Protein Chips for HPV Detection

Abstract

Embodiments of the invention provide methods, 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. Detection of HPV DNAs, genomes, and/or oncoproteins by protein chips immunological assays can be used in early clinical screening for HPV infection and general diagnosis for cervical cancer and can be advantageous performed in a multiplexed test. Comparative detection of altered levels of HPV proteins and host proteins can performed in one or more assays. 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 making an array comprising a plurality of antibodies that specifically bind to a plurality of recombinant HPV proteins selected from the group consisting of a papillomavirus E6 gene product, a papillomavirus E7 gene product, a papillomavirus L1 gene product, a papillomavirus truncated L1 gene product, and a papillomavirus L2 gene product comprising the steps of:
 providing a recombinant construct encoding a fusion protein comprising said papillomavirus gene product and an affinity tag selected from the group consisting of a HIS tag, a GST tag, and an MBP tag;   expressing said recombinant construct in a host cell;   incubating an extract prepared from said host cell with an affinity resin under conditions in which said affinity resin specifically binds said affinity tag;   eluting said recombinant HPV protein from said affinity resin, wherein
 said expressing of said recombinant construct in said host cell results in a level of protein expression such that said eluting produces a composition comprising said recombinant HPV protein at a concentration of from 1 mg/L to 10 mg/L, wherein
 said recombinant HPV protein is present in said composition at a purity of at least 90% as determined by SDS PAGE, and 
 wherein said recombinant HPV protein is present in said composition in a substantially soluble, monomeric form, as determined by size exclusion chromatography; 
 
   preparing said plurality of antibodies that bind specifically to said plurality of recombinant HPV proteins; and   adsorbing said plurality of antibodies to a substrate at a defined location on said substrate.   
     
     
         2 . The method of  claim 1 , wherein said expressing, incubating and eluting steps are carried out without the use of a denaturant. 
     
     
         3 . The method of  claim 1 , wherein said construct does not encode a chaperone. 
     
     
         4 . The method of  claim 1 , wherein said papillomavirus gene product comprises one or more of the group consisting of an HPV-6 gene product, an HPV-11 gene product, an HPV-16 gene product, an HPV-18 gene product, an HPV-31 gene product, an HPV-33 gene product, an HPV-35 gene product, an HPV-39 gene product, an HPV-42 gene product, an HPV-43 gene product, an HPV-44 gene product, an HPV-45 gene product, an HPV-51 gene product, an HPV52 gene product, an HPV-53 gene product, an HPV-54 gene product, an HPV-55 gene product, an HPV-56 gene product, an HPV-58 gene product, an HPV-59 gene product, and an HPV-66 gene product. 
     
     
         5 . The method of  claim 1 , wherein said papillomavirus gene product comprises one or more gene products selected from the group consisting of an HPV-16 gene product and an HPV-18 gene product. 
     
     
         6 . The method of  claim 1 , further comprising adsorbing one or more antibodies that specifically bind to one or more human proteins selected from the group consisting of p I61NK4a, pRB, p53, E2F, E2F activated cell cycle protein, cyclin dependent kinase, CDK4, CDK6, Ki-67 (MIB-I), MYC protein, cyclin-A, cyclin-B, cyclin-E, telomerase-TERC, MCM2, TOP2A, heat shock protein 40 (HSP40), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), CA9/MN, laminin5, bm-3a, CDK N2, topoisomerase 2A, microsome maintenance protein-2, microsome maintenance protein-4, microsome maintenance protein-5, survivin, VEGF, p27 (kipl), and p21 (waf) to said substrate at a second defined location on said substrate. 
     
     
         7 . A method for screening a human subject for a papillomavirus infection comprising:
 obtaining a sample from said human subject, wherein said sample comprising proteins;   contacting said sample with an array, wherein said array is made according to the method of  claim 1 ;   determining the presence, absence, or amount of one or more proteins in said sample that specifically binds to one or more antibodies of said array; and   screening said human subject for papillomavirus infection based on the determined presence, absence, or amount of said one or more proteins in said sample.   
     
     
         8 . The method of  claim 7 , wherein said one or more proteins comprise one or more proteins selected from the group consisting of a papillomavirus E6 gene product, a papillomavirus E7 gene product, a papillomavirus L1 gene product, a papillomavirus truncated L1 gene product, and a papillomavirus L2 gene product. 
     
     
         9 . The method of  claim 7  wherein said papillomavirus infection comprises an infection by one or more viruses selected from the group consisting of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-42, HPV-43, HPV-44, HPV-45, HPV-51, HPV-52, HPV-53, HPV-54, HPV-55, HPV-56, HPV-58, HPV-59, and HPV-66. 
     
     
         10 . The method of  claim 7  wherein said papillomavirus infection comprises an infection by HPV-16 or HPV-18. 
     
     
         11 . A method for screening a human subject for a papillomavirus infection comprising:
 obtaining a sample from said human subject, wherein said sample comprising proteins;   contacting said sample with an array, wherein said array is made according to the method of  claim 6 ;   determining the presence, absence, or amount of one or more proteins in said sample that specifically binds to one or more antibodies of said array; and   screening said human subject for papillomavirus infection based on the determined presence, absence, or amount of said one or more proteins in said sample.   
     
     
         12 . The method of  claim 11 , wherein said one or more proteins comprise one or more proteins selected from the group consisting of a papillomavirus E6 gene product, a papillomavirus E7 gene product, a papillomavirus L1 gene product, a papillomavirus truncated L1 gene product, a papillomavirus L2 gene product, p16INK4a, pRB, p53, E2F, E2F activated cell cycle protein, cyclin dependent kinase, CDK4, CDK6, Ki-67 (MIB-I), MYC protein, cyclinA, cyclin-B, cyclin-E, telomerase-TERC, MCM2, TOP2A, heat shock protein 40 (HSP40)′ heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), CA9/MN, 1-aminin5, brn-3a, CDK N2, topoisomerase 2A, microsome maintenance protein-2, microsome maintenance protein-4, microsome maintenance protein-5, survivin, VEGF, p27 (kipl), and p21 (waf). 
     
     
         13 . The method of  claim 11  wherein said papillomavirus infection comprises an infection by HPV-16 or HPV-18.