A300 688

To determine antibody specificity of mCT monoclonal antibodies, a sandwich ELISA method was employed using the -NT or -CT peptides. Briefly, 100 µl/well of mCT in the concentration of 1:20,000 diluted with coating buffer: (pH = 9.5; 44mM Sodium bicarbonate and 6mM Sodium carbonate) and incubated in an opaque ELISA 96 well plate (Nunc F96 MAXISORP, cat #442,404) for 2 h at room temperature. Following three washes with 0.05% PBS-Tween 20, antibodies were blocked with PBS solution containing 1.5% BSA and 0.05% Tween 20 (blocking solution) for 2 h at room temperature. Next, the wells were washed three times and incubated with 200 ng/mL of -NT or -CT peptides in PBS (Sigma Aldrich) overnight at 4 C°. The following day, after three additional PBS-T washes, either NT-SIRT1 reactive (Millipore #07-131; 1:2000) or CT-SIRT1 reactive (Bethyl Laboratories #A300-688; 1:2000 both in blocking solution) were added (100 µl/well) and incubated 2 h at room temperature then washed four times. Finally, the plate was incubated with a secondary anti-rabbit HRP antibody (Cat# ab7090, Abcam, UK; 1:20,000) for 1 h at 37 C°, washed five times and signal developed using TMB (Cat# 0410 Southern Biotech, AL, US) for 15–30 min at room temperature, followed by 12 M HCL stop buffer. Plates were read using TECAN infinite plate reader and M200PRO software (450 nm).

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.