Development of conjugated secondary antibodies for wildlife disease surveillance

Abstract

Disease monitoring in free-ranging wildlife is a challenge and often relies on passive surveillance. Alternatively, proactive surveillance that relies on the detection of specific antibodies could give more reliable and timely insight into disease presence and prevalence in a population, especially if the evidence of disease occurs below detection thresholds for passive surveillance. Primary binding assays, like the indirect ELISA for antibody detection in wildlife, are hampered by a lack of species-specific conjugates. In this study, we developed anti-kudu (Tragelaphus strepsiceros) and anti-impala (Aepyceros melampus) immunoglobulin-specific conjugates in chickens and compared them to the binding of commercially available protein-G and protein-AG conjugates, using an ELISA-based avidity index. The conjugates were evaluated for cross-reaction with sera from other wild herbivores to assess future use in ELISAs. The developed conjugates had a high avidity of >70% against kudu and impala sera. The commercial conjugates (protein-G and protein-AG) had significantly low relative avidity (<20%) against these species. Eighteen other wildlife species demonstrated cross-reactivity with a mean relative avidity of >50% with the impala and kudu conjugates and <40% with the commercial conjugates. These results demonstrate that species-specific conjugates are important tools for the development and validation of immunoassays in wildlife and for the surveillance of zoonotic agents along the livestock-wildlife-human interface.

Keywords: adaptive immunity; avidity; conjugates; diagnostics; enzyme-linked immunosorbent assay (ELISA); passive disease surveillance; wildlife species.

Figure 1

Images showing the required molecular weights of kudu and impala IgG as well as anti-kudu and anti-impala IgY binding to their respective IgG. (A) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) image from the ammonium sulfate precipitated immunoglobulin fractions from kudu and impala sera. The protein bands at 50 and 25 KDa correspond to the heavy and light chains of IgG. Kudu serum was used as the serum control. (B) Western blot image indicating the binding of impala immunoglobulin G (IgG) to the chicken anti-impala IgY directly from theammonium sulfate precipitated egg yolk without affinity chromatography before conjugation. (C) Western blot image indicating the binding of kudu IgG to the chicken anti-kudu IgY directly from the ammonium sulfate precipitated egg yolk without affinity chromatography before conjugation. (D) Western blot image showing binding of impala (left) and kudu (right) IgG against the corresponding chicken affinity-purified IgY before conjugation. Red arrows with solid rectangles highlight the molecular weight of interest.

Figure 2

Bar charts with error bars (standard deviation) showing the differences in mean optical densities (OD) for the developed and commercial conjugates: (A) impala sera against anti-impala IgY, protein AG, and protein G conjugates, and (B) kudu sera against anti-kudu IgY, protein AG and protein G conjugates. The red bars represent wells without the chaotrope and the blue bars represent wells that received dissociating chaotrope. For each species, 10 replicates were used for the experiments.

Figure 3

Scatter plot with error bars (standard deviation) showing the avidity index for each of the conjugates (red anti-impala, blue anti-kudu, yellow protein AG, and green protein G) determined for different wildlife species (Table 1). The avidity between the conjugate and different sera was calculated as the reduction in color between wells without a chaotropic agent (CT) and those with CT and presented as the AI for each serum. The silhouettes in color connect species and conjugate colors to denote the species used as a control for each conjugate: impala for anti-impala IgY, kudu for anti-kudu IgY, cattle for protein AG, and goat for protein G. The horizontal dotted lines indicate the avidity index of the respective controls, and the colors correspond to the conjugates. Species were grouped into subfamilies as described by Hassanin, et al. (38); however, ordering of the species was not done by phylogenetic relationships.

Figure 4

Box plots showing the avidity index for the wildlife species grouped by (A) the tribe they belong to and (B) their subfamilies. These species were classified based on the work described by Hassanin, et al. (38) and Gatesy, et al. (39). Red indicates anti-impala IgY and pink is anti-kudu IgY conjugate.