title: Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines.
abstract: Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.
authors.affiliation: Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 1619 Campus Delivery, Fort Collins, CO 80523, USA.
authors.forename: Britta A
authors.initials: BA
authors.lastname: Wood
authors.forename: Kevin P
authors.initials: KP
authors.lastname: O'Halloran
authors.forename: Sue
authors.initials: S
authors.lastname: Vandewoude
mesh_terms: D000818:Animals; D002415:Cats; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques / Q000379:methods*; D007118:Immunoassay / Q000379:methods; D007371:Interferon-gamma / Q000032:analysis* / Q000378:metabolism; D016753:Interleukin-10 / Q000032:analysis* / Q000378:metabolism; D018664:Interleukin-12 / Q000032:analysis* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008863:Microspheres; D012680:Sensitivity and Specificity
chemical_list: D016753:Interleukin-10; D018664:Interleukin-12; D007371:Interferon-gamma