Development of an enteric pathogen multiplex immunoassay to measure antibody responses in blood and saliva for integrated serology applications.

Background: Enteric infections are a leading cause of global morbidity and mortality, with the highest burden of disease in young children in low- and middle-income countries. Comprehensive profiling of enteric infections, which requires intensive sampling and molecular and culture-based detection methods, may miss recent and historical infections. High-throughput antibody-based testing of blood or non-invasive specimens like saliva can fill this gap, especially in vulnerable populations.

Methods: We developed a bead-based multiplex immunoassay (MIA) that includes enteric pathogen targets from bacteria (Shigella, Enterotoxigenic Escherichia coli [ETEC], Campylobacter, typhoidal Salmonella, non-typhoidal Salmonella); viruses (norovirus, rotavirus, hepatitis E virus, adenovirus, astrovirus); and protozoa (Cryptosporidium, Giardia), as well as select targets for respiratory viruses and vaccine preventable diseases (VPDs). Coupling was optimized for peptide and lipopolysaccharide (LPS) antigens separately and assay performance characterized for both serum and salivary applications.

Results: The ability to assess IgG and IgA antibody responses in serum ranging over 4 orders of magnitude was demonstrated, allowing for assessment of various phases of infection. The mean inter-operator assay precision was excellent (6.7 % CV in serum IgG and 10.6 % CV in saliva IgG). The MIA was highly correlated (r = 0.72-0.96, p < 0.0001) with reference ELISA antibody titers in selected antigens.

Conclusions: Improved scalability of enteric pathogen detection through an MIA may be combined with existing serosurveillance for VPDs and respiratory viruses and complement broad enteric pathogen burden and vaccine studies. This framework for integrated serology provides methods for assessing disease burden to inform prevention and treatment guidelines and guide targeted interventions.