ABSTRACT. A monoclonal antibody, 6E9 established from mice injected with dolphin peripheral blood lymphocytes (PBLs) was characterized. In addition to its reactivity against 89.4% of dolphin PBLs, 6E9 reacted with 33.1% of bovine PBLs of which 22% were CD5 + , 11.1% were CD5 . 6E9 recognized a 34 kD protein on the surface of dolphin and bovine PBLs. Analysis of the protein's Nterminal amino acid sequence indicated that 6E9 recognizes bovine major histocompatibility complex (MHC) class II antigens. These results suggested that 6E9 recognized MHC class II antigens on bovine PBLs. As we have already produced an anti-dolphin MHC class I monoclonal antibody, analysis of immune system using these monoclonal antibodies will advance our understanding of the evolution of the mammalian immune system.-KEY WORDS: dolphin, MHC class II antigen, monoclonal antibody.J. Vet. Med. Sci. 60(3): 291-293, 1998 mice were purchased from Clea Japan Inc. (Tokyo, Japan).Cows were maintained at Nihon University. Production of 6E9 mAb: BALB/c mice were immunized with dolphin peripheral blood lymphocytes (PBLs) isolated by the Ficoll-Paque gradient (1.077) centrifugation method. The immunization schedule consisted of intraperitoneal injections of live 10 7 cells at 2-month intervals. After three injections, splenocytes from immune mice were fused with P3-X63-Ag8.653 myeloma cells [6]. The screening of hybridoma supernatants was based on the reactivity to dolphin PBLs, as analyzed by immunofluorescence staining. A hybridoma, termed 6E9 selected by this screening procedure, was further subcloned by limiting dilution and was cultured in RPMI 1640 (Gibco, Grand Island, New York) containing 10% heat-inactivated fetal calf serum (FCS), 1% L-glutamine, 1% nonessential amino acids, 1% Na-pyruvate, and 5 × 10 5 M 2-mercaptoethanol (ME), referred to as complete medium.Immunofluorescence tests: For immunofluorescence staining, cells were incubated at 4°C for 30 min with 30 µl of hybridoma supernatant. The cells were then washed with cold PBS (pH 7.2) containing 2% heat-inactivated newborn calf serum (NCS) and 0.05% NaN 3 , and mixed with 10 µl of fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse Ig mAb (Dakopatts, Cophenhagen, Denmark) diluted in PBS containing 2% NCS and 0.05% NaN 3 . The expression of the 6E9 surface antigens on bovine PBLs was compared with that of anti-bovine CD4 (CC30), CD5 (CC17), and CD8 (CC63) antibodies using two-color immunofluorescence tests. Namely, cells were stained with these mAbs followed by FITC-conjugated rabbit anti-mouse Ig mAb, and then, cells were stained with biotin-conjugated 6E9 and then vizualized with phycoerythrin (PE)-conjugated avidin (Caltag Laboratories, San Francisco, CA). Background staining was assessed using FITC-conjugated anti-mouse Ig mAb and PE-conjugated avidin. The Dolphins are very interesting mammals because they have returned to the sea from the land, after acquiring pulmonary respiration system. We are attempting to characterize their immune functions and as the first step t...