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The flow rate was maintained at 1 mL/min collecting 1 mL fractions.
All CSF samples had a CRAG LFA performed at bedside, collecting 1 drop of CSF into an Eppendorf tube with 1 drop of sample diluent.
Positive fractions were pooled, dialyzed, and loaded onto a 1 ml heparin HiTrap column (GE Healthcare), eluted with a NaCl gradient, collecting 1 ml fractions.
The bound enzyme was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) at a flow rate of 0.5 ml/min collecting 1 ml fractions.
Remaining bound rhPln.D1 was eluted from the column with a 50 ml gradient to 100% Elution Buffer at 2.5 ml/min collecting 1 ml fractions and monitoring absorbance at 280 nm.
Colony-forming units were counted by collecting 1 ml of conidia-containing cell lysates, diluting 10×, 100×, and 1000× in PBS, and plating 100 μl of each dilution onto MYPD agar plates.
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