Increased oxidative stress and inflammatory responses are common effects of exposure to environmental agents. There are many putative protein markers of environmental insult, although there is limited knowledge of the usefulness of these proteins for monitoring low-dose exposures in humans. In addition to putative protein markers, the proteomic and DNA microarray analyses proposed in Projects 1 and 2 are expected to identify new candidate biomarkers for exposure to side-stream smoke. The purpose of this ELISA microarray core is to evaluate the usefulness of candidate protein biomarkers for assessing environmental exposures. For Project 1, we will screen -50 proteins in -120 human plasma samples to identify proteins that are altered in response to cigarette smoke. Although most of these analyses will be undertaken using commercial reagents, this core will also develop sandwich ELISA's for up to 10 biomarkers for which commercial assays are unavailable. For Project 2, a subset (-20) of the proteins screened in humans will be analyzed in mice exposed to cigarette smoke, lipopolysaccharide, CCI4 or paraquat. The mouse data will identify markers that show selectivity for cigarette smoke exposure. The data for known markers will also provide baseline information for comparison with novel biomarkers such as nitrated proteins identified in this U54 program. Since the ELISA microarray format is a highly efficient system for evaluating multiple affinity reagents, once we have identified the best candidate biomarkers of exposure, we will screen over 500 human plasma samples using the most promising markers identified so far. Since the on-site multiplexed sensor that will be developed in Project #3 is limited to 4 or 5 assays, these ELISA microarray tests are essential to winnow down the number of candidate biomarkers and thereby identify the most useful set of proteins for this instrument. Furthermore, since ELISA analyses are routinely used as a quantitative clinical test, any ELISA's we develop can be directly translated to a standard clinical test. Therefore, this core will facilitate the develop of routine clinical tests and of a on-site sensor for evaluating exposure to environmental stimuli such as side-stream cigarette smoke.
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