title: Development of DNA-designed avian IgY antibodies for detection of Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal cattle.
abstract: Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.
authors.affiliation: Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. nikbakht@ut.ac.ir
authors.forename: Gholamreza Nikbakht
authors.initials: GN
authors.lastname: Brujeni
authors.forename: Darioush
authors.initials: D
authors.lastname: Gharibi
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology; D002645:Chickens; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology; D008297:Male; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; C016512:IgY; D007136:Immunoglobulins