Background Abnormal cytokine expression is postulated to be a factor in the immunopathology of Systemic Lupus Erythematosus (SLE), leading to inflammation and organ damage. Several studies have shown elevated levels of IL-6, IL-17, IL-21 and TNF-α in adult SLE serum and plasma using ELISAs1,2. A spike-and-recovery assessment is used to measure the analyte recovery difference between a natural sample matrix and the standard diluent. Generally 80-120% is considered an acceptable range of recovery. A previous spike-and-recovery study of SLE serum and plasma has shown poor recovery of high mobility group box 1 (HMGB1) protein3.
Objectives To validate and assess the assay response of juvenile-onset SLE (JSLE) serum and plasma in IL-6, IL-17, IL-21 and TNF-α detection kits.
Methods JSLE plasma and serum samples were assayed in spike-and-recovery and linearity-dilution experiments. Three spike-and-recovery ELISA assessments with IL-21 (Biolegend), IL-6 and TNF-α (R&D Systems) were performed, each including 3 JSLE plasma samples and 1 control sample. IL-17 spike-and-recovery ELISA (eBioscience) was executed with 2 JSLE serum and 2 JSLE plasma samples. A spike-and-recovery assessment with IL-17 was performed on 5 JSLE serum samples using a single Bio-Plex Assay (Bio-Rad). Percentage recovery of respective cytokines in spiked and unspiked samples were then calculated and compared against the concentration of spiked sample diluents.
Results Recovery percentages in JSLE samples were less than 80% in TNF-α, IL-17 and IL-21 assays (Fig. 1), apart from IL-17 and TNF-α at 1 in 8 dilution which yielded 80% and 113% respectively. Measuring IL-21 in JSLE and control samples showed poor recovery out of acceptable range at all 4 dilutions. IL-6 concentrations fell within the acceptable range and demonstrated good responses in JSLE and control samples.
Conclusions Interference was observed in the assay response of JSLE serum and plasma in IL-17, IL-21 and TNF-α assays but not IL-6 ELISA. Recovery increased with sample matrix dilution, indicating that dilution reduced the interference, however this must be balanced against the risk of diluting out the analyte. It can be inferred that constituents in the sample matrix may be affecting the detection of these cytokines. JSLE plasma/serum cytokine concentrations measured by ELISA and single plex assays must be interpreted with caution. Methods such as using specialist buffers or alternative assays should be considered to minimise interference and to increase robustness of future studies. Spike-recovery experiments using a multiplex assay platform are currently underway.
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Disclosure of Interest None Declared
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