Procedures
Using standardized phlebotomy procedures, up to 30 ml of peripheral blood was drawn from each of the patients. Samples utilized in this study were obtained from subjects after study enrollment but prior to treatment initiation (baseline). Blood samples were collected without anticoagulant into red top vacutainers and allowed to coagulate for 20-30 minutes at room temperature. Sera were separated by centrifugation, and all specimens were immediately aliquoted, frozen and stored in a dedicated-80°C freezer. No more than 2 freeze-thaw cycles were allowed before testing for each sample [
18].
The Aushon Multiplex Platform (Aushon Biosystems, Billerica, MA) was used to simultaneously quantitate the serum levels of 115 candidate analytes. The assay comprises a multiplex sandwich ELISA of monoclonal capture antibodies spotted in custom planar arrays in 96-well micro-titer plates. After serum incubation and washing, a second biotinylated monoclonal antibody to a different site from the capture epitope was introduced and streptavidin-horseradish peroxidase (HRP) was subsequently bound to the biotin site. Luminol Enhancer/Peroxidase solution was added and the HRP catalyzed the oxidation of luminol to 3-aminophthalate resulting in light emission at 428 nm. A chemiluminescent image was acquired and processed using a 4-parameter curve fit program (SearchLight Array Analyst Software) to compare the experimental sample values to a recombinant calibration curve run in parallel wells to derive absolute concentrations adjusted for dilution and quality values.
The analystes tested were human IL-6, IL-7, IL-10, IL-16, TNF alpha trimer, IL-1 beta, IFN alpha, IL-4R, IL-18, RANK-L, IL-1 alpha, IL-2R, IL-6R, MPIF-1, Leptin, MIG, GDNF, MIP-1 alpha, MIP-1 beta, MIP-1 delta, ITAC, GM-CSF, MCP-4, MIP-3 alpha, MIP-3 beta, MMP-1, SP-C, Amphiregulin, RANK, MCP-2, IP-10, OPG, FGF basic, KGF, HGH, GCSF, MMP-8, MMP-13, TGF alpha, TGF beta 1, TGF beta 2, TRAIL, TARC, MDC, Eotaxin-2, Beta Defensin 2, GRO gamma, TPO, HGF Beta, NGF, ErbB2, EGF, E-Selectin, P-Selectin, E-Cadherin, PAPP-A, VEGF-R1, VEGF- R2, PECAM-1, MCP-1, c peptide, CD40L, TSP-2, H-CC4, BDNF, RAGE, sAPPb, Thrombomodulin, ENA-78, GRO alpha, CD30, ICAM-3, MMP-7, CG alpha, IL-1RII, TNF-RII, IGFBP-3, EGFR, Human Resistin, MMP-9, ICAM-1, VCAM-1, PAI-1 total, IgE, MPO, TIMP-1, TIMP-2, PDGF-AA, NGAL, Acrp-30, MIP-4/PARC, SHBG, CD14, Clusterin, CRP, NAP-2, Fibronectin, PDGF-BB, VEGF-D, VEGF, TWEAK, PDGF-AB, M-CSF, SDF-1 beta, OPN, Ang-2, IL-17A, RBP4, alpha-2 macroglobulin, Apo-A1, Von Willebrand Factor, A-SAA, IL-23, Visfatin, Fibrinogen.