Characterization of monoclonal antibodies to an immunodominant protein of the etiologic agent of human granulocytic ehrlichiosis

Immunodominant proteins in the range of 42-45 kD are important for the sero diagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human iso lates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclona l antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA) , and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent o f HGE, Ehrlichia equi and E. phagocytophila. Following screening and subclo ning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which wer e determined to be of the isotypes IgG,, IgG,, and IgG,,, respectively. The se results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocyto phila, and the etiologic agent of HGE. Western blot analysis showed reactiv ity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathoge ns could be detected when undiluted supernatants from hybridoma cultures we re allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggestin g that these two antibodies may compete for the same epitope of the 44-kD p rotein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immu nologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.