on the clinical sample and comparing the results of the cytological papanicolaou smear assay with the results of the one or more immunohistochemistry assays.
13 . A method of detecting papillomavirus infection in a human subject, comprising:
conducting an immunohistochemistry assay on a clinical sample of the human subject using two or more antibodies capable of binding to a HPV viral protein selected form the group consisting of E6, E7, L1 proteins, and combinations thereof; and
comparing the staining of the human cell by the two or more antibodies, wherein positive staining of the human cell by at least one of the two or more antibodies indicates papillomavirus infection in the human subject.
14 . The method of claim 13 , further comprising comparing the staining of the nuclear portion of the human cell from the clinical sample with the staining of the cytoplasmic portion, wherein positive staining of the cytoplasmic portion of the epithelial tissue sample indicates dysplasia progression by HPV infection.
15 . The method of claim 13 , wherein the staining of the human cell from the clinical sample is compared using the two or more antibodies including an anti-HPV E6 antibody and an anti-HPV E7 antibody.
16 . The method of claim 13 , wherein the staining of the human cell from the clinical sample is compared using the two or more antibodies including an antibody capable of binding to an early HPV viral protein and an antibody capable of binding to a late HPV viral protein.
17 . The method of claim 13 , wherein the immunohistochemistry assay is used to detect HPV infection at various disease stages, the disease stage is selected from the group consisting of early stage HPV infection, late stage HPV infection, early stage cervical cell lesion, late stage cervical cell lesion, low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), cervical intraneoplasm stage 1, (CIN1), cervical intraneoplasm stage 2 (CIN2), cervical intraneoplasm stage 3 (CIN3), developed cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and combinations thereof.
18 . A kit for detecting papillomavirus infection in a human subject, comprising:
an anti-HPV monoclonal antibody for performing an immunological assays on a clinical sample of the human subject, capable of staining a nuclear portion of one or more human cells from the clinical sample to compare the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cells.
19 . The kit of claim 18 , wherein the anti-HPV antibody is selected from the group consisting of an anti-HPV E7 monoclonal antibody, an anti-HPV E6 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E7 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E6 monoclonal antibody, a combination of an anti-HPV E6 antibody and anti-HPV E7 monoclonal antibody, and combinations thereof.
20 . The kit of claim 18 , wherein the immunological assay is selected from the group consisting of ELISA (enzyme linked immunoabsorbant assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, protein chip assays, radioimmunoprecipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays, immunohistochemistry for tissues and/or cervical cells, and immunocytological assays followed by flow cytometry, and combinations thereof.
21 . The kit of claim 18 , wherein the anti-HPV monoclonal antibody is used to detect HPV infection at various disease stages, the disease stage is selected from the group consisting of early stage HPV infection, late stage HPV infection, early stage cervical cell lesion, late stage cervical cell lesion, low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), cervical intraneoplasm stage 1, (CIN1), cervical intraneoplasm stage 2 (CIN2), cervical intraneoplasm stage 3 (CIN3), developed cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and combinations thereof.