title: Production and characterization of monoclonal antibodies to human interleukin-12.
abstract: Interleukin-12 is a cytokine that plays a central role in mediating cell mediated immunity via enhancement of a TH1 cell response. IL-12, unusually for a cytokine, has a heterodimeric structure made up of 35 kDa and 40 kDa subunits. The aim of this study was to produce and characterize monoclonal antibodies to recombinant human IL-12. Twenty-two monoclonal antibodies to IL-12 were successfully produced and subunit specificity determined using recombinant human IL-12 and chimeric murine/human IL-12. All antibodies were shown to react with the p40 subunit by ELISA and immunoblotting with three of the MAbs being found to cross-react with murine IL-12. Using two individual bioassays for IL-12, seven of the MAbs were shown to neutralize biological activity of IL-12. Ten of the antibodies were found to be of use in immunocytochemistry, reacting with LPS-stimulated peripheral blood monocytes. The approaches and difficulties encountered in characterizing antibodies to a heterodimeric cytokine are discussed together with possible reasons for the failure to generate antibodies to the p35 subunit.
authors.affiliation: ICRF Cancer Medicine Research Unit, St James's University Hospital, Leeds, UK.
authors.forename: R W
authors.initials: RW
authors.lastname: Carter
authors.forename: P M
authors.initials: PM
authors.lastname: Patel
authors.forename: A J
authors.initials: AJ
authors.lastname: Stanley
authors.forename: E
authors.initials: E
authors.lastname: Ingham
authors.forename: M
authors.initials: M
authors.lastname: Wadhwa
authors.forename: C
authors.initials: C
authors.lastname: Bird
authors.forename: R
authors.initials: R
authors.lastname: Thorpe
authors.forename: P J
authors.initials: PJ
authors.lastname: Selby
authors.forename: R E
authors.initials: RE
authors.lastname: Banks
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas; D015151:Immunoblotting; D007132:Immunoglobulin Isotypes; D018664:Interleukin-12 / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins; D018664:Interleukin-12