Quantifying Plasma Proteins in A-T

Plasma samples from a subset of individuals with classic A-T (n = 28), mild A-T (n = 3), and unaffected healthy controls (n = 3) were collected under IRB protocol numbers IRB 20-017524 from Children’s Hospital of Philadelphia and NA_00051764 from Johns Hopkins School of Medicine. We used Abcam in vitro SimpleStep ELISA kits (human gp130 (ab246548), Annexin A2 (ab264612), PAI1 (ab269373), and VEGFR3 (ab252350)) to quantify the concentration of plasma proteins of interest. Kits were used according to their individual standard issued protocols. Standard curve dilutions were created from the protein stock provided in the kits and following recommended dilutions. Plasma samples were then diluted 1:50 (PAI-1, VEGFR3) or 1:1000 (gp130). Samples were incubated on a pre-coated, 96-well antibody plate with antibody cocktail, shaking for one hour. Wells were washed three times and then incubated for a maximum of 10 min with development solution while wrapped in metal foil. Stop solution was immediately added and optical density at 450 nm was read on a Multiskan SkyHigh Microplate Spectrophotometer. A linear standard curve was fitted and used to calculate the protein concentration in each sample.