Detection method for human pappilomavirus (HPV) and its application in cervical Cancer

Abstract

Embodiments of the invention provide methods, assays, and kits for detecting HPV infection and HPV associated epithelial cell abnormalities, most notably those associated with pre-malignant and malignant epithelial cell lesions. Detection of HPV DNAs, genomes, and/or oncoproteins by nucleic acid hybridization assays and immunological assays can be used in early clinical screening for HPV infection and diagnosis for cervical cancer. The polypeptides, recombinant proteins, antibodies, nucleic acids, and various detection methods thereof are particularly useful for diagnosing carcinomas of the uterine cervix and those at risk of developing cervical cancer.

1 . A method of screening a human subject infected with a human papillomavirus, comprising:
 obtaining a clinical sample from the human subject;   conducting a nucleic acid hybridization assay on the clinical sample;   detecting the presence of a papillomavirus genome in the clinical sample from the human subject;   conducting one or more immunological assays on the clinical sample;   detecting the presence of an antibody to an early papillomavirus viral protein or the presence of the early papillomavirus viral protein in the clinical sample using a first recombinant protein of the early papillomavirus viral protein; and   detecting the presence of an antibody to a late papillomavirus viral protein or the presence of the papillomavirus late viral protein in the clinical sample using a second recombinant protein of the late papillomavirus viral protein.   
     
     
         2 . 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 for the presence of the papillomavirus genome and the results of the one or more immunological assays. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid hybridization assay is selected from the group consisting of polymerase chain reactions, nucleic acid hybridization assays, DNA chip assays, radioactive nucleic acid hybridization and detection assays, and non-radioactive nucleic acid hybridization and detection assays. 
     
     
         4 . The method of  claim 1 , wherein the presence of the papillomavirus genome is detected using a nucleic acid probe with sequence homology to conservative DNA sequences from a gene selected from the group consisting of papillomavirus late genes, L1 genes, L2 genes, papillomavirus early genes, E2 genes, E6 genes, E7 genes, and combinations thereof. 
     
     
         5 . The method of  claim 1 , wherein the human papillomavirus is 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 the one or more immunological assays are selected from the group consisting of ELISA, antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, protein chip assays, radioimmunopercipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays. 
     
     
         7 . The method of  claim 1 , wherein the early papillomavirus viral protein is selected from the group consisting of HPV-16 E6 proteins, HPV-16 E7 proteins, HPV-18 E6 proteins, HPV-18 E7 proteins, and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the late papillomavirus viral protein is selected from the group consisting of HPV-16 L1 proteins, HPV-18 L1 proteins, and combinations thereof. 
     
     
         9 . 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, and combination thereof. 
     
     
         10 . A method of screening a human subject of high risk human papillomavirus infection, comprising:
 obtaining a clinical sample from the human subject;   obtaining a first recombinant protein purified from a first protein expression system with a first expression vector having a portion of nucleic acid sequence corresponding to the full length nucleic acid sequence of an early papillomavirus gene;   obtaining a second recombinant protein purified from a second protein expression system with a second expression vector having a portion of nucleic acid sequence corresponding to the full length nucleic acid sequence of a late papillomavirus gene;   conducting one or more immunological assays on the clinical sample;   detecting the presence of an antibody to a viral oncoprotein or the presence of the viral oncoprotein in the clinical sample using the first recombinant protein; and   detecting the presence of an antibody to a viral capsid protein or the presence of the viral capsid protein in the clinical sample using the second recombinant protein.   
     
     
         11 . The method of  claim 10 , wherein the first recombinant protein is 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, and combinations thereof. 
     
     
         12 . The method of  claim 10 , wherein the second recombinant protein is selected from the group consisting of recombinant HPV-16 L1 proteins, recombinant HPV-18 L1 proteins, and combinations thereof. 
     
     
         13 . The method of  claim 10 , wherein the clinical sample is selected from the group consisting of cervical cells, cervical tissues, cervical swabs, body fluids, serum, blood, tumors, and combination thereof. 
     
     
         14 . The method of  claim 10 , 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 immunological assays. 
     
     
         15 . The method of  claim 10 , wherein the early papillomavirus gene is selected from the group consisting of papillomavirus E6 genes, papillomavirus E7 genes, and combinations thereof. 
     
     
         16 . The method of  claim 10 , wherein the late papillomavirus gene is selected from the group consisting of papillomavirus L1 genes, papillomavirus L2 genes, and combinations thereof. 
     
     
         17 . The method of  claim 10 , wherein the one or more immunological assays are selected from the group consisting of ELISA, antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, protein chip assays, radioimmunopercipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays. 
     
     
         18 . The method of  claim 10 , wherein the high risk human papillomavirus is selected from the group consisting of HPV-16 and HPV-18.