Hepatocyte Nuclear Factor 1 Alpha (HNF1a), encoded by the HNF1A gene, is a key transcription factor predominantly expressed in the liver, pancreas, and kidney. It regulates the expression of numerous genes involved in glucose and lipid metabolism, epithelial cell differentiation, and organ homeostasis. Mutations in HNF1A are closely linked to maturity-onset diabetes of the young type 3 (MODY3), a monogenic form of diabetes. HNF1a interacts with FOXA3, a pioneer transcription factor, to synergistically enhance the transcription of target genes related to metabolic processes.Thus a functional ELISA assay was conducted to detect the interaction of recombinant rat HNF1a and recombinant human FOXA3. Briefly, HNF1a was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FOXA3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HNF1a 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 rat HNF1a and recombinant human FOXA3 was shown in Figure 1, the EC50 for this effect is 0.2245µg/mL.