Prion Protein (PRNP) is a membrane-anchored glycoprotein highly expressed in the central nervous system. In its native conformation, this protein participates in important physiological processes including neuronal development, synaptic function, and copper ion binding. The unique aspect of PRNP lies in its ability to undergo conformational changes into a pathogenic form that causes neurodegenerative disorders known as transmissible spongiform encephalopathies.These diseases,which include Creutzfeldt-Jakob disease in humans and mad cow disease in cattle, are characterized by progressive brain damage and sponge-like tissue degeneration. Stress Induced Phosphoprotein 1 (STIP1) binds PRNP through direct physical interaction, modulating PRNP's neuroprotective functions and potentially influencing prion disease pathogenesis.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human STIP1 and recombinant human PRNP. Briefly, STIP1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PRNP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-STIP1 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 PRNP is lmmobilized at 2 ug/mL(100 uLwell), the concentration of STIP1 that produces 50% optimal bindingresponse is found to be approximately 0.79ug/mL.