Lack of serologic assays validated for non-domestic hoofstock species (Order Artiodactyla) makes diagnosis and control of infectious diseases challenging. As one example, diagnosis of Johne's disease in captive and free-ranging artiodactylids is limited to culturing the organism from tissue or feces. This procedure is slow and costly. However, the need for a labeled, species-specific secondary antibody (conjugate)precludes use of an ELISA assay to detect antibody in animals as diverse as artiodactylids. Protein G reportedly binds IgG in most mammals to varying degrees and thus may potentially serve as a universal conjugate. A Johne's disease ELISA with a conjugate capable of recognizing antibody from a multitude of species was investigated for use in artiodactyl. Twelve species representing three families within Order Artiodactyla were included (addax, antelope, bison, bontebok, kudu/nyala, llama, oryx, sheep, white-tail deer, elk, muntjac, impala), plus bovine (positive control) and chicken (negative control). A protocol for immunoglobulin (Ig) enrichment from serum was optimized in bovids and utilized for all other species. Binding assays were performed to assess the ability of protein G to bind Ig from artiodactyl species. A prototype ELISA assay for the detection of antibodies to Mycobacterium paratuberculosis in artiodactyl species, using the protein G conjugate, was developed. Sera from animals with documented paratuberculosis infections and from animals considered free of paratuberculosis infection were obtained. The prototype artiodactyl protein G ELISA was able to distinguish among samples containing M. paratuberculosis antibodies and those that did not. With further assay optimization and use of appropriate species' controls, ELISA OD cutoffs to classify negative and positive serum samples may be determined. Characterization of the binding ability of protein G to Ig of these species indicates its potential use as a conjugate in ELISAs for any disease, regardless of the causative organism (solid phase antigen).