Abstract
Cytokines are cell signalling proteins that mediate a number of different physiological responses. The accurate measurement of cytokine profiles is important for a variety of diagnostic and prognostic scenarios in relation to animal health and welfare. Simultaneous quantification of cytokine profiles in a single sample is now possible using fluorescent microsphere immunoassays (FMIA). We describe the development and validation of a novel multiplex assay using the Bio-Plex® 200 system to quantify cytokines in five different porcine tissues (brain, placenta, synovial tissue and fluid, plasma). The cytokine profiles are both tissue, and research hypothesis, -dependent but include Interleukin-1beta (IL-1β), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10) and Tumor necrosis factor (TNF-α).This methods paper is reported in two parts: the development of a FMIA for porcine tissues and validation of pre-treatment for optimal cytokine recovery in porcine brain, placenta, synovial tissue and plasma. Validation steps are critical in ensuring an assay is suitable for novel sample types.This technique advances traditional ELISAs by:•FMIA provides insight into the profiles of multiple porcine cytokines in certain situations (e.g. disease, parturition).•Use of the Bio-Plex® 200 system to investigate novel sample types, including brain, placenta and synovial tissue.•Multiplexing utilises a fraction of the sample volume compared with multiple ELISAs.
Highlights
Our standard curve range and assay sensitivity is similar to previous fluorescent microsphere immunoassays (FMIA) and covers a wider range than many commercial ELISA kits [2,15] and we have shown its use with a wider range of sample types
The standard curve range of IL-1β, in this multiplex has a reduced sensitivity compared to the other analytes, this has been observed previously [3], which allows us to measure porcine plasma without need for dilution
This assay will reduce sample volume, time and cost associated with single ELISAs yet providing a more comprehensive picture of the suite of cytokines produced in different tissues, allowing investigation into immunological response to specific challenges
Summary
S.A. Halla,*, S.H. Isona,b, C. Owlesc, J. Coea, D.A. Sandercocka, A.J. Zanellad a SRUC, West Mains Road, Edinburgh, United Kingdom b World Animal Protection, United Kingdom c University of Nottingham, School of Biosciences, United Kingdom d Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Campus Pirassununga, Brazil
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