In situ detection of early stages and late stages HPV einfection

• US 20090311668A1
• Filed: 06/10/2009
• Published: 12/17/2009
• Est. Priority Date: 06/13/2008
• Status: Active Grant

• Current Assignee: Heer Medical Technology Development Co., Ltd.
• Original Assignee: Onco Health Corporation
• Inventors: Cheng, Shuling
• Granted Patent: US 8,859,218 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 16/084 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: G01N 2333/025 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: G01N 2469/10 Detection of antigens from microorganism in sample from host
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/571 for venereal disease, e.g. syphilis, gonorrhoea herpes G01N33/56994; chlamydia G01N33/56927

Abstract

Embodiments of the invention provide methods, monoclonal antibodies, polyclonal antibodies, assays, and kits for detecting HPV infection and HPV related cancer diagnosis, including infection by various HPV genotypes, early and/or late stage HPV-associated or HPV-specific cancers. The anti-HPV antibodies are used in performing immunological assays on clinical samples. Various immunological assays and kits for detecting HPV infection, cervical cancer, other HPV related cancers, early stage precancerous lesions as well as late stage cancer progression are also provided.

Claims

1. A method of detecting papillomavirus infection in a human subject, comprising:

providing a clinical sample from the human subject, the clinical sample containing a mixture of morphologically abnormal and normal human cells are processed and applied into a thin layer of cells on a slide;

conducting an immunocytochemistry assay using one or more antibodies generated against one or more purified recombinant papillomavirus proteins, wherein at least one antibody is capable of binding to a papillomavirus early viral protein; and

staining in situ one or more papillomavirus viral proteins from one or more papillomavirus types present in the clinical sample on the slide containing the mixture of morphologically abnormal and normal human cells.

2. The method of claim 1, wherein the one or more purified recombinant papillomavirus proteins are selected from the group consisting of recombinant HPV-16 E6 proteins, recombinant HPV-16 E7 proteins, recombinant HPV-18 E6 proteins, recombinant HPV-18 E7 proteins, recombinant HPV-16 L1 proteins, recombinant HPV-18 L1 proteins combinations thereof.

3. The method of claim 1, wherein the papillomavirus early viral protein is selected from the group consisting of HPV-16 E6 protein, HPV-16 E7 protein, HPV-18 E6 protein, HPV-18 E7 protein, and combinations thereof.

4. The method of claim 1, wherein the one or more papillomavirus, viral proteins are selected from the group consisting of papillomavirus E6 protein, papillomavirus E7 protein, papillomavirus L1 protein and combinations thereof.

5. The method of claim 1, wherein the one or more papillomavirus types are selected from the group consisting of high risk HPV types, low risk HPV types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, and combinations thereof.

6. The method of claim 1, wherein one antibody among the one or more antibodies is capable of binding to two or more papillomavirus viral proteins.

7. The method of claim 6, wherein the two or more papillomavirus viral proteins comprises the papillomavirus early viral protein and a papillomavirus late viral protein and the one antibody is capable of binding to both the papillomavirus early viral protein and the papillomavirus late viral protein.

8. The method of claim 1, wherein the one or more antibodies are selected from the group consisting of antibodies capable of binding to HPV E6 proteins, antibodies capable of binding to HPV E7 proteins, antibodies capable of binding to HPV E6 and E7 proteins, antibodies capable of binding to HPV L1 proteins, antibodies capable of binding to HPV E6 proteins from different HPV types, antibodies capable of binding to HPV E7 proteins from different HPV types, antibodies capable of binding to HPV L1 proteins from different HPV types, antibodies capable of binding to HPV E6 and E7 proteins of the same HPV types, antibodies capable of binding to HPV E6 and E7 proteins of different HPV types, antibodies capable of binding to HPV E6, E7, and L1 proteins, and combinations thereof.

9. The method of claim 1, wherein the immunocytochemistry assay is used to detect a diseased stage caused by HPV infection, the disease stage is selected from the group consisting of early stage HPV infection, late stage HPV infection, early stage cervical lesion, late stage cervical 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, 2, 3 (CIN1, CIN2, CIN3, respectively), invasive cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and combinations thereof.

10. The method of claim 1, 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 immunocytochemistry assays.

11. The method of claim 1, further comprising comparing the staining pattern and intensities of the mixture of the morphologically abnormal and normal human cells with the morphology of these cells.

12. The method of claim 1, wherein the staining in situ on the slide the one or more papillomavirus proteins from the one or more papillomavirus types present in the clinical sample comprises under a microscope detecting the presence of an agent tagged on the one or more antibodies, wherein the agent is selected from the group consisting of a colormetric agent, a fluorescent chromogen, and combinations thereof.

13. The method of claim 1, wherein the conducting the immunocytochemistry assay further comprises using two or more anti-HPV antibodies pre-labeled with different agents in one binding reaction to detect more than one HPV proteins.

14. The method of claim 1, wherein the clinical sample is selected from the group consisting of cervical cells, cervical tissues, cervical swabs, body fluids, serum, blood, tumors, cell cultures, biopsies, and combination thereof.

15. The method of claim 1, wherein the clinical sample is obtained from general population and the immunocytochemistry assay is conducted for the detection of cervical cancer.

16. A method of detecting papillomavirus infection in a human subject, comprising:

providing a clinical sample from the human subject, the clinical sample is processed into a liquid-based solution containing a mixture of morphologically abnormal and normal human cells;

staining the mixture of the morphologically abnormal and normal human cells using one or more antibodies generated against one or more purified recombinant papillomavirus proteins, wherein the one or more antibodies are each tagged with an agent, and at least one antibody is capable of binding to a papillomavirus early viral protein;

conducting one or more flow cytometry assays to detect each individual cells by separating each cell from the mixture of the morphologically abnormal and normal human cells; and

detecting the presence of one or more human papillomavirus viral proteins from one or more papillomavirus types in the mixture of the morphologically abnormal and normal human cells contained in the liquid-based solution from the clinical sample.

17. The method of claim 16, wherein the one or more antibodies further comprises two or more anti-HPV antibodies each pre-labeled with different agents and used in one binding reaction to detect the presence of two or more human papillomavirus viral proteins.

18. A kit for detecting papillomavirus infection in a human subject, comprising:

an anti-HPV monoclonal antibody, capable of binding to a papillomavirus early protein for conducting an immunocytochemistry assay on a clinical sample from the human subject processed into a solution containing a mixture of morphologically abnormal and normal human cells and applied into a thin layer of cells on a slide for staining in situ one or more papillomavirus viral proteins from one or more papillomavirus types present in the clinical sample on the slide containing the mixture of morphologically abnormal and normal 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.

20. The kit of claim 18, wherein the one or more papillomavirus types are selected from the group consisting of high risk HPV types, low risk HPV types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, and combinations thereof.