Acid phosphatase locus 1 (ACP1) is a low molecular weight protein tyrosine phosphatase that has been shown to be an important regulator of insulin receptor signaling. EFNA1 is a kind of receptor tyrosine kinase which can Interact with ACP1. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNA1 and recombinant human ACP1. Briefly, EFNA1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ACP1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EFNA1 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 450/630 nm immediately. When recombinant human ACP1 is immobilized at 2 ug/ml (100 ul/well), the concentration of EFNA1 that produces 50% optimal binding response is found to be approximately 0.21 ug/ml.