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 enzymelinked 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 l 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.

Microsphere immunoassay for the detection of cytokines in domestic cat (Felis catus) plasma: Elevated IL-12/23 in acute feline immunodeficiency virus infections

“…Methods for coupling capture antibody to microspheres and confirmation of coupling are identical to those previously described (Wood et al, 2011), with the exception that microsphere concentrations were determined using a hemocytometer. For each analyte, the concentration of capture antibody used was 5 μg per 10 6 microspheres (optimal concentration reported in (Wood et al, 2011)).…”

“…For each analyte, the concentration of capture antibody used was 5 μg per 10 6 microspheres (optimal concentration reported in (Wood et al, 2011)).…”

“…Here we a) describe the development and validation of a MIA to simultaneously quantify domestic cat cytokines interferon gamma (IFNγ), interleukin (IL)-10, and IL-12/IL-23 p40 (IL-12/23) in plasma (modified from (Wood et al, 2011)), b) demonstrate the use of this assay with plasma samples collected from domestic cats inoculated with FIV and/or PLV, and c) compare cytokine concentrations to mRNA expression. IFNγ, IL-10, and IL-12/23 were selected because reagents are commercially available, and their mRNA expression during the acute stage of infection may be relevant to immunodeficiency virus pathogenesis (Dean and Pedersen, 1998; Ritchey et al, 2001; Avery and Hoover, 2004; TerWee et al, 2008).…”

Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

“…Breeds represented were domestic shorthair (n = 24), domestic longhair (3), Maine Coon (2), and Himalayan (1). Cats had been hospitalized because of urinary (n = 8), inflammatory (7), gastrointestinal (5), cardiac (3), metabolic (3), or other (2) diseases or trauma (2). Twenty of the 30 cats remained alive 28 days after hospital discharge.…”

Determination of Human Anticytokine Autoantibody Profiles Using a Particle-Based Approach

“…Human GM-CSF, INFα, IFNg, IL-12p70, and IL-17A were coupled to magnetic beads and were individually validated using both commercial antibodies [13] specific to each cytokine as well as human plasma (1:100 dilution) [14] with known anticytokine autoantibody profiles. The intensity of antibody bound to its target bead was measured with the standard Bio-Plex assay and compared to beads incubated with plasma from healthy blood bank donors or beads incubated without primary antibody (Table I).…”