Citation:

Augustine, S., T. Eason, K. Simmons, C. Curioso, S. Griffin, M. Ramudit, AND T. Plunkett. Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens. Journal of Visualized Experiments . JoVE, Somerville, MA, 115:e54415, (2016).

Impact/Purpose:

This article presents a method to measure the presence of human salivary antibodies to multiple environmental pathogens simultaneously. This method is capable of achieving more data with less sample; reduced costs and labor; and can be customized to measure many targets of interest. Furthermore, this assay will be used to measure antibody responses in swimming related exposures and infections and to develop risk assessment models.

Description:

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. The principal objective of this work is to develop an 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 the Luminex xMAP solution-phase assay. Beads were coupled to 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 detection 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, analyzed on a Luminex 100 platform, 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 Luminex assay may be capable of identifying previous exposures and infections which can be especially useful in surveillance studies involving large human populations.

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