title: Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN-γ.
abstract: OBJECTIVE
  Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ).
  
  
  METHODS
  Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples.
  
  
  RESULTS
  Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations.
  
  
  CONCLUSIONS
  Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.
authors.affiliation: Equine Clinic, University of Veterinary Medicine, Hannover, Germany. mcdurang@tiho-hannover.de
authors.forename: Maria Carolina
authors.initials: MC
authors.lastname: Duran
authors.forename: Saskia
authors.initials: S
authors.lastname: Willenbrock
authors.forename: Jessika-M V
authors.initials: JM
authors.lastname: Müller
authors.forename: Ingo
authors.initials: I
authors.lastname: Nolte
authors.forename: Karsten
authors.initials: K
authors.lastname: Feige
authors.forename: Hugo
authors.initials: H
authors.lastname: Murua Escobar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay / Q000662:veterinary*; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006736:Horses; D007159:Immunologic Tests; D007371:Interferon-gamma / Q000097:blood* / Q000276:immunology; D018664:Interleukin-12 / Q000097:blood* / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D018664:Interleukin-12; D007371:Interferon-gamma