Forsman. In this study, we obtained seven MAbs that recognize (R)-Oxiracetam at least five different epitopes carried by and as well. These MAbs can be utilized for antigenic analyses of organisms as well as for the diagnosis of tularemia and tularemia-like diseases. Twenty-six strains (15 Japanese strains and 11 non-Japanese strains), the U112 strain, and the 029 strain were kindly provided by H. Fujita, Ohara Research Laboratory, Fukushima, Japan. Two strains (ATCC 25017 and ATCC 25018), and subsp. were propagated in our laboratory. All strains were propagated on Difco Eugon agar (Becton, Dickinson and Company, Sparks, MD) with chocolatized 8% sheep blood in a biosafety level-3 laboratory. The MAb against LPS (FB11) (Biodesign International, Saco, ME) was used as a reference, and fluorescent isothiocyanate (FITC)-labeled antirabies computer virus monoclonal antibody (Fujirebio Diagnostics, Inc. Malvern, PA) was used as an isotype control. All animal experiments were approved by the animal research committee of the National Institute of Infectious Diseases. Hybridoma clones secreting MAbs (M11D3, M11H7, M13B10, M14B11, M15C6, S11E7, and U22F2) were obtained by the fusion of mouse myeloma cells (P3-X63-Ag8.653) and spleen cells from BALB/c mice, which had been immunized with the formalin-inactivated GIEM Miura (Japanese) strain, the Schu (non-Japanese) strain, or the U112 strain, as described elsewhere (14). Characteristics of the MAbs (Table ?(Table1)1) were based on MAbs TCF10 obtained from hybridoma supernatant or mice ascitic fluids. Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that this MAbs acknowledged at least five different epitopes carried by LVS (Fig. ?(Fig.1).1). The banding patterns obtained with the Schu and GIEM Miura strains were not different from those obtained with the LVS strain (data not shown). MAb M14B11 stained ladder-like bands having molecular masses greater than 15 kDa. Identical ladder-like bands were obtained with MAbs M11H7 and M15C6 (data not shown). These three MAbs also reacted with purified LPS (Fig. ?(Fig.1),1), a major protective antigen of (17). On the other hand, MAb M11D3, M13B10, and S11E7 reactions produced single bands with molecular masses of 40, 17, and 10 kDa, respectively, while MAb U22F2 reactions produced 41- and 43-kDa bands (Fig. ?(Fig.1).1). These four MAbs did not react with proteinase K-digested antigen (data not shown), suggesting that this MAbs recognized protein components. proteins of 10, 17, 40, 41, and 43 kDa were found to (R)-Oxiracetam be recognized (R)-Oxiracetam by the sera from tularemia patients (4, 12). In addition, immunoreactive membrane components of might play important roles in both the invasion of host cells and escape from phagolysososmes (6, 11). Although it is usually unclear whether our MAbs identify these essential components, they may help to analyze the pathogenicity of LVS, U112, and 029 (lanes 1 to 3, respectively) were reacted with MAbs M11D3, M13B10, M14B11, S11E7, and U22F2 and normal mouse serum (unfavorable control). The reaction of MAb M14B11 against purified LPS from Schu (lane 4) is also shown. The positions of the molecular size markers are (R)-Oxiracetam indicated (in kilodaltons). TABLE 1. Summary of the characteristics of monoclonal antibodies (26)(1)(3)strains utilized for immunization of mice. bMolecular mass of antigen appeared in Western blotting following SDS-PAGE. cDetermined by indirect fluorescence assay: +, positive; ?, unfavorable. dDetermined by microagglutination test: +, positive; ?, unfavorable. eDetermined with a mouse monoclonal antibody isotyping test.