Activity assay kits

The safety of the QβS100A9 vaccine was determined by detecting plasma biomarkers related to liver and kidney injury. For liver damage, we determined the concentrations of the enzymes AST and ALT using the corresponding activity assay kits (Abcam). For kidney damage, we determined the concentration of KIM-1 using the Mouse KIM-1 ELISA Kit (Abcam). Plasma samples collected and tested at weeks 0 and 12 post-vaccination. The plasma samples were pooled from each group and tested in quadruplicate.

Sirt6/Sirt1/Sirt2/Sirt3 activity was tested by Activity Assay Kits (Abcam) according to the manufacturer’s instruction with minor modification. Briefly, compounds of indicated concentrations were added into the reaction system, 2.5 μL 10X buffer + 2.5 μL Fluoro-substrate peptide (0.1 mM) + 0.5 μL NAD⁺ (8 mM) + 2.5 μL Developer + 1 μL recombinant Sirt6/Sirt1/Sirt2/Sirt3, and the mixture was brought up to 25 μL with ddH₂O. Fluorescence intensity was detected using a microplate reader at Ex/Em = 490 nm/530 nm for Sirt6/Sirt2 activity or at Ex/Em = 350 nm/450 nm for Sirt1/Sirt3 activity for 125 minutes. All experiments were conducted at room temperature.