Defensin Alpha 3, Neutrophil Specific (DEFa3) is a cationic antimicrobial peptide belonging to the alpha-defensin family, primarily expressed in neutrophils and epithelial tissues. It exhibits potent broad-spectrum activity against Gram-positive/negative bacteria, fungi, and enveloped viruses by disrupting microbial membranes. Beyond direct pathogen clearance, DEFa3 modulates immune signaling and enhances phagocyte function, reinforcing host innate immunity. DEFa3 binds with DEFa4 (DEF4), forming heterodimers to synergistically amplify antimicrobial and immunomodulatory effects. Briefly, DEFa3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to DEFa4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-DEFa3 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.Measured by its binding ability in a functional ELISA. When Recombinant DEFa4 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of DEFa3 that produces 50% optimal bindingresponse is found to be approximately 1.10488 µg/mL.