Tubulin Beta 3 (TUBb3) is a primarily neuron-expressed β-tubulin isotype critical for microtubule dynamics, axonal guidance, and neurodevelopment. As a key cytoskeletal component, it maintains neuronal structural integrity and intracellular transport, serving as a differentiation marker. Aberrant TUBB3 expression correlates with neurological disorders and chemoresistance. Its interaction with kallikrein-related peptidase 5 (KLK5) may modulate KLK5's proteolytic activity in pathological contexts such as tumor metastasis or neural plasticity.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human TUBb3 and recombinant mouse KLK5. Briefly, TUBb3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to KLK5-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TUBb3 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 human TUBb3 and recombinant mouse KLK5 was shown in Figure 1, the EC50?for this effect is 0.03 μg/mL.