Skip to main content
. 2017 Aug 11;7(14):3398–3414. doi: 10.7150/thno.20919

Figure 3.

Figure 3

Binding specificity of hJF5 to A. fumigatus in infected mouse lungs. A. Experimental workflow for the in situ binding of mAb hJF5: (1) Neutrophil depletion by i.p. injection of 100 μg of the anti-Gr-1 antibody 17 h before injection with the pathogen; (2) Intra-tracheal infection with A. fumigatustdTomato; (3) After 48 h, the lungs were explanted, fixed, and fungal biomass stained in situ with DyLight650 conjugates of hJF5 or the human IgG1 isotype control antibody ET901. B. Micrographs of infected lungs showing cytoplasmic fluorescence (false colour green) of the genetically modified strain of the pathogen A. fumigatustdTomato, and co-localisation of cell-wall-bound mannoprotein antigen with the probes hJF5-DyLight650 (red fluorescence) or ET901-DyLight650 control (red fluorescence). Note red fluorescence of tissue probed with hJF5-DyLight650, but lack of fluorescence with the control antibody. Scale bar = 50 μm. C. Experimental workflow for hJF5 binding in vivo: (1) Neutrophil depletion by i.p. injection of 100 μg anti-Gr-1 antibody (clone RB6-8C5) 17 h before injection with the pathogen; (2) Intra-tracheal infection with A. fumigatustdTomato; (3) i.v. injection of hJF5-DyLight650 or ET901-DyLight650 antibodies 24 h after fungal infection; (4) Another 24 h later, the lungs were explanted and (5) longitudinal sections of explanted lung tissues were examined under confocal microscopy. D. Micrographs of A. fumigatustdTomato infected lungs (shown in false colour green) following i.v. injection with hJF5-DyLight650 (red) or ET901-DyLight650 (red). Note red fluorescence of lungs with hJF5-DyLight650, but lack of fluorescence with the control. Scale bar = 50 μm. E. An individual hypha is shown at higher magnification, with green fluorescence of A. fumigatustdTomato cytoplasm (white arrow) and binding of hJF5-DyLight650 (red) to the target mannoprotein antigen in the hyphal cell wall (white arrowhead). Note the lack of red fluorescence of the outer cell wall with the isotype control, further demonstrating the specificity of the hJF5 antibody. Scale bar = 2 μm.