Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses

Wagner, Bettina; Freer, Heather

doi:10.1016/j.vetimm.2008.10.313

Cited by 121 publications

(90 citation statements)

References 14 publications

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“…OspA is a component of all currently available Lyme vaccines for dogs. In serological diagnostics, antibodies to OspA therefore have been interpreted as vaccination markers in dogs (8,11,37,38). However, our current data show that OspA antibodies also form in response to infection.…”

Section: Resultsmentioning

confidence: 65%

Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

Wagner

Freer

Rollins

et al. 2012

Clin. Vaccine Immunol.

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ABSTRACTLyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

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Section: Resultsmentioning

confidence: 65%

Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

Wagner

Freer

Rollins

et al. 2012

Clin. Vaccine Immunol.

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“…Commercially available cytokine kits and assays developed by individual research groups (e.g., for porcine [4] and horse [21] cytokines) provided valuable insights into the innate immune response of a host against infection. For example, microsphere-based assays have been used to characterize the cytokine response in the lymph nodes of chronically infected HIV patients (3) and cytokine production during acute HIV-1 infections (17).…”

Section: Discussionmentioning

confidence: 99%

Development and Validation of a Multiplex Microsphere-Based Assay for Detection of Domestic Cat ( Felis catus ) Cytokines

Wood

O’Halloran

VandeWoude

2011

Clin Vaccine Immunol

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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.

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“…After 48 hr of culture at 37°C supernatants were collected and analyzed for IL-10, IFN-c, and IFN-a concentration, using a fluorescent bead-based multiplex assay. 25 IL-6 concentrations were quantified by ELISA. 31 …”

Section: Pbmc Cytokine Secretion Assaymentioning

confidence: 99%

Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

et al. 2015

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Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5 · 10 11 vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2-or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2-and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2-or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA.

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Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses

Cited by 121 publications

References 14 publications

Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

Development and Validation of a Multiplex Microsphere-Based Assay for Detection of Domestic Cat ( Felis catus ) Cytokines

Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

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