Pregnancy-associated plasma protein A (PAPPA), also known as pappalysin-1, is a secreted protease whose main substrate is insulin-like growth factor binding proteins. PAPPA's proteolytic function is activated upon collagen binding. It is thought to be involved in local proliferative processes such as wound healing and bone remodeling. Low plasma level of this protein has been suggested as a biochemical marker for pregnancies with aneuploid fetuses (fetuses with an abnormal number of chromosomes). Besides, Plasminogen (Plg) has been identified as an interactor of PAPPA, thus a binding ELISA assay was conducted to detect the interaction of recombinant human PAPPA and recombinant human Plg.
Briefly, PAPPA were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to Plg-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PAPPA 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 PAPPA and Plg was shown in Figure 1, and this effect was in a dose dependent manner.