Abstract
BackgroundFluorescence-enhancing microarray on plasmonic gold film is an attractive alternative to traditional enzyme-linked immunosorbent assay (ELISA) for cytokine detection because of the increased sensitivity. The assay chemistry is similar to an ELISA sandwich assay, but owing to the gold substrate, cytokine measurements are 10 to 100 times more sensitive and can be multiplexed. Plasmonic protein microarrays are, as other immunoassays, affected by the presence of heterophilic antibodies and rheumatoid factor may lead to analytical errors with serious implications for patient care. Here, we present a plasmonic gold substrate protein microarray for high-sensitivity detection of cytokines with simultaneous assessment of rheumatoid factor interference on a single chip.ResultsPaired serum and synovial fluid samples from patients with rheumatoid arthritis (n = 18), osteoarthritis (n = 9) or healthy controls (n = 10) were arrayed on near-infrared fluorescence enhancing plasmonic gold chips spotted with cytokine-specific capture antibody and isotype control antibody. Possible rheumatoid factor interference was visualised by a non-specific signal from the isotype control antibody, and pre-treatment of samples with heat-aggregated animal IgG eliminated this background contamination. The platform was optimised using the cytokine IL-20. The protein microarray platform allowed for the detection of human IL-20 at levels <1 pg/ml with reliable IL-20 quantification over a 5-log dynamic range. Samples for which rheumatoid factor caused artefacts were identified and a method for eliminating rheumatoid factor interference was developed and validated. IL-20 protein levels were significantly higher in synovial fluid samples from patients with rheumatoid arthritis compared to osteoarthritis (p < 0.001), while serum levels of IL-20 did not differ between patients with rheumatoid arthritis, osteoarthritis or healthy controls.ConclusionUsing novel plasmonic gold chips, we developed a highly sensitive and accurate assay platform to detect lowly expressed cytokines in biological fluids, allowing for the elimination of rheumatoid factor interference in as little as 5 μl sample volume. The detection limit was below 1 pg/ml for IL-20 and linearity was achieved over a 5-log dynamic range. This technology is highly advantageous for cytokines where sensitivity or sample volume is critical or where assessment of rheumatoid factor interference needs addressed and eliminated.
Highlights
Fluorescence-enhancing microarray on plasmonic gold film is an attractive alternative to traditional enzyme-linked immunosorbent assay (ELISA) for cytokine detection because of the increased sensitivity
We further wanted to apply the technology on biological fluids from patients with rheumatoid arthritis (RA) where samples are often scarce in volume and may be compromised by the presence of heterophilic antibodies
Detection of IL-20 protein levels using plasmonic gold substrates Highly similar standard curves were obtained from recombinant human-IL-20 expressed in E.Coli or HEK-293 (Fig. 1a), suggesting that post-translational modifications do not interfere with detection of IL-20
Summary
Fluorescence-enhancing microarray on plasmonic gold film is an attractive alternative to traditional enzyme-linked immunosorbent assay (ELISA) for cytokine detection because of the increased sensitivity. We present a plasmonic gold substrate protein microarray for high-sensitivity detection of cytokines with simultaneous assessment of rheumatoid factor interference on a single chip. Cytokine measurements using enzymelinked immunosorbent assay (ELISA), the current standard procedure for protein quantification [2], is often limited by the requirement of high sample volume (at least 50 μL), narrow dynamic range and low throughput due to the analysis of a single analyte per sample [3]. Compared with ELISA, protein microarray technology on fluorescence-enhancing plasmonic gold films recently described by Tabakman et al [4] provides improved sensitivity and a broader dynamic range while allowing for high-throughput analysis of multiple determinants within a single sample [5,6,7]. IL-20 is involved in tumour growth and metastasis and represents a promising target in the treatment of breast, bladder and lung cancers [11,12,13]
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