Lysyl-tRNA Synthetase (KARS)?is an essential enzyme that catalyzes the attachment of lysine to its cognate tRNA during protein synthesis, ensuring accurate translation of the genetic code. As a member of the aminoacyl-tRNA synthetase (aaRS) family, KARS plays a critical role in maintaining cellular protein homeostasis. Beyond its canonical function, KARS exhibits non-canonical roles in immune signaling, angiogenesis, and viral defense. It exists in both cytoplasmic and mitochondrial forms, contributing to protein synthesis in multiple cellular compartments.?KARS binds to AIMP1 as part of the multi-tRNA synthetase complex (MSC), where this interaction helps regulate enzyme activity and coordinate cellular processes related to protein synthesis.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human KARS and recombinant mouse AIMP1. Briefly, KARS was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to AIMP1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-KARS pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant human KARS and recombinant mouse AIMP1 was shown in Figure 1, the EC50?for this effect is 0.015ug/mL.