Novel methods for point-of-care diagnosis of nerve agent exposure (Abstract)

Methods to unequivocally and rapidly assess exposure to nerve agents are highly valuable from a military and security perspective. Within this framework we currently follow two different approaches towards rapid point-of-care diagnosis. Regarding the first approach we hypothesized that proteins in the skin might represent an ideal source of persistent biomarkers for exposure to organophosphates, since the skin is a predominant route of entry for various nerve agents. Upon exposure of human callus to VX and various organophosphate pesticides, LC-tandem MS analysis of pronase and trypsin digests of isolated keratin proteins demonstrated that tyrosine and to a lesser extent serine residues had been selectively modified. These results will form a firm basis for the development of non-invasive methods, e.g. based on tapestripping, for assessment of exposure to organophosphates. In the second approach, the development of a generic and rapid point-of-care immunochemical method for assessment of nerve agent exposure is pursued. The first step in this assay consists of the selective removal of native, non-modified HuBuChE with a Sepharose-based solid phase containing a reactive, immobilized OP moiety. Already inhibited HuBuChE will not be affected by this treatment and is subsequently detected by using commercially available, relatively inexpensive anti-HuBuChE antibodies in a competitive ELISA. In its current form, the assay is compatible with human plasma. It is envisaged that the design of the assay is easily adaptable to a dipstick-type format.