Development of a Multiplex Bead Assay for the Detection of Canine IgG4 Antibody Responses to Guinea Worm

Abstract

Increased levels of guinea worm (GW) disease transmission among dogs in villages along the Chari River in Chad threaten the gains made by the GW Eradication Program. Infected dogs with preemergent worm blisters are difficult to proactively identify. If these dogs are not contained, blisters can burst upon submersion in water, leading to the contamination of the water supply with L1 larvae. Guinea worm antigens previously identified using sera from human dracunculiasis patients were coupled to polystyrene beads for multiplex bead assay analysis of 41 non-endemic (presumed negative) dog sera and 39 sera from GW-positive dogs from Chad. Because commercially available anti-dog IgG secondary antibodies did not perform well in the multiplex assay, dog IgGs were partially purified, and a new anti-dog IgG monoclonal antibody was developed. Using the new 4E3D9 monoclonal secondary antibody, the thioredoxin-like protein 1-glutathione-S-transferase (GST), heat shock protein (HSP1)-GST, and HSP2-GST antigen multiplex assays had sensitivities of 69-74% and specificities of 73-83%. The domain of unknown function protein 148 (DUF148)-GST antigen multiplex assay had a sensitivity of 89.7% and a specificity of 85.4%. When testing samples collected within 1 year of GW emergence (n = 20), the DUF148-GST assay had a sensitivity of 90.0% and a specificity of 97.6% with a receiveroperating characteristic area under the curve of 0.94. Using sera from two experimentally infected dogs, antibodies to GW antigens were detected within 6 months of exposure. Our results suggest that, when used to analyze paired, longitudinal samples collected 1-2 months apart, the DUF148/GST multiplex assay could identify infected dogs 4-8 months before GW emergence.

Guinea Worm Disease: A Neglected Diseases on the Verge of Eradication

“…In consideration of possible cross-reactions with other nematodes, ELISA and western blot detection of IgG4 has been observed to obtain the best sensitivity and specificity [35]. A possible confirmation of these findings can be assumed by recent studies by Priest et al for the realization of multiplex bead assay for seroprevalence assessment among dog populations in endemic areas [36,37]. Nevertheless, serological tests are not included among tools for either seroprevalence assessment or as confirmation tests [38].…”