Clinical characteristics of patient groups
Samples from HC (n = 10, 5 female, 5 men) were from BioreclamationIVT (West Sussex, UK), while samples from patients with RA (n = 18, 12 female, 6 men) and OA (n = 9, 6 female, 3 men) were from DVBiologics (Costa Mesa, CA, USA). Mean age (±SD) for both RA and OA groups was 65 (±13). Of the 18 RA patients, 17 were RF positive (mean titer ± SD; 26 ± 12). All RA patients were CRP positive (mean ± SD; 11 ± 5 mg/dl). Disease Activity Score in 28 Joints was >2.6 for all RA patients indicating no remission cases. Validation of the RF removal protocol was performed on 3 additional RA samples (DVBiologics).
Written informed consent was obtained for all subjects according to the Declaration of Helsinki and the General Assembly Revision of 2008. The study has been approved by the Ethics Committee of Copenhagen and Frederiksberg.
Protein array reagents
The following reagents were from Sigma–Aldrich (St. Louis, MO, USA); 16-mercaptohexadecanoic acid, cysteamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, succinic anhydride and N-hydroxysuccinimide. Triethylamine, dimethylformamide and bovine serum albumin (BSA) were from Fisher Scientific (Pittsburgh, PA, USA). Six-armed-poly(ethyleneglycol)–amine was from SunBio (South Korea). PBS (Cat#70011-036) and Tween-20 were from Life Technologies (Grand Island, NY, USA).
Sandwich antibody pairs for IL-20 were produced by Novo Nordisk A/S. Capture anti-IL-20 antibody was of the IgG1 isotype. Unspecific binding was assessed using a IgG1 isotype control from R&D Systems (MAB002, Minneapolis, MN, USA). IRDye-800 streptavidin conjugate was from LiCor Biosciences (Lincoln, NE, USA). Mouse, goat and bovine IgG were from Jackson ImmunoResearch (West Grove, PA, USA).
Purified cytokine antigen standards and sandwich antibody pairs for IL-1β (MAB601, BAF201), IL-10 (MAB2172, BAF217), IL-6 (MAB206, BAF206), and TNFα (MAB610, BAF210) were purchased from R&D systems. All capture antibodies were of the IgG1 isotype.
rh-IL-20 protein standards
Rh-IL-20 derived from E.Coli cells was purchased from R&D systems (Cat#1102-IL-025) or produced at Novo Nordisk A/S. Rh-IL-20 produced in HEK-293 cells was from Novo Nordisk A/S. The activity of E.coli expressed Met-IL-20 and HEK-293 expressed IL-20 was determined by a IL-20R1/IL-20R2 Reporter Gene Assay (STAT-Luc) showing EC50 of 896 and 819 pg/ml, respectively (data not shown). The protein standards were diluted from 1 nM to 10 fM (17600–0.176 pg/ml) using 10-fold dilution steps in 5 % BSA in PBS.
Chemically modified plasmonic gold slides were prepared as described . Capture antibody or isotype control antibody (3 μM in PBS + 1 % glycerol) were printed in triplicate spots using Bio-Rad VersArray Chipwriter and solid pins (Arrayit, USA) at 25 °C and 60 % humidity, resulting in microarray feature diameters of ~400 μm. Slides were blocked overnight at 4 °C in 5 % BSA in PBS. Samples (5 μl) were diluted 4-fold in PBS and applied to each micro-well and incubated for 4 h at RT. Slides were washed twice in 0.5 % Tween-20 in PBS, once with PBS, and 5 nM human biotinylated polyclonal antibody in 10 % BSA in PBS was incubated over each micro-well for 1 h at RT. Slides were washed as before and 2 nM IR800 conjugated streptavidin in 10 % BSA in PBS solution added to the slides for 1 h at RT. After another wash, slides were rapidly immersed in deionized water and dried with compressed air.
Fluorescence measurement and data analysis
Slides were scanned using Odyssey scanner (Li-cor Biosciences) as described . Fluorescence intensities were background-subtracted using the global background subtraction method (GenePixPro V6), before calculating the median pixel intensities for features printed in triplicates. Standard curves were obtained using 5-parameter logistic curve. Limit of detection (LOD) was defined as the concentration at three standard deviations above the mean fluorescence of the blank. Lower limit of quantification (LLOQ) was defined as the lowest concentration at which the recovery of each calibrator was within 70-130 % and the inter-run coefficients of variation (CV) was <20 % . The assay range was defined as the concentration range in which the cytokine measurements were linear (r2 > 0.99), inter and intra-run CV was 20 %, respectively, and spike recoveries between 70-130 %. All cytokine measurements are reported as median and range.
Statistical analysis was performed using GraphPad Prism V.6 by applying a Mann–Whitney test. Wilcoxon matched-pairs test was used to compare IL-20 levels in matched serum and synovial fluid of each subject. P-values <0.05 were considered statistically significant.
Serum samples from three of the HCs were spiked with 20 or 2000 pg/ml of rh-IL-20 (produced in E.Coli by Novo Nordisk A/S). Spike recoveries were obtained by subtracting the observed concentration of the un-spiked sample (observed un-spiked) from the observed concentration of the added sample (observed spiked), and divided by the known concentration of the spiked sample (expected spiked).
Removal of RF interference
By using an isotype control capture antibody, non-specific signal due to RF can be identified [20, 21]. To remove RF from biological samples showing RF-interference, we applied a modified protocol of Kragstrup et al. , where samples were diluted in an assay diluent containing a mix of animal IgG (mouse, bovine and goat, each at 50 μg/ml in PBS) previously heat-aggregated at 60 °C for 30 min. Serum samples were diluted 4-fold in the assay diluent containing animal IgG and incubated 30 min at RT. Samples were added to the gold slides and IL-20 detection performed as described above. Cytokine measurements were unaffected by addition of animal IgGs to serum samples, as spike-in recoveries for samples with and without animal IgG-treatment (n = 3, RA donors) were within the accepted range of 70–130 % (IL-6, 99 % (94–103 %); IL-1β 93 % (92–95 %); TNFα, 98 % (84–112 %) and IL-10, 88 % (86–89 %).
For the analysis of RA, OA and HC samples, RF interference assessment was performed and interference identified for two RA samples for which the RF elimination protocol was applied.