Filaggrin (FLG), a key keratinocyte differentiation protein encoded by the?FLG?gene, is critical for skin barrier formation. Synthesized as a large precursor, profilaggrin, it is cleaved into functional filaggrin monomers in the stratum granulosum of the epidermis. These monomers bind to keratin filaments, promoting their aggregation to form a dense, protective cytoskeletal structure. Filaggrin also contributes to skin hydration by breaking down into natural moisturizing factors. FLG interacts with SPINK5, which encodes the serine protease inhibitor LEKTI; SPINK5 regulates proteases that process profilaggrin into filaggrin, maintaining proper barrier function.Briefly, SPINK5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FLG-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-SPINK5 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 FLG is lmmobilized at 2 μg/mL(100 uLwell), the concentration of SPINK5 that produces 50% optimal bindingresponse is found to be approximately11.830 μg/mL.