Additional file 1: Figure S1. Schematic of the Development of our Biotinylated TDP-43 Phage Capture ELISA System. A schematic of the entire process utilized to develop our biotinylated TDP-43 phage-capture ELISA system for heightened detection of TDP-43 variants is shown. Stage 1A—Previously described AFM based negative and positive biopanning protocol to isolate the capture scFvs reactive with TDP-43 variants isolated from ALS brain tissue . Starting with an initial scFv library, we first eliminated scFvs reactive with undesired targets including BSA, aggregated alpha-synuclein and healthy human brain tissue using immunotubes. AFM analysis was utilized to monitor the process. We next completed negative panning against TDP-43 immunoprecipitated from healthy and FTD brain tissue (motor cortex) using a mica surface to conserve limited sample availability. The remaining phages were then utilized in positive biopanning against TDP-43 immunoprecipitated from ALS brain tissue. Any eluted phages following this process should be specific for ALS related TDP-43 variants. Stage 1B—To isolate a detection phage that is reactive with all forms of TDP-43 we utilized the AFM biopanning process described in Fig. 1. Stage 2—Both the potential ALS TDP-43 reactive phages isolated in the previous study and the detection phages acquired in the current study were first screened in indirect ELISAs against ALS, FTD and healthy brain tissue. Any bound phage particles were detected with an anti-M13-HRP secondary antibody. ALS TDP-43 specific phages should generate signals only in the wells containing ALS tissue, whilst the detection phages should produce signals with all three tissue types. Stage 3 - Finally, following biotinylation of the 2700 coat protein on our detection phage to heighten our sensitivity, our complete phage capture ELISA system was used to analyze individual human brain tissue and plasma samples. Some of the utilized illustrations were adapted from previously published graphics [32, 38].
|Date made available||Jan 25 2017|
|Publisher||figshare Academic Research System|