Development of sandwich ELISA for detection and quantification of human and murine a disintegrin and metalloproteinase17

Abstract

ADAM17 (a disintegrin and metalloproteinase-containing protein 17) is a membrane-bound metalloproteinase, implicates in many physiological processes, including cell migration and proliferation. Of particular note, most of the studies so far are restricted on the analysis of ADAM17 mRNA levels. In this study we generated, utilizing hybridoma technology, three monoclonal antibodies (mAbs) (A 300, A 309 and A 318) against the extracellular domain of human ADAM17 to enable quantification of protein expression. The specificity of these mAbs against ADAM17 was tested by enzyme-linked immunoadsorbent assay (ELISA), flourescence-activated cell sorting (FACS) and western blotting. In order to quantify human and murine ADAM17 expression two pairs of these mAbs (biotinylated A 309 in combination with A 300 and biotinylated A 300 in combination with A 318), were used to develop sandwich ELISA. A panel of monoclonal antibodies was generated for first time to measure mouse ADAM17 with a sensitivty of 2 ng/ml. Such systems provide a useful tool to quantify protein levels of ADAM17 and are valuable tools for diagnostic purposes.