[13, 24]. Results Characterization of mAb MEST-3 Aiming to study the biological role of GIPCs, and since expression of these glycoconjugates with terminal
galactofuranose residues, which are recognized by MEST-1, is restricted to P. brasiliensis (Pb), H. capsulatum (Hc) and A. fumigatus (Af), we decided to develop a mAb directed to GIPC Pb-2, from P. brasiliensis, which structure Manpα1→3Manpα1→2IPC is expressed in a wide variety of fungi, and therefore a mAb directed to such structure would be highly desirable to detect a large number of pathogenic fungi. Among a few clones showing reactivity with GIPC Pb-2, a clone secreting an IgG2a monoclonal antibody was established, and termed MEST-3. By HPTLC-immunostaining (Figure 1B, lanes 1-3) it was observed Evofosfamide mw that MEST-3 reacts with Pb-2 from
yeast and Ruxolitinib concentration mycelium forms of P. brasiliensis, and other GIPCs containing the same structure as Pb-2, such as Hc-Y2 from yeasts of H. capsulatum (Figure 1B, lane 7), Ss-Y2 from yeasts of S. schenckii (Figure 1B, lane 9), Af-2 from hyphae of A. fumigatus (Figure 1B, lane 4), and An-2 from hyphae of A. nidulans (Figure 1B, lane 5). Moreover, lanes 6 and 8 of Figure 1A-B confirm that mycelium forms of H. capsulatum and S. schenckii do not express GIPCs bearing the epitope recognized by MEST-3, as described before [8, 9, 22, 23]. Also, by solid-phase radioimmunoassay (RIA), it was verified that this website mAb MEST-3 was able to detect as low as 5 ng of purified Pb-2, Hc-Y2, SS-Y2 and Af-2 (Figure 1C). Conversely, no reactivity of MEST-3 with GIPCs, presenting
the structures Manp(α1→3) [Galf(β1→6)]Manp(α1→2)IPC (Pb-3, Hc-Y3, Af-3); Manα1→2IPC (MIPC) and Manα1→3Manα1→6IPC (Ss-M2), was detected by HPTLC-immunostaining or RIA. Figure 1 Reactivity these of fungal GIPCs with MEST-3. Fungal GIPCs were purified by a combination of chromatography in DEAE-Sephadex, silica gel 60, HPLC and preparative HPTLC. HPTLC was developed in solvent A. Panel A, stained with orcinol/H2SO4 and panel B, immunostaining with MEST-3. Lane 1, GIPC Pb-2 from mycelium form of P. brasiliensis; lane 2, acidic GSLs from mycelium form of P. brasiliensis; lane 3, acidic GSLs from yeast form of P. brasiliensis (Pb); lane 4, acidic GSLs from hyphae of A. fumigatus (Af); lane 5, acidic GSLs from hyphae of A. nidulans (An); lane 6, acidic GSLs from mycelium form of H. capsulatum (Hc); lane 7, acidic GSLs from yeast form of H. capsulatum; lane 8, acidic GSLs from mycelium form of S. schenckii (Sc); lane 9, acidic GSLs from yeast form of S. schenckii; lane 10, acidic GSLs from the edible mushroom Agaricus blazei (Ab). Arrows indicates chromatographic migration of Pb-2, Af-2, An-2, Hc-Y2 and Ss-Y2. Panel C, GIPCs (first well 0.