Michisato Murata

Production and Characterization of Monoclonal Strain-Specific Antibodies against Prototype Strains of Rickettsia tsutsugamushi

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain‐specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti‐Gilliam, four anti‐Karp and five anti‐Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain‐ or type‐specific polypeptides and do not show any cross‐reaction among strains.

Effect of Molar Ratio of Fluorescent Dye to IgG on the Detection of Lymphocyte Surface Immunoglobulins by Immunofluorescence

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S‐Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. The percentage of S‐Ig bearing cells increased markedly when labeled anti‐human H‐ or L‐chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S‐Ig was caused by increased non‐specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. This non‐specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mice showed less non‐specific adsorption than those from humans. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.