Synaptosomal Associated Protein 25kDa (SNAP25) is a protein that plays a crucial role in the process of neurotransmitter release at the synaptic cleft. It is a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family, which is essential for the fusion of synaptic vesicles with the presynaptic membrane, leading to the release of neurotransmitters into the synaptic cleft. SNAP25 is specifically located on the plasma membrane of neurons and is involved in the regulation of exocytosis, a fundamental process in synaptic transmission. Syntaxin 1A (STX1A) binds to SNAP25 to form the SNARE complex, which is essential for the release of neurotransmitters.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human SNAP25 and recombinant human STX1A. Briefly, STX1A was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to SNAP25-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-STX1A 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 SNAP25 is lmmobilized at 2 ug/mL(100 uLwell), the concentration of STX1A that produces 50% optimal bindingresponse is found to be approximately 0.026ug/mL.