Characterization of monoclonal antibodies against antigens shared with neutrophils and macrophages rainbow trout Oncorhynchus mykiss

Four monoclonal antibodies (MAbs: Mm, MGm, MGcp1 and MGcp2) were produced a gainst peritoneal exudate cells of rainbow trout, Oncorhynchus mykiss. The MAbs were characterized by immunofluorescence assay, flow cytometry, immuno electron microscopy and Western blotting, and utilized to isolate macrophag es and neutrophils by magnetic cell sorting. The immunofluorescence assay a nd flow cytometry showed that each of the MAbs was specific for both macrop hage and neutrophil populations and these cells were observed in leucocytes from various origins, i.e. the head kidney, peripheral blood and peritonea l exudate. Mm and MGm reacted with membrane antigens, while MGcp1 and MGcp2 reacted with intracellular antigens. The MAb-positive cells were identifie d as both macrophages and neutrophils by electron microscopy using immunogo ld labelling. A Western blot analysis revealed that the MAbs detected diffe rent determinants (Mm; 205,151 and 107 kDa: MGm; 205, 143, 107, 76, 55 and 52 kDa: MGcp1; no reaction: MGcp2; 64 kDa). Macrophages and neutrophils wer e efficiently isolated by a magnetic cell separation system with Mm and MGm . These antibodies will be useful for identifying and isolating rainbow tro ut macrophages and neutrophils, which facilitate studies of trout leucocyte s.