Imager 6000

Competitive ELISAs using overlapping linear PfCSP peptides (peptides 16–61; Genscript) that span the R1 and repeat region of PfCSP were performed on the Meso Scale Discovery (MSD) U-Plex Assay platform. Peptides were all 15 amino acids in length, overlapping by 11 residues, and numbered according to their position on the protein. Briefly, streptavidin-coated plates (Meso Scale Discovery, MSD) were blocked with 5% BSA in PBS for 30 min at room temperature (RT), washed five times (wash buffer, 0.05% Tween-20 in PBS), then coated with biotinylated-recombinant PfCSP (0.2μg/mL, Genscript) in PBS with 1% BSA, and allowed to incubate for 1h at RT. Either PfCSP specific monoclonal antibodies (all at 10 ng/mL except iGL-CIS43 at 100ng/mL), or polyclonal mouse sera (pooled per group then diluted 1:250) were preincubated with varying concentrations (0 – 1,000 mg/mL) of selected PfCSP peptides in PBS with 1% BSA/0.05% Tween-20 for 2hrs at 37C, then added onto the rPfCSP- coated plates. Plates were incubated for 1 h at RT, washed five times, then incubated for an additional 1h at RT with 1 μg/mL of appropriate secondary (either anti-human or anti-mouse) IgG SULFO-TAG (Meso Scale Discovery) in PBS with 1% BSA/0.05% Tween-20. After washing, plates were read using 1X MSD Read T Buffer (Meso Scale Discovery) on an MSD SECTOR © Imager 6000 instrument.

Cells were cultured on Matrigel in neural induction medium with or
without DOX. After 24 h, supernatant was collected and cells were scraped in
lysis buffer. WNT3A protein levels were assayed using a Mesoscale Discovery
system (MSD) WNT3A kit following the manufacturer’s protocol. Plates were
analyzed on a SECTOR Imager 6000 instrument with MSD Workbench™ v.3
software. Human WNT3A recombinant protein (R&D Systems) was used as
standard.