Development of A First-Responder Fluorescence Reader for Microarray Cytokine Assay of Human Immune Response to Disease
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1004-P03-26
Development of A First-Responder Fluorescence Reader for Microarray Cytokine Assay of Human Immune Response to Disease David B Fenner, David I Rosen, Anthony A Ferrante, Amy E Stevens, Chad E Bigelow, and Steven J Davis Photonics, BioMed and Bio Sciences, Physical Sciences Inc., 20 New England Business Center, Andover, MA, 01810 ABSTRACT Development of field portable apparatus and methods for cytokine assay of human saliva by fluorescent-reporting microarray plates is described. Multiplexed assay of 12 cytokines for minimally-processed saliva is read with a CCD-based imager under LED excitation. Immune responsive cytokines are measured at levels significant for indication of human disease state. The motivational context for the new apparatus development, the general optical design issues, saliva protocol, and image analysis are described. INTRODUCTION The practical utility of technologies for early detection of disease has been limited in many cases by the absence of non-invasive procedures and instruments suitable for first responders and the field hospital. Biomarkers that are in the disease pathway and deployable apparatus are needed to assess adverse health effects in the field. For early stage treatment, detection of disease or injury need not provide specific diagnoses. Hence the potential usefulness of ascertaining immune system imbalance as indicated by endogenous proteins in bodily fluids. Cytokines are a large number of small proteins released by cells into blood and saliva. Some cytokines signal or trigger inflammation in response to infectious challenge, while others indicate stress. A recent study of monkeys infected with the 1918 flu reported greatly elevated blood levels of selected cytokines beginning soon after infection [1]. Saliva is considerably more practical than blood for biomarker analysis and a growing body of evidence supports correlation of patterns in cytokine profiles with various disease, stress and injury states [2,3]. It would appear that cytokine profiles are distinct between viral infection, oral cancer, HIV, and physiological stress in humans [4-9]. Current methods for cytokine assay require full laboratory facilities, such as bead assay with flow cytometry or ELISA with fluorescence microscopy. Bioanalysis with stationary-site sandwich recognition is a technique whereby fluorescent labels report antibody-antigen bound pairs at microarray spots and are scanned or imaged for quantitative assay. Microarrays on a plate with multiple array replicas, each in its own well, provide highly multiplexed assay of a large number of human cytokines. Analysis of blood is the common method but saliva is more readily accessible and able to provide quick indication of immunological challenge. However, protocols for saliva need development [10] and cross-correlation study against blood assay. New methods utilizing saliva and field portable instruments are needed for point-of-care use. We report development of a highly portable system configured with microarray cytokine capture set, dye
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