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Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin , a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Persefoni Klimentzou, Angeliki Drougou, Birgit Fehrenbacher, Martin Schaller, Wolfgang Voelter, Calypso Barbatis, Maria Paravatou-Petsotas and Evangelia Livaniou

Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research "Demokritos," Aghia Paraskevi, Athens, Greece (PK,MP-P,EL); Pathology Department, Hellenic Red Cross Hospital, Athens, Greece (AD,CB); Universitaets-Hautklinik, Tuebingen, Germany (BF,MS); and Interfakultaeres Institut fuer Biochemie, Eberhard-Karls-Universitaet Tuebingen, Tuebingen, Germany (WV)

Correspondence to: Livaniou Evangelia, Immunopeptide Chemistry Laboratory, Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research "Demokritos," Aghia Paraskevi Attikis, Athens 15310, Greece. E-mail: livanlts{at}rrp.demokritos.gr

Prothymosin (ProT) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProT immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProT or the synthetic fragments ProT[1-28], ProT[87-109], and ProT[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProT[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProT in tumorigenesis. (J Histochem Cytochem 56:1023–1031, 2008)

Key Words: prothymosin • polyclonal antibody • ELISA • Western blot • immunocytology • immunohistochemistry • cancer prostate tissues

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