Maxisorp 96 well microplates

For the saturation binding tests, fibronectin, vitronectin and laminin were immobilized in wells of MaxiSorp 96-well microplates (Sarstedt) by overnight incubation at 4 °C (5 pmol protein in total volume of 50 μl per well). After this step and all subsequent steps, the wells were washed three times with 1 % BSA in PBS. The unoccupied surfaces in each well were blocked with 3 % BSA in PBS at 4 °C (overnight). Solutions of biotinylated fungal cell wall-associated proteins (50 μl) were added to the wells and the plate was incubated at 37 °C for 1.5 h. In an alternative competitive variant of this assay, the microplate-immobilized ECMP (3 pmol fibronectin or 1.25 pmol of vitronectin or laminin in total volume of 50 μl) competed with soluble ECMP, added in increasing molar excess, for binding to biotinylated fungal cell wall-associated proteins. In both types of assay, the binding level was detected with the SA-HRP/TMB detection system.

Hyphal or pseudohyphal forms of C. albicans, C. parapsilosis or C. tropicalis which adhered to the wells of MaxiSorp 96-well microplates (Sarstedt, Nümbrecht, Germany), were obtained from 1 × 10⁶ cells propagated in RPMI 1640 medium at 37 °C for 3 h. Each step of the following binding assay was preceded by washing the fungal cells three times with PBS buffer containing 1 % BSA. The unoccupied surfaces of the microplate wells were blocked with 3 % BSA in PBS at 4 °C overnight. The wells without fungal cells (with the surface blocked with BSA) served as controls. Solutions of biotinylated fibronectin, vitronectin and laminin (50 μl) were added to the cells and incubated at 37 °C for 1.5 h with gentle shaking. The amounts of bound biotinylated proteins were determined with the use of a SA-HRP/TMB detection system as described previously [23 (link)]. The values obtained for control samples were subtracted from the total binding values.