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Quantification of Nucleosomes in Serum,,,by the Cell Death Detection ELISAplus

ipuncture and centrifugation to more than two hours resulted in elevated values, the delayed addition of 10mM EDTA led to falsely low values. These effects were avoided by careful handling of the blood samples, centrifugation within two hours and addition of EDTA immediately after centrifugation [16].

Discussion

The modifications of the Cell Death Detection ELISAplus including standardization of ABTS incubation time, establishment of a standard curve and introduction of a new scaling with AUs improved the reproducibility of the measured values considerably. This was demonstrated by an intra- and inter-assay imprecision within the limits demanded for hand-performed assays (CVintra-assay less than 10% and CVinter-assay less than 15 %). Further, the analytical specificity showed that only complexes of nucleosomes, peroxidase-labeled anti-DNA antibodies and biotinylated anti-histone antibodies were detected by the CDDE.

The relative scaling in AU takes into account the unknown proportionality of mono- and oligonucleosomes in circulation with probably different accessibilities to antibody binding sites. The measured amount of peroxidase-labeled anti-DNA antibodies does not necessarily reflect the exact concentration of the nucleosomes, suggesting that the use of an absolute scaling in ng/ml nucleosomes probably would have led to inappropriate results [16].

The standardization of the pre-analytic handling of the serum samples was a prerequisite for the reliable and reproducible determination of the concentration of nucleosomes in serum. Particularly, the addition of 10mM EDTA (pH 8) to serum immediately after centrifugation inhibited further fragmentation of the DNA by endonucleases such as the Ca2+- and Mg2+-dependent DNase I [17] and
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