Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX $ Miriam Zat’ovic ˇova ´ a , Kvetoslava Tara ´bkova ´ a , Elis ˇka S ˇ vastova ´ a , Adriana Gibadulinova ´ a , Vojtech Mucha a , Ly ´dia Jakubı ´c ˇkova ´ a , Zuzana Biesova ´ a , Monika Rafajova ´ a , Marta Ortova Gut b , Seppo Parkkila c , Anna-Kaisa Parkkila d , Abdul Waheed e , Willam S. Sly e , Ivan Horak b , Jaromı ´r Pastorek a , Silvia Pastorekova ´ a, * a Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Du ´bravska ´ cesta 9, 845 05 Bratislava, Slovak Republic b Department of Molecular Genetics, Institute of Molecular Pharmacology and Medical Centre of Free University of Berlin, Berlin, Germany c Institute of Medical Technology, University of Tampere and Tampere University Hospital, Finland d Department of Neurology, University of Tampere and Tampere University Hospital, Finland e Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA Received 15 May 2003; received in revised form 25 July 2003; accepted 11 August 2003 Abstract Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/ 38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure – function relationships in the CA IX molecule and a better understanding of the role 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2003.08.011 Abbreviations: CA, carbonic anhydrase; IHC, immunohistochemistry; MAb, monoclonal antibody; PG, proteoglycan; GST, glutathione-S transferase. $ According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital Roman letters and numbers, while their genes are written in Italic letters and Arabic numbers. * Corresponding author. Tel.: +42-1-2-5930-2404; fax: +42-1-2-5477-4284. E-mail address: virusipa@savba.sk (S. Pastorekova ´). www.elsevier.com/locate/jim Journal of Immunological Methods 282 (2003) 117 – 134