Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

Abstract

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.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

“…To help determine immunoprevalence, researchers have used a variety of methods including three times the mean (77) and mean plus 3 times the standard deviation (SD) of the control beads (16, 78). Cutoff point criteria 1 (CC1) is defined as the mean plus 3 SDs (μ + 3σ) of the control beads.…”

Rapid Salivary IgG Antibody Screening for Hepatitis A

“…Antigen coupling and confirmation using animal-derived antibodies. Beads were activated and coupled, as previously described, and serial dilutions of primary capture antibodies were used to confirm that the beads were coupled properly, thus ensuring that the dynamic range of the assay could be defined (30,31). Briefly, coupled bead stocks were diluted in phosphate-buffered saline, pH 7.4, with 1% bovine serum albumin (PBS-BSA) to a final concentration of 100 beads/l.…”

“…Beads (5 ϫ 10 3 ) from each bead set and an equal volume of diluted saliva were loaded onto each well, resulting in a final dilution of 1:8 in a total volume of 100 l per well. The loaded filter plates were processed as previously described, and reporter fluorescence was measured using a Luminex 100 analyzer and expressed as median fluorescence intensity (MFI) of at least 100 beads per bead set (30,31). MFI readings are produced for every sample and serve as a proxy for antibodies present against the targeted pathogens.…”

“…It has emerging applications in research and clinical settings, and in fact, several studies have shown the efficacy of salivary antibodies as biomarkers of hepatitis A virus infections (27)(28)(29). Our team developed a bead-based, multiplex salivary antibody immunoassay to measure the prevalence of antibodies to multiple waterborne pathogens associated with drinking and recreational water contamination simultaneously (30,31). Application of the assay has allowed us to measure immunoprevalence (32), immunoconversions (incident infections), coinfections (33), and asymptomatic infections (34) from exposure to various waterborne pathogens in visitors to Boquerón Beach, Puerto Rico.…”

Use of Pathogen-Specific Antibody Biomarkers to Estimate Waterborne Infections in Population-Based Settings

“…The Norwalk virus assay developed in Griffin et al (2011) was subsequently validated using samples from a human volunteer challenge study [53•]. A similar salivary immunoassay is being applied to measure the incidence of norovirus infections following recreational water exposures at beaches in Puerto Rico, Iowa, and Wisconsin where saliva has been collected as part of the Environmental Protection Agency's National Epidemiologic and Environmental Assessment of Recreational Water Study [55]. …”