Article Figures & Data
Figures
- Fig. 1.
Kinetics of activation and binding of C3 fragments toC. neoformans cells incubated with 40% NHS in the presence or absence of anti-GXM MAbs. The indicated antibody concentrations are micrograms of MAb per milliliter of reaction mixture volume. Binding of C3 fragments was determined by incorporation of trace amounts of125I-labeled C3 into the reaction mixture.
- Fig. 2.
Immunofluorescence analysis of the sites for binding of C3 to C. neoformans cells incubated for 2, 4, 8, and 16 min with 40% NHS in the presence (50 μg/ml) or absence of anti-GXM MAbs. Sites of C3 deposition were determined by use of FITC-labeled antiserum to C3. All images were collected under identical conditions of image acquisition, including the number of image integrations (five) and camera gain (−3 db), with the exception of selected (∗) cells incubated with NHS for 2 min, in which case the number of image integrations was increased to 20. The fluorescence found with some samples was so intense that digital deconvolution of the images could not completely remove haze found in the center of the cell, e.g., cells incubated with NHS in the presence of MAb 386.
- Fig. 3.
Kinetics of activation and binding of C3 fragments toC. neoformans cells incubated with 40% NHS in the absence of anti-GXM MAbs or in the presence of an isotype switch (IgG1→IgG2b→IgG2a) family of MAbs derived from MAb 439 (top row) and MAb 471 (bottom row). The indicated antibody concentrations are micrograms of MAb per milliliter of reaction mixture volume. Binding of C3 fragments was determined by incorporation of trace amounts of125I-labeled C3 into the reaction mixture.
- Fig. 4.
Kinetics of alternative pathway-mediated activation and binding of C3 fragments to C. neoformans cells incubated with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAbs. Binding of C3 fragments was determined by incorporation of trace amounts of 125I-labeled C3 into the reaction mixture.
- Fig. 5.
Immunofluorescence analysis of the sites for binding of C3 to C. neoformans cells incubated for 2, 4, 8, and 16 min with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAbs. Sites of C3 deposition were determined by use of FITC-labeled antiserum to C3. Unless otherwise indicated, images were collected under identical conditions of image acquisition, including the number of image integrations (five) and camera gain (−3 db). ∗, 20 image integrations; ∗∗, 50 image integrations. As in the case with images shown in Fig. 2, the fluorescence found with some samples was so intense that digital deconvolution of the images could not completely remove haze found in the center of the cell.
- Fig. 6.
Stereoscopic images of sites for binding of C3 toC. neoformans cells incubated for 4 min with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAb 3C2. Sites of C3 deposition were determined by use of Oregon Green 514-labeled antiserum to C3. Conditions for collection of the immunofluorescence image were optimized for the intensity of fluorescence exhibited by each cell type (NHS-Mg-EGTA, 10 image integrations and camera gain of 0 db; NHS-Mg-EGTA plus MAb 3C2, 30 image integrations and camera gain of 9 db).
Tables
- Table 1.
Serotype specificity and molecular characteristics of MAbs reactive with C. neoformans GXM
MAb groupa MAb Isotype Serotype specificity Molecular structure References A B C D VH family JH D size VL JL II 439 IgG1 + + + + 7183 2 7 κ5.1 1 2,5, 15, 50 471 IgG1 + + + + 7183 2 7 κ5.1 1 2,5, 50 3C2 IgG1 + + + + 7183 2 7 κ5.1 1 2,5, 50 III 339 IgG1 + + − + 10 4 6 κ21 5 2,5, 50 1255 IgG1 + + − + 10 4 6 κ21 5 2,5, 15, 50 IV 302 IgG1 + − − + VGam 2 6 κ4/5 1 2,5, 15, 50 386 IgM + ± ± + VGam 3 4 κ4/5 1 2,5, 50 ↵a Based on differences in antibody variable region usage, which determines antibody molecular structure (5).
- Table 2.
Binding of MAbs to encapsulated cryptococci as determined by Scatchard analysis
MAb group MAb No. of MAb molecules/cell Under saturating conditions At 50 μg/4 × 105 yeast cells II 439 5.1 × 107 4.5 × 107 471 4.7 × 107 4.3 × 107 3C2 6.1 × 107 5.5 × 107 III 1255 9.4 × 106 8.7 × 106 339 2.3 × 107 2.2 × 107 IV 302 5.8 × 107 5.4 × 107 386 2.2 × 106 2.2 × 106 - Table 3.
Binding of trypsin-generated metastable C3b to C. neoformans cells in the presence or absence of anti-GXM MAbs (125 μg of MAb/106 yeast cells)
MAb added MAb group Mean no. of molecules of C3b bound/cell ± SEM None 65,000 ± 2,700 439 II 63,000 ± 3,800 471 64,000 ± 4,200 3C2 73,000 ± 4,100 1255 III 69,000 ± 3,600 339 67,000 ± 3,200 302 IV 60,000 ± 3,700 386 61,000 ± 3,100
