Aptamer-capture based assays for human neutrophil elastase

Human neutrophil elastase (HNE) is a multifunctional serine protease, involved in infection defense, inflammatory process regulation, and physiopathological processes of several diseases. We developed aptamer-capture based assays for human neutrophil elastase with different substrates and solid supports to meet different demands, such as simplicity, sensitivity, and high throughput. Aptamers against HNE were immobilized on magnetic beads or microplates as affinity ligands to capture HNE, and then the enriched HNE catalyzed the conversion of chromogenic substrates or fluorogenic substrates to products. The measurement of the generated enzymatic products enabled the final detection of HNE. In the assay using chromogenic substrates and aptamer modified magnetic beads, 0.4 pM HNE could be successfully detected. The sensitivity of the assay was further improved by using fluorogenic substrates, and a detection limit of HNE at 20 fM was achieved. The use of aptamer-coated microplates instead of aptamer modified magnetic beads in the assays also allowed the sensitive detection of HNE, offering advantages in fast sample handling and measurement. The established assays for HNE displayed good specificity, and proteins including serum albumin, transferrin, immunoglobulin G, thrombin, porcine pancreatic elastase, trypsin, proteinase K, chymotrypsin, lysozyme, cathepsin G, and proteinase 3 did not cause interference in the detection of HNE.