title: Preparative separation of foreign antigens for highly efficient presentation to T cells in vitro.
abstract: A method is described for the separation and purification of proteins from complex mixtures of foreign antigens in a form suitable for stimulating T cells in vitro. The technique involves electrophoretic separation of proteins followed by elution, concentration and adsorption of the polypeptide subunits to latex microspheres. Alternatively, where a specific antibody is available, proteins may be affinity-purified from a heterogeneous mixture of antigens, using antibody-coated latex microspheres. Nanogram quantities of protein coupled to latex were shown to be highly efficient stimulators of antigen-specific T cells as tested by in vitro proliferation and cytokine release assays. The utility of this technique was demonstrated using poliovirus capsid proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and coupled to latex microspheres for specificity analysis of T cell clones. Antigen reactivity of the T cell clones was confirmed using recombinant baculoviruses expressing individual poliovirus proteins. Furthermore, recombinant proteins coupled to latex microspheres were used for efficient stimulation and in vitro propagation of T cell clones specific for the simian immunodeficiency virus (SIV) envelope (env) protein. Although the technique is illustrated in this report using viral antigens, it has also proved to be an efficient method for the separation of bacterial antigens in studies of polyclonal T cell responses to Bordetella pertussis antigens.
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Herts., UK.
authors.forename: K
authors.initials: K
authors.lastname: Katrak
authors.forename: B P
authors.initials: BP
authors.lastname: Mahon
authors.forename: W C
authors.initials: WC
authors.lastname: Jones
authors.forename: S
authors.initials: S
authors.lastname: Bräutigam
authors.forename: K H
authors.initials: KH
authors.lastname: Mills
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification*; D002213:Capsid / Q000276:immunology; D002846:Chromatography, Affinity; D002999:Clone Cells / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D015686:Gene Products, env / Q000276:immunology; D007840:Latex; D008213:Lymphocyte Activation; D008251:Macaca / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008863:Microspheres; D017955:Poliovirus / Q000276:immunology; D015302:Simian Immunodeficiency Virus / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D015686:Gene Products, env; D007840:Latex