SIRT1 Fragment Detection by Indirect ELISA

Indirect ELISA was carried out for KJD serum samples to detect NT/CT SIRT1 fragments, according to the method in Batshon et al., [29 (link)]. Diluted serum samples (1:2000) were used against two standard curves in two separate opaque plates; CT reactive standard curve with a full-length human SIRT1 protein ranging from 0.2 to 100 ng/mL (i.e. CT plate); or an NT reactive standard curve with human 75SIRT1 ranging from 0.2 to 100 ng/mL (i.e. NT plate). NT-SIRT1 reactive (Millipore #07-131; 1:2000) or CT-SIRT1 reactive (Bethyl Laboratories #A300-688; 1:2000 both in blocking solution containing PBS with 1.5% BSA and 0.05% Tween 20). Diluted serum (1:2000) and standard curves were allowed to adhere and incubated the next day with blocking for 3 h. After three PBS-T washes samples were incubated with primary antibody overnight. Following three washes, the plate was incubated with a secondary anti-rabbit HRP antibody (Cat# ab7090, Abcam, UK) for 1 h at room temperature and washed five times with PBS-Tween. Substrate signal was developed using TMB (Cat# 0410 Southern Biotech, AL, US) for the NT-plate or SuperSignal ELISA Femto (Thermo Fischer Scientific) kit for the CT plate. Data were analyzed using TECAN infinite M200PRO software (450 nm)and an NT/CT SIRT1 ratio calculated, following standard curve comparisons.