Production and Characterization of the Monoclonal Antibodies to Rickettsia tsutsugamushi

Abstract

Twenty six hybridoma clones which secrete monoclonal antibodies to Rickettsia tsutsugamushi, Gilliam, Karp and Kato strains, were produced by fusing mouse myeloma cell line P3X63Ag8.653 with spleen cells from BALB/c mice immunized with the rickettsial strains. In order to clarify the immunological characteristics of Rickettsia tsutsugamushi, reactivity of these monoclonal antibodies against the three strains was examined by immunofluorescent antibody test. The results obtained are as follows:
1) Ten clones produced Gilliam strain specific antibodies.
2) Three clones produced Karp strain specific antibodies.
3) Three clones produced Kato strain specific antibodies.
4) Five clones produced antibodies which reacted to Karp and Kato strains, but not to Gilliam strain.
5) Five clones produced antibodies which reacted to Gilliam, Karp and Kato strains.
Reactivity of these monoclonal antibodies against Irie strain was examined by immunofluorescent antibody test. And it was clarified that Irie strain was Gilliam-Kato type.
Analysis by polyacrylamide gel electrophoresis and immnuoblotting experiments revealed that Rickettsia tsutsugamushi has over 30 bands, including major bands corresponding to molecular sizes of 61 kilodaltons (K) and 46K when samples for electrophoresis were dissolved at 21°C for 15 hours, and including major bands corresponding to molecular sized of 61K and 60K when samples were dissolved at 100°C for 5 min. Immunoblotting experiments with the monoclonal antibodies against each strain demonstrated the 61K, 60K, 46K and 44K proteins showed antigenic activities. The 60K and 46K proteins appeared to be strain specific antigen, whereas 61K and 44K proteins to be common antigens of Rickettsia tsutsugamushi.
A major polypeptide which showed strain-specific antigenicity appeared at the 46K position in samples solubilized at 21°C for 15 hours but moved to the 60K position in samples heated at 100°C for 5minutes. The finding suggests this strain specific antigen is a heat modifiable protein. But a major polypeptide which showed common antigenicity appeared at the 61K position samples solubilized at 21°C for 15 hours did not move after samples were heated at 100°C for 5 minutes.