Development and validation of an anti-p16 monoclonal antibody for the detection of high risk HPV-associated lesions in cervical biopsies

GABRIELA CAMPOREALE, SUSANA VIGHI, GONZALO DE PRAT GAY

Congreso; Franco-Argentinean Immunology Congress; 2011

Human papillomavirus (HPV) infection is an essential factor for the development of cervical lesions that may lead to cancer. Cervical cancer is an important public health problem in developing countries. However, the diagnosis of HPV is currently based on microscopic observation of the cervix and morphological analysis of lesions in endocervical smears and biopsies, being these methods highly dependent on the criteria and training of the observer. As HPV oncoproteins induce cell cycle deregulation, proteins of the cell cycle may be used as indicators of HPV infection. These proteins may be used in immunochemical techniques to help in the diagnosis of cervical neoplasia. Here, we aimed to develop monoclonal antibodies (mAbs) to proteins that are known to be surrogate markers of high risk HPV-associated neoplasias. p16 is a cyclin dependent kinase inhibitor regulated by retinoblastoma protein (pRb). In high risk HPV-related cervical lesions there is a functional inactivation of pRb by HPV E7 oncoprotein, leading to p16 upregulation. We expressed and purified recombinant p16 protein and produced mAbs using standard technique. From the panel of mAbs obtained, we characterized one anti-p16 mAb that specifically recognized endogenous p16 protein in HPV cancer cell lines, in agreement with reported data, as judged by enzyme-linked immunoassays and Western blots. Importantly, in conventional immunohistochemistry our mAb specifically immunostained paraffin-embedded sections of cervical cancer biopsies, HPV positive, and was not reactive in normal cervical epithelium, in correlation with a commercial anti-p16 mAb immunostaining used as control. Our results suggest that our anti-p16 mAb has high sensitivity for immunohistochemical detection on high risk HPV-related neoplasias, constituting a useful tool to improve diagnostic accuracy at low cost.