Indirect ELISA for A. fumigatus CWF Antibodies

For the indirect ELISA, the process was similar to that described by Schubert et al. [29 (link)]. Briefly, various concentrations of A. fumigatus CWFs in PBS were coated overnight on microtiter plate wells (Corning, New York, NY, USA). The following day, the plate was washed with PBS containing 0.1% Tween 20 (PBST) and the wells were blocked with 3% BSA in PBST. Then, serial dilutions of purified 1D2 and 4E4 (from 1:100 to 1:102,400) were added to each well (100 μL) for 1 h with the control being an unrelated IgM isotype antibody (6B10) [40 (link)]. After washing, 100 μL of goat anti-mouse IgM-HRP (Abcam, Cambridge, UK) diluted at 1:2000 was added to each well, followed by washing and detection with tetramethybenzidine (TMB) substrate. The reaction was stopped by the addition of 100 μL of 1 M hydrochloric acid/well and the absorbance was read by a microplate spectrophotometer at 450 nm (Thermo Fisher Scientific, Waltham, MA, USA). With the exception of coating and washing, all other dilution steps in the ELISAs were in PBST containing 3% BSA. All laboratory ELASAs in this study were performed at RT with triplicate determinations unless otherwise specified. The optimal reaction concentrations of all antibodies were determined by titration.