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The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I. 1 and G II. 4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.