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Abstract
Bartonellosis is spotlighted recently as an emerging zoonosis and Bartonella henselae is reported to be the main infectious agent. In Korea, however, few studies have been made on the epidemiology and microbiology on bartonellosis. Thus, this study was conducted to produce a new monoclonal antibody that can be used for identifying B. henselae. In order to prepare monoclonal antibodies against B. henselae, we inoculated mice with the isolated strain from Korean patient and performed cell fusion experiment. The selected hybridoma clones produced monoclonal antibodies which showed positive immunofluorescence staining of bacteria and specific protein bands in western blot analysis. In order to examine whether these antibodies could be used for the identifying and quantifying Bartonella, we performed confocal microscopy and flow cytometry using the new antibodies. These monoclonal antibodies can be used as a useful tool in further researches on the biology of Bartonella.
Figures and Tables
Figure 1
The immunofluorescence antibody test of hybridoma clones secreting the monoclonal antibodies against Bartonella. Culture supernatant of hybridoma clones were tested for their reactivity against B. henselae that was included in the commercial antibody test kit.
Figure 2
Western blot analysis of the 24 hybridoma clones secreting the monoclonal antibodies against Bartonella. The Korean isolate of B. henselae was grown in blood agar plate and harvested. After separation by SDS-PAGE, the antigenic bands were transferred to polyvinylidene difluoride membrane and cut into small strips. Each strip was analyzed using the supernatants of the hybridoma clones as a primary antibody. M, Protein size marker; P, positive control (B. henselae infected mouse serum); N, negative control (supernatant of the V653 cell cultured media); lane 1~28, the culture supernatants of each hybridoma clones.
Figure 3
The reactivity of a monoclonal antibody against ECV 304 cells infected with B. henselae was examined with the confocal microscope. The culture supernatant of a selected hybridoma clone (#1) was used as a primary antibody and the results were analyzed using the confocal laser scanning microscopy at the indicated times (0, 1, 3, 6 and 24 h after infection). ECV cell bodies were stained with Evans blue and bacterial cells were stained with IFA.
Figure 4
The reactivity of a monoclonal antibody against ECV 304 cells infected with B. henselae was examined with flow cytometry. The culture supernatant of a selected hybridoma clone (#1) was used as a primary antibody and the results were analyzed using the flow cytometer at the indicated times (0, 1, 3, 6 and 24 h after infection). The percentages of positive cells (M1) were indicated at each figure.
References
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