کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
8991111 1555806 2005 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک علوم دامی و جانورشناسی
پیش نمایش صفحه اول مقاله
HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis
چکیده انگلیسی
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 chromatography. 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 relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (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 relative 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Veterinary Parasitology - Volume 131, Issues 3–4, 10 August 2005, Pages 227-234
نویسندگان
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