title: Incorporation of antigens from Mannheimia haemolytica culture supernatant, and recombinant bovine C3d into ISCOM matrix using neutravidin-biotin interaction.
abstract: The aim of this study was to incorporate antigens from Mannheimia haemolytica culture supernatant, and an immune modulatory molecule, recombinant bovine C3d (rBoC3d), into immune stimulating complexes (ISCOMs) using neutravidin-biotin interaction. Biotinylated ISCOM matrix was generated using a commercial kit. The biotinylated ISCOM matrix was incubated with neutravidin and then centrifuged in a sucrose density gradient. The rBoC3d was expressed as an in vivo biotinylated protein and with a c-Myc tag (EQKLISEEDL) engineered to facilitate detection. The neutravidin-coated ISCOM matrix was incubated with biotinylated antigens from M. haemolytica culture supernatants and rBoC3d. To test the association among the neutravidin-coated ISCOM matrix, biotinylated antigens and rBoC3d, an analytical sucrose density gradient (10-40%, w/w) was performed. The experimental formulations were run in SDS-PAGE gels under reducing conditions. For Western immunoblot analysis, polyclonal bovine antiavidin, monoclonal anti-c-Myc, monoclonal antileukotoxin, and anti-GS60 antibodies were used to detect the presence of neutravidin, rBoC3d, leukotoxin, and GS60 antigens, respectively. By taking advantage of the biotin-neutravidin interaction, not only leukotoxin but also the recombinant immunomodulatory molecule, rBoC3d, was incorporated into ISCOM particles.
authors.affiliation: CONICET, Rivadavia, Buenos Aires, Argentina. pmoore@balcarce.inta.gov.ar
authors.forename: D P
authors.initials: DP
authors.lastname: Moore
authors.forename: D C
authors.initials: DC
authors.lastname: Hodgins
authors.forename: M A
authors.initials: MA
authors.lastname: Firth
authors.forename: B A
authors.initials: BA
authors.lastname: McBey
authors.forename: P E
authors.initials: PE
authors.lastname: Shewen
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology* / Q000302:isolation & purification; D001360:Avidin / Q000378:metabolism*; D001709:Biotechnology; D001710:Biotin / Q000378:metabolism*; D002417:Cattle; D015933:Complement C3d / Q000276:immunology*; D003470:Culture Media / Q000737:chemistry*; D016882:ISCOMs / Q000276:immunology*; D016978:Mannheimia haemolytica / Q000166:cytology / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D003470:Culture Media; D016882:ISCOMs; D011994:Recombinant Proteins; C093483:neutravidin; D001360:Avidin; D001710:Biotin; D015933:Complement C3d