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Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 8/31/2004 Publication Date: 9/30/2004 Citation: Jenkins, M.C. 2004. Use of recombinant antigen in serodiagnosis of protozoan infection: advantages and limitations in diagnosing bovine neosporosis. Meeting Abstract. Interpretive Summary: Technical Abstract: Researchers have utilized DNA cloning to produce recombinant parasite proteins in both prokaryotic and eukaryotic hosts as a means of producing large quantities of specific antigen for sensitive enzyme-linked immunosorbent assay (ELISA) or immunoblot (IB) assay. This approach has also been used to overcome the well-documented cross-reactivity between native proteins of closely related protozoa that are capable of infecting a single host species. Although using one to several recombinant proteins will in theory increase the sensitivity and specificity of a serological assay, care must be taken in the preparation of the antigen to ensure the nearly complete absence of contaminating non-recombinant protein. In our studies, an unacceptably high background binding of serum from known Neospora caninum-negative cows has been observed with NiNTA-affinity purified recombinant NcGRA6 protein expressed as histidine-tagged protein Escherichia coli. Analysis of 'purified' NcGRA6 protein revealed several co-purifying E. coli proteins that avidly bound to the NiNTA agarose and eluted with recombinant His-tagged protein. A secondary reverse-phase HPLC separation of the NcGRA6 eluates fractionated the recombinant N. caninum protein from contaminating E. coli proteins. In ELISA, HPLC-purified NcGRA6 exhibited a high signal:noise ratio and greater agreement with ISCOM ELISA compared to NiNTA-purified NcGRA6 when used to assay sera from cattle with confirmed N. caninum infection. The usefulness of the 'improved' NcGRA6 ELISA compared to other serological assays for N. caninum infection will be discussed. |
