Glucosidase Alpha, Neutral AB (GaNAB) is an enzyme in the glycoside hydrolase family. It hydrolyzes alpha - glucosidic bonds, participates in carbohydrate metabolism, and is involved in endoplasmic reticulum quality control. GaNAB binds to Heat Shock 70kDa Protein 5 (HSPA5), collaborating to ensure proper glycoprotein folding and maintain cellular protein homeostasis.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GaNAB and recombinant mouse HSPA5 .Briefly, biotin-linked GaNAB were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to HSPA5-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 GaNAB and HSPA5 was shown in Figure 1, the EC50?for this effect is 3.21ug/mL.