Figure. The binding activity of ITIH4 with Grb2.
Inter Alpha-Globulin Inhibitor H4 (ITIH4) is secreted into the blood, where it is cleaved by plasma kallikrein into two smaller forms. ITIH4 has been detected only in liver, and it seems to be upregulated during surgical trauma. It may also play a role in liver development or regeneration. Besides, Growth Factor Receptor Bound Protein 2 (Grb2) has been identified as an interactor of ITIH4, thus a binding ELISA assay was conducted to detect the interaction of recombinant human ITIH4 and recombinant human Grb2. Briefly, ITIH4 were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to
Grb2-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ITIH4 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 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 ITIH4 and Grb2 was shown in Figure 1, and this effect was in a dose dependent manner.