Evaluation of anti-bovine anti-equine and recombinant protein A/G horseradish peroxidase conjugates for cross reactivity to wildlife serum antibodies using ELISA

Abstract

Primary binding assays like the indirect immunofluorescence assay or the enzymelinked immunosorbent assay (ELISA) are serological methods that have been used to effectively screen for many diseases in humans and animals. These assays depend on reagents that can recognise antibodies (immunoglobulins) of the target species. The indirect ELISA for example requires an enzyme conjugated anti-immunoglobulin, which is specific to the target species, also referred to as an anti-species conjugate. Conjugated antispecies immunoglobulins are commercially available for all common domestic species and even for the predominant wildlife species in the northern hemisphere. The African wildlife species are, however, extremely diverse and anti-species immunoglobulins are not readily available, which renders primary binding assays that depend on these conjugated molecule largely unavailable for these species. The purpose of this study was to evaluate the cross-reactivity of commercially available polyvalent anti-bovine IgG: HRP and anti-horse IgG: HRP and binding of recombinant protein A/G: HRP with antibodies in the serum of various herbivore African wildlife species. Serum from 27 herbivore and hoof-stock wildlife species were obtained and a direct ELISA was performed on 10 animals from each species for each of the three selected conjugated molecule. Binding reactions between wildlife serum immunoglobulins and rabbit anti-bovine: HRP, as well as recombinant protein A/G: HRP were expressed relative to the response to bovine serum immunoglobulin and cross-reaction with the antihorse IgG: HRP was expressed relative to equine serum immunoglobulin. A relative affinity index as well as a usefulness index was calculated for each of the conjugated molecules for each of the wildlife species. Thirteen wildlife species performed better than or equal to the bovine control with the recombinant protein A/G: HRP while the rest of the species performed significantly lower than the bovine control. In contrast, the conjugated rabbit anti-bovine IgG: HRP -was found not to be useful for any of the wildlife species tested in this study. The goat anti-horse IgG: HRP was found to bind equivalent to the equine control in four species, however the enzyme-conjugated recombinant protein A/G: HRP performed better in all four these species. The calculation of a usefulness index using the colour change (optical density) in relation to a known homologues control serum and the relative avidity index (a ratio of the measured effect of a chaotropic agent) could become a useful tool for evaluating and comparing the cross-reactivity between conjugated anti-species immunoglobulins or binding of other conjugated molecule with the antibodies in the serum of heterologous species.