Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies Figen Caliskan a, * , Emel Ergene b , Ibrahim Sogut c, d , Ibrahim Hatipoglu d , Aynur Basalp d , Hulya Sivas b , Gungor Kanbak e a Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, Eskisehir, Turkey b Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey c Istanbul Bilim University, Vocational School of Health Services, 34394 Istanbul, Turkey d TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, Gebze, Kocaeli, Turkey e Department of Biochemistry, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey article info Article history: Received 24 January 2013 Received in revised form 8 September 2013 Accepted 11 September 2013 Available online xxxx Keywords: Scorpion toxin Androctonus crassicauda Cytotoxicity Apoptosis Immunization Monoclonal antibodies abstract Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA frag- mentation assay, respectively. Caspase 3 and caspase 9 activities were measured spec- trophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies. Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC 50 value of 5 mg/ml. Exposure of the cells to 0.1 and 0.5 mg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM. * Corresponding author. Eskis ¸ ehir Osmangazi Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, F-5 Blok, Kat2, Mes ¸ elik Kampüsü, Mes ¸ elik, Eskis ¸ ehir 26480, Turkey. E-mail addresses: fcalis@ogu.edu.tr, fcalis@gmail.com (F. Caliskan). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.toxicon.2013.09.009 Toxicon xxx (2013) 1–12 Please cite this article in press as: Caliskan, F., et al., Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.09.009