Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2005
Publication Date: 8/10/2005
Citation: Jenkins, M.C., Fetterer, R.H., Schares, G., Bjorkman, C., Mcallister, M., Dubey, J.P. 2005. Hplc purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis. Veterinary Parasitology. 131:227-234. Interpretive Summary: Bovine neosporosis is caused by the protozoan parasite, Neospora caninum. This disease is responsible for the majority of abortions in dairy cattle worldwide. The ability to identify which cattle are infected with N. caninum is the first step in controlling the spread of this disease, and thereby prevent fetal loss in dairy cattle. Using DNA cloning technology, our laboratory has cloned the gene coding for a N. caninum protein, and inserted this gene into harmless Escherichia coli. The E. coli produced large amounts of this N. caninum protein, which was purified by two methods. The secondary purification improved the usefulness of the recombinant N. caninum protein to detect antibodies in serum from cows infected with N. caninum. This improved method of purification should enhance the reliability of the recombinant protein-based serological assay for N. caninum in dairy cattle.
Technical Abstract: The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatograpy. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase high performance liquid chromatograpy (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein- immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.