Active Apolipoprotein A4 (APOA4)

Apolipoprotein A4 (APOA4) is a lipid-binding protein, which is primarily synthesized in the small intestine, packaged into chylomicrons, and secreted into intestinal lymph during fat absorption. In the circulation, apoA-IV is present on chylomicron remnants, high-density lipoproteins, and also in lipid-free form. ApoA-IV is involved in a myriad of physiological processes such as lipid absorption and metabolism, anti-atherosclerosis, platelet aggregation and thrombosis, glucose homeostasis and food intake. Besides, Apolipoprotein H (APOH) has been identified as an interactor of APOA4, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human APOA4 and recombinant bovine APOH. Briefly, APOA4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to APOH-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-APOA4 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 450/630 nm immediately. The binding activity of recombinant human APOA4 and recombinant bovine APOH was shown in Figure 1, the EC50 for this effect is 0.16 ug/mL.