Optimizing Cytokine Measurement in Biological Samples

Blood samples were collected from 4 healthy volunteers using the following blood collection tubes; normal clotting tube (SST II Advance, BD Biosciences) for serum and sodium heparin (NH), EDTA, or sodium citrate (NC) tubes for collecting plasma (all BD Biosciences) in the morning on 2 following days. All samples were kept on room temperature and were spun within 1 hour at 700 × g at room temperature. Cell free plasma or serum was aliquoted and stored at -80°C until analysis. Before analysis all thawed samples were centrifuged through a polypropylene centrifuge tube containing a 0.22 μm nylon membrane (Spin-X column; Corning, Corning, NY, USA) to remove debris. Non-specific heterophilic antibodies, such as natural polyspecfic antibodies, idiotypic antibodies and natural rheumatoid factors, were pre-absorbed, without loss of cytokine concentration, from all samples with protein-L pre coated ELISA plates (Pierce, Rockford, IL, USA) as described previously [14 (link)]. 100 μl of sample well was incubated for 1 hour at room temperature under continuous shaking. As this incubation step removes 60-80% of the total immunoglobulin fraction without depleting natural occurring cytokines [14 (link)]samples were then diluted with 10% v/v normal rat and mouse serum (1:1 ratio; Rockland, Gilbertsville PA, USA) and incubated for 10 additional minutes at room temperature to block any residual interfering proteins which are able to bind (a) specifically to rat or mouse immunoglobulins. Animal serum batches were tested upfront, and only used when they did not show any cross-reactivity within the assay.
All samples obtained were approved for collection by the medical ethical committee of the UMC Utrecht. Informed consent was obtained from each individual who donated samples.