Blood samples were collected weekly prior to treatment and levels of IgG AAb were measured using an ELISA developed in our laboratory (Fig. 1C). Briefly, NUNC maxisorp plates (ThermoFisher) were coated with rHSP25 at a concentration of 500 ng/well in carbonatebicarbonate buffer overnight (ON) at room temperature. The wells were blocked with 1% BSA/PBST, washed in phosphate buffered saline tween (PBST), and incubated with plasma at a final dilution of 1:2,000 in 1% BSA for two hrs followed by 3 more washes in PBST. A horse radish peroxidase (HRP) labeled goat anti-mouse IgG (H&L) antibody (catalogue #115-035-062; Jackson Immunoresearch, West Grove, PA) was used as a detection antibody at a dilution of 1:5,000 and incubated for one hr at room temperature. Finally, substrate solution (3,3',3.5'-Tetramethylbenzidine Liquid Substrate, TMB; Millipore Sigma; Oakville, ON) was added to each well and incubated for 10 mins avoiding direct light. The reaction was stopped by 2N H 2 SO4 and the optical density quantified at 450 nm, using Synergy Mx plate reader (BioTek).
To establish an internal standard for the measurement of HSP25 auto-antibodies, plasma from a healthy control subject was diluted 500 times in 1% BSA/PBST and arbitrarily defined as 50 absorbance units (a.u.).
To establish an internal standard for the measurement of HSP25 auto-antibodies, plasma from a healthy control subject was diluted 500 times in 1% BSA/PBST and arbitrarily defined as 50 absorbance units (a.u.).