Oral Presentation International Veterinary Immunology Symposium 2016

Development of cytometric bead array assays to quantify sheep cytokines (#44)

Gabriela Simonova 1 , Eunike McGowan 1 , Melinda M Dean 1 , Gary Entrican 2 , Sean Wattegedera 2 , John-Paul Tung 1
  1. Australian Red Cross Blood Service, Kelvin Grove, QLD, Australia
  2. Moredun Research Institute, Pentlands Science Park, Bush Loan, Midlothian, Edinburgh, Scotland, UK

Background: Multiplex technologies (e.g. cytometric bead array (CBA)) are available for the simultaneous detection of inflammatory markers from humans, mice, rats, dogs, pigs and horses, but not sheep. Our group specialises in ovine transfusion models and quantitation of inflammatory markers is crucial to understand adverse transfusion outcomes. Prior to development of a multiplex sheep cytokine assay, we aimed to develop single analyte CBA assays to quantify sheep IL-1β, IL-6, IL-8 and IL-10.

Methods: Whole blood from a Merino ewe was stimulated with lipopolysaccharide (100ng/mL; 6 hours; 37°C). Plasma was harvested and stored (-80°C). Functional Bead Conjugation Buffer Set and Human Master Buffer Kit (BD Biosciences) were used. Antibodies for capture and detection were screened to select antibody pairs. Antibody-conjugated beads were incubated with serial dilutions of sheep recombinant cytokines (IL-1b, IL-6 and IL-8: 8 - 8,000 pg/mL; IL-10: 20 - 20,000 biological units (bU)/mL) or the plasma sample for 1 hour at room temperature (RT). Specific paired antibody was then added (1hr, RT), followed by a PE-conjugated secondary (1hr, RT). Beads were washed before analysis via flow cytometry. Limit of detection (LOD), intra- and inter-assay variations were determined for each assay by evaluating mean fluorescence intensities across three repeated assay runs.

Results: Mean LODs across runs were IL-1b: 333.3 pg/mL; IL-6: 270.8 pg/mL; IL-8: 354.2 pg/mL; and IL-10: 26.7 bU/mL. Intra-assay coefficients of variations (CVs) were IL-1b: 9-12%; IL-6: 10-12%; IL-8: 21-29%; and IL-10: 12-14%. Inter-assay CVs were IL-1b: 16%; IL-6: 12%; IL-8: 18%; IL-10: 9%. 

Conclusions: CBA was a suitable platform for development of assays to quantify sheep cytokines. Further studies will investigate whether combining these single analyte assays into a multiplex assay will maintain sensitivity and specificity. A multiplex assay for quantification of sheep cytokines will provide a fast and cost-effective tool for sheep immunological research.