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  • Title: Methodological and preanalytical evaluation of a RAGE immunoassay.
    Author: Wittwer C, Lehner J, Fersching D, Siegele B, Stoetzer OJ, Holdenrieder S.
    Journal: Anticancer Res; 2012 May; 32(5):2075-8. PubMed ID: 22593491.
    Abstract:
    BACKGROUND: Soluble receptor of advanced glycation end products (sRAGE) is a promising biomarker for the prognosis and the monitoring of cancer and of acute diseases such as trauma and sepsis. MATERIALS AND METHODS: We investigated the methodological characteristics of an ELISA for sRAGE (R&D Diagnostics) including intra- and inter-assay imprecision, dilution linearity and differences in various serum and plasma materials. Furthermore, the influence of various preanalytical factors such as time and storage temperature before and after centrifugation prior to definite deep freezing, as well as multiple freeze-thaw cycles, were tested. By the use of sera from 30 healthy individuals, a reference range and the dependency on patient characteristics was established. RESULTS: Intra-assay imprecision (coefficients of variation (CV): 6.0-11.5%) and inter-assay imprecision (5.9-7.8%) were in an acceptable range of manual assays. Linearity testing yielded satisfying results with dilution recoveries of 99-131%. Results of serum, EDTA-plasma (recovery of 85.9-114.7%), and heparin-plasma samples (88-102%) were quite comparable, while results from citrate-plasma were slightly lower (78-96%). There was no influence of the time to centrifugation after 6 and 24 hours (recoveries 87-102%) at storage temperatures of 4°C and 25°C. Similarly, results were the same when samples were kept at 4°C and 25°C after centrifugation for up to 7 days (recoveries 88-109%). Repeated freeze-thawing of samples did not affect the results obtained for the RAGE protein (recoveries 92-104%). The median value of healthy individuals was 1.10 ng/ml, with 90% limits of 0.52 to 1.49 ng/ml. CONCLUSION: sRAGE ELISA is a very robust and safe assay which produces reliable quantitative results for sera and plasma measurements.
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