250 HYBRIDOMA AND HYBRIDOMICS Volume 23, Number 4, 2004 © Mary Ann Liebert, Inc. A Novel Monoclonal Antibody Against Canine Monocytes/Macrophages PAULO HENRIQUE PALIS AGUIAR, 1,2 ROBERTO ROBSON BORGES DOS SANTOS, 2 DANIELA FARIAS LARANGEIRA, 2 MARTA ALMEIDA DOS SANTOS, 1 STELLA MARIA BARROUIN-MELO, 1,2 TÂNIA MARIA CORREIA SILVA, 1 JOSÉ O. MENGEL, 1 WASHINGTON LUIS CONRADO DOS SANTOS, 1,3 and LAIN PONTES-DE-CARVALHO 1,3 ABSTRACT The production and partial characterization of a monoclonal antibody, the IgG 1 IH1, which recognizes an antigen distributed in canine monocytes/macrophages, is reported here. The distribution and apparent mo- lecular weight of the antigen recognized by the IH1 MAb was determined in peripheral blood leukocytes, pe- ripheral blood monocyte-derived macrophages and tissue sections of spleen, liver and skin, using Western blotting, immunocytochemistry, immunohistochemistry and flow cytometry. The IH1 MAb-recognized anti- gen was detected in Western blotting under non-reducing conditions spread out as a large band covering the position corresponding to the migration of molecules with molecular weights from 55 to 73 kDa. The IH1 MAb labeled blood monocytes, tissue macrophages in lymph nodes, and in the mantle zone of the spleen, and Kupffer cells in the liver. It did not react with human cells. In flow cytometric analysis, the IH1 MAb reacted with a subpopulation of monocytes. The MAb described herein may become a valuable tool for diagnosis and research on canine diseases. INTRODUCTION B ESIDES THEIR IMPORTANCE as companion and working do- mestic animals, dogs have become a useful animal model in biomedical research. Dogs are used in studies on transplan- tation and on infections diseases such as leishmaniasis and many diseases that also occur in human beings. For instance, dogs spontaneously develop a variety of diseases such as al- lergic asthma, (1) penfigus, systemic lupus eritematosus, and congenital or acquired immunodeficiences, (2) providing an ad- equate model for research. Monocytes/macrophages are the major target cells for most intracellular pathogens. (2) These cells also play important roles in graft rejection (3) and in the development of the immune re- sponse in zoonosis such as leishmaniasis. (4) Nevertheless, the panel of cell markers for the identification of different macro- phage populations in dog is restricted. Macrophages are widely distributed throughout body tissues, showing a high plasticity and constituting a heterogeneous cell population. Depending on their localization, macrophages show differences in their morphology, function and antigen expres- sion. Moreover, macrophages may be in different maturation and activation stages. (5) Monoclonal antibodies with specificity against canine leuko- cytes have been reported previously. However, only a limited panel of canine cell markers are available. (6–10) These antibod- ies react with many cell types, especially lymphocytes and/or granulocytes. However, antibodies that react with canine mono- cytes and macrophages are rare. (11–13) The production and partial characterization of a monoclonal antibody binding to canine monocytes/macrophages but not to granulocytes and lymphocytes is reported here. Its successful uses in immunohistochemical and flow cytometric analysis are also reported. MATERIALS AND METHODS Animals and tissues Two normal 24–36-month-old outbreed dogs, weighing 15 kg, were used throughout the study. The dogs were kept sepa- rately, under standard care and feeding conditions, in the ken- 1 Laboratorio de Patologia e Bio-Intervenção, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Bahia, Brazil. 2 Departamento de Patologia e Clínica, Escola de Medicina Veterinária Universidade Federal da Bahia, Bahia, Brazil. 3 Escola Bahiana de Medicina, Fundação para Desenvolvimento da Ciência, Bahia, Brazil.