title: Development of an indirect ELISA for detection of E. Coli antibodies in cow serum using a recombinant OmpT as antigen.
abstract: The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 μg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT was E. coli specific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection of E. coli with different serotype strains.
authors.affiliation: a School of Life Science & Biotechnology , Heilongjiang Bayi Agricultural University , Heilongjiang Province , People's Republic of China.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Tong
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Wu
authors.forename: Liquan
authors.initials: L
authors.lastname: Yu
authors.forename: Ziyao
authors.initials: Z
authors.lastname: Fan
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Rui
authors.initials: R
authors.lastname: Hu
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Ma
authors.forename: Baifen
authors.initials: B
authors.lastname: Song
authors.forename: Zhanbo
authors.initials: Z
authors.lastname: Zhu
authors.forename: Yudong
authors.initials: Y
authors.lastname: Cui
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000097:blood / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D015870:Gene Expression; D006735:Horseradish Peroxidase / Q000737:chemistry; D008414:Mastitis, Bovine / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D010447:Peptide Hydrolases / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C499588:ompT protein, E coli; D006735:Horseradish Peroxidase; D010447:Peptide Hydrolases