Phospholipase C Delta 4 (PLCd4) is a specialized phospholipid-hydrolyzing enzyme that catalyzes PIP₂cleavage to generate secondary messengers which activate the inositol trisphosphate receptor (ITPR1), triggering calcium release from the endoplasmic reticulum. This functional coupling establishes PLCd4 as a central regulator of calcium signaling pathways, with particularly crucial roles in mammalian reproduction. Its testis-specific expression drives the calcium oscillations essential for egg activation during fertilization, and complete PLCd4 deficiency results in male infertility through profound sperm functional defects. Beyond reproduction, emerging evidence implicates PLCd4 in diverse physiological and pathological processes including cancer progression and cellular differentiation. The enzyme's activity is tightly regulated through conserved structural domains, enabling its precise integration into complex calcium-dependent signaling networks that underscore its multifaceted cellular functions.To assess the biological activity of PLCd4, a functional binding ELISA was performed to evaluate the interaction between recombinant human PLCd4 and recombinant human ITPR1 . Briefly, biotin-linked PLCd4 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to ITPR1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of PLCd4 and ITPR1 was shown in Figure 1, the EC50 for this effect is 0.11µg/mL.