Application of salivary antibody immunoassays for the detection of incident infections with Norwalk virus in a group of volunteers

Abstract

Norovirus infection is the most common cause of acute gastroenteritis in developed countries. Developing an assay based on a non-invasive biomarker for detecting incident norovirus infections could improve disease surveillance and epidemiological investigations. This project involved analysis of IgA and IgG norovirus-specific antibody responses in saliva samples from a Norwalk virus (Genogroup I, genotype 1 norovirus) challenge study involving infected and symptomatic, and non-infected asymptomatic individuals. Saliva was collected at the challenge, and two weeks and 40 days post-challenge. Samples were analyzed using the Luminex fluorometric and Meso Scale Discovery (MSD) electrochemiluminescence immunoassays. Recombinant P domains of Norwalk virus capsid protein, as well as similar recombinant proteins of two genogroup II noroviruses (VA387 and VA207) were used as antigens. Immunoconversions were defined as >4-fold increase in antibody responses to the norovirus antigens. Various sample pre-treatment options, buffers, saliva dilution ratios, and data adjustment approaches to control for sample-to-sample variability in saliva composition were compared using the Luminex assay. The results suggest that adjusting responses to the norovirus antigens for responses to the protein purification tag, glutathione-S-transferase (GST), significantly improved the odds of producing a correct immunoconversion test result. IgG-based tests were more accurate compared to IgA-based tests. At optimal conditions, both Luminex and MSD assays for Norwalk-specific IgG antibodies correctly identified all infected and non-infected individuals. There was no evidence of cross-reactivity of anti-Norwalk virus antibodies with genogroup II noroviruses. These results suggest that salivary antibody responses can be used for the detection of incident infections with Norwalk virus in prospective surveys.

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

“…Oral fluid contains saliva (enriched with secre-tory IgA) and crevicular fluid (flows from between the gum margins and teeth) and is enriched with serum antibodies [50]. Some oral fluid sampling techniques are specifically designed to collect samples enriched with crevicular fluid for measurements of systemic antibody responses [51, 52, 53•]. …”

“…Griffin et al (2011) applied the Luminex xMAP microsphere-based technology (Luminex Corp., Austin, TX) assay to measure antibodies to multiple pathogens within a single saliva sample volume [52••]. 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].…”

“…Typically, it is necessary to assay saliva at relatively low dilutions, where matrix effects (e.g., inhibition, high background signal) can be pronounced in some pathogen-specific antibody assays [57]. For each pathogen-specific antibody target, it is critical to optimize the conditions that may influence assay performance and sensitivity and specificity [53•]. …”

“…Multiplex immunoassays, such as those based on the Luminex (Luminex Corp., Austin, TX) microbead suspension fluorescence immunoassay platform, require a low sample volume to analyze multiple pathogen-specific antibody analytes simultaneously. They are also less labor intensive because more data are generated per test/analyte and thus are more cost-effective [52••, 53•, 66–69]. Another immunoassay platform that is used and allows multiplexing is the Meso Scale Discovery (MSD; Rockville, M D) electrochemiluminescence (ECL) platform.…”

“…Platforms that facilitate multiplexing can be used to expand the range of available options for testing the signal of pathogen-specific antibody responses as well as background signals. The adjustment of the pathogen-specific antibody signal for background signals, such as those produced by total IgA or total IgG or by antigen tags such as glutathione-S-transferase (used during antigen purification), can improve the performance of antibody assays [52••, 53•]. Multiplexing of these target signals can also reduce excess use of biospecimen sample volume because all signals can be measured in one sample volume in a single reaction well.…”