ABSTRACT
UNLABELLED: There is an emerging trend in immunotoxicological studies to use the multiplex technologies for testing the safety and the efficacy of new pharmaceuticals by using cytokines profiling as biomarker. The Luminex 200 xMAP (multi-analyte profiling) technology provides simultaneous measurement of multiple cytokines in small sample volumes, expressing rapidly the differences between various test compounds. The aim is to develop and validate the Luminex 200 multiplex immunoassays by correlation with ELISA (enzyme-linked immunosorbent assays) for implementation in evaluating cytokine profiling in immunotoxicological studies in vitro. METHODS: Human peripheral whole blood from healthy subject diluted 1+4 with RPMI 1640 was cultured 48 hours in 28 experimental variants: control, in presence of mitogens, bioflavonoid extracts (from Crataegus monogyna and Echinacea purpurea) as cytoprotectors and with a toxic compound [Pb(NO3)2]), separately or variously combined. IL-1beta and IL-2 were comparatively performed by xMAP and ELISA immunoassays from the same sample to initialize validation of multiplex cytokine panel: IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, usually performed by Luminex 200 system in our immunotoxicological studies. The results indicate similarly typed trends of cytokine values obtained by both methods, with comparable relative changes in presence of mitogens, bioflavonoids and toxic, respectively. Although xMAP absolute cytokine values were higher than ELISA values, the correlation between multiplexed assay and ELISA was good for IL-1beta and IL-2 with positive correlation coefficients near to 1. Conclusions. Quantitative differences between absolute values for IL-1beta and IL-2 obtained by xMAP and ELISA assays are found, but the relative values are comparable and the two methods keep similar trends in similar exposure conditions. The performance parameters of the xMAP assay and the good correlation coefficients with the "gold standard" ELISA recommend to validate the multiplex assay for analyzing cytokine profiles in immunotoxicological studies in vitro.
Subject(s)
Cytokines/analysis , Immunoassay/methods , Immunoassay/standards , Reagent Kits, Diagnostic/standards , Biomarkers/analysis , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Flavonoids/immunology , Humans , Interleukin-1beta/analysis , Interleukin-2/analysis , Leukocytes, Mononuclear , Sensitivity and SpecificityABSTRACT
BACKGROUND: Pancreatic cancer, owing to its raising incidence and aggressiveness, is a major challenge, both for research and for clinical management. As pancreatic cancer has a complex pathophysiology, in addition to improving the methods of early diagnosis, sensitive and specific biomarkers are a prerequisite. OBJECTIVE: As there is no specific tumor marker for pancreatic cancer diagnosis, extensive genomics/transcriptomics and proteomics studies have been developed with the aim of finding candidate markers and contributing to high-throughput systems for large cohort screening. METHODS: A literature review was done to study these biomarkers in relation to diagnosis, prognosis and therapy targets in pancreatic cancer. RESULTS/CONCLUSION: For early diagnosis improvement, only a panel of soluble biomarkers could provide the appropriate combination between high sensitivity and specificity. Prognostic upgrading would benefit from biomarker discovery and validation performed on tumor tissue. New technology could delineate molecular targets for innovative therapy in pancreatic cancer.
ABSTRACT
UNLABELLED: Angiogenic markers such as VEGF/basic FGF (bFGF) can enlarge the diagnostic biomarkers panel for pancreatic cancer. MATERIALS & METHODS: Serum samples from 32 stage I-IV pancreatic cancer patients and 20 controls were analyzed for soluble VEGF/bFGF by ELISA and xMAP array. RESULTS: VEGF/bFGF serum levels were significantly increased in patients compared with controls (p < 0.0001). We report a correlation with tumor diameter (p < 0.01/p < 0.05), stage (p < 0.001), Ki67LI (p < 0.005/p < 0.05) and carbohydrate 19-9 antigen (p < 0.005/p < 0.001). VEGF/bFGF levels analyzed by xMAP array were comparable with the pattern (patient/control) outline obtained by ELISA tests. We obtained a good correlation between these two soluble angiogenic markers (p < 0.001). CONCLUSION: Data obtained for angiogenic markers qualifies them as important candidates in the pancreatic cancer biomarker panel.
