4-Aminobutyrate Aminotransferase (ABAT) is a mitochondrial enzyme that catalyzes the conversion of γ-aminobutyric acid (GABA) to succinic semialdehyde in the GABA shunt pathway, playing a crucial role in neurotransmitter metabolism. It maintains GABA homeostasis by regulating its degradation, with dysfunction linked to neurological disorders like epilepsy and autism spectrum disorders. ABAT also participates in cellular energy metabolism through the TCA cycle. ABAT functionally interacts with Glutamate Decarboxylase 2 (GAD2) by forming a metabolic coupling where GAD2 synthesizes GABA from glutamate, and ABAT subsequently degrades it, jointly controlling synaptic GABA levels.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse ABAT and recombinant rat GAD2. Briefly, ABAT was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to GAD2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ABAT 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 mouse ABAT and recombinant rat GAD2 was shown in Figure 1, the EC50 for this effect is 0.03µg/mL.