Figure 2.

Quantification of mono- and dual-staining of single motif capsules. Capsules of each single motif strain were stained individually by each antibody fragment from the three Ab families (18B7, 2H1 and 3E5) and antibody staining patterns were analyzed by immunofluorescence. A, left: representative immunofluorescence images for the 18B7 family binding to strain Mu-1 (Motif 2). Right: pixel intensities of 1D slices of fluorescence for the representative cells. The diameter of each cell was normalized in this analysis to compare the relative capsular penetrance of each antibody fragment. B, quantification of capsular binding distances with respect to the cell body for each antibody fragment are overlaid to demonstrate the differences in penetrance between mAb, Fab and scFv. Fluorescence intensity plots for mAbs, Fabs, scFv and cell body are colored red, black, green, and blue, respectively. The intensity plots represent the average of five measurements for each condition. C, summary of capsular penetrance measurements for all cryptococcal strains and antibody formats. Reliable measurables of capsular penetrance for weak binding Fabs could not be obtained. See Fig. S2 for representative immunofluorescence staining of each antibody fragment with each single motif strain. D–G, each single motif strain was co-stained with mAb 18B7 and scFv 18B7. mAb and scFv were incubated with cells at equal molarities of Fv. mAb and scFv localization on each capsule were visualized by TRITC (red) and FITC (green) fluorescence, respectively. Pixel intensities through 1D slices for each fluorescent channel are quantified and displayed to right of each image. White arrows represent the 1D slice that was quantified. Black arrows indicate clear examples of increased penetrance of scFv with respect to mAb. For each motif, the structure of the triad is presented next to the motif. The mannan backbone and xylose and glucuronic acid sidechains are represented as green circles, orange stars, and blue-white diamonds, respectively, according to Symbol Nomenclature for Glycans (SNFG). Representative immunofluorescence capsular staining patterns are displayed for single motif cryptococcal cells expressing (D) Motif 1, (E) Motif 2, (F) Motif 3, and (G) Motif 4. For images marked with asterisks, the exposure for the red channel was increased to visualize the localization of the relatively weak binding mAb. The intensity plots for the increased exposure images are shown as dotted lines and overlaid with the intensity plots taking at the same exposure for both mAb and scFv, shown with solid lines. See Fig. S3 for additional representative images of co-staining with the other mAb or Fab and scFv pairs. White scale bars on all microscopy images indicate 10 μm.