U plex assay platform

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.

U-PLEX is a protein multiplex assay which utilizes biotinylated antibodies coupled with proprietary linkers with electrochemiluminescent labels. This allows for detection and quantification of proteins. The U-PLEX Assay platform was purchased from Meso Scale Diagnostics (MSD, Rockville, MD, USA) and used according to the manufacturer’s protocol. Sample preparations and measurements were performed by the Emory Multiplexed Immunoassay Core (EMIC). After 1 h of incubation at room temperature with 5% MesoScale Discovery Blocker A (R93AA-1) and 3 washes with 150 μL PBS/0.05% Tween, 25 μL of brain homogenate or standard were added, and the plate was incubated for 1 h at room temperature, 500 rpm. After 3 PBS Tween washes, 25 μL of secondary antibody was added to each well and the plate was incubated 1 h at room temperature. Finally, 25 μL of streptavidin Sulfo-TAG/well was added after 3 PBS Tween washes, and the plate was incubated 1 h at room temperature. A MesoScale Quickplex Plate Scanner was used for protein quantification. The analytes tested included the following proteins from both the ischemic core and the peri-infarct regions: MCP-1, MIP-1a, MIP-1b, TNF, IL-4, IL-6.