Puromycin Sensitive Aminopeptidase (PSA) also known as cytosol alanyl aminopeptidase or alanine aminopeptidase (AAP) is used as a biomarker to detect damage to the kidneys, and that may be used to help diagnose certain kidney disorders. It is found at high levels in the urine when there are kidney problems. PSA has been proposed to function in a variety of processes, including metabolism of neuropeptidase, regulation of the cell cycle, and hydrolysis of proteasomal products to amino acids. Besides, Protein Disulfide Isomerase A3 (PDIA3) has been identified as an interactor of PSA, thus a binding ELISA assay was conducted to detect the interaction of recombinant human PSA and recombinant human PDIA3. Briefly, PSA were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to PDIA3-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PSA 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 PSA and PDIA3 was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of PSA with PDIA3