Lonized Calcium-binding Adapter Molecule 1 (IBA1), a 17-kDa EF-hand calcium-binding protein, serves as a gold-standard marker for microglia in the central nervous system (CNS). Predominantly expressed in microglia and marginally in peripheral macrophages, it mediates calcium-dependent cytoskeletal reorganization, governing microglial migration, phagocytosis of damaged neurons, and pro-inflammatory cytokine secretion. This protein is pivotal for CNS immune surveillance and homeostasis; its elevated expression is tightly linked to microglial activation in neuroinflammatory conditions and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Additionally, IBA1 coordinates with actin-regulatory proteins to modulate microglial morphological transitions during immune responses. IBA1 interacts with ARPC4 to facilitate actin polymerization and modulate microglial migratory capacity in neuroinflammation.Thus a functional ELISA assay was conducted to detect the interaction of recombinant IBA1 and recombinant ARPC4. Briefly, IBA1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ARPC4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IBA1 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 IBA1 and recombinant ARPC4 was shown in Figure 1, the EC50?for this effect is 17.38μg/mL.