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

proved the comparability of the results by establishing a standard curve, standardizing the test performance and the preanalytic handling of blood samples [16]. Despite the presence of interfering factors in circulation, the concentration of nucleosomes in serum might reflect the rate of cell death particularly in active, well perfused tumors and the rate of induced cell death in individuals during antitumor therapy.

Materials and Methods

The Cell Death Detection ELISAplus is based on a quantitative sandwich-enzyme-immunoassay principle: Monoclonal mouse antibodies directed against DNA (single-stranded [ss] and double-stranded [ds] DNA) and histones (H1, H2A, H2B, H3 and H4) detect specifically mono- and oligonucleosomes.

The sample was placed in a streptavidin-coated microtiter plate and incubated with a mixture of biotinylated anti-histone antibodies, peroxidase-labeled anti-DNA antibodies and incubation buffer (1% BSA, 0.5% Tween, 1mM EDTA in PBS) for two hours. The antibodies were bound to the histone- and DNA-component of the nucleosomes, and the immunocomplexes were fixed to the microtiter plate by streptavidin-biotin interaction. After the incubation period, unbound antibodies were removed by a washing step. The peroxidase-linked complexes retained were incubated with ABTS (2,2-Azino-di[3-ethylbenzthiazoline-sulfonate]) for 30 minutes, resulting in color development proportional to the number of nucleosomes captured in the antibody sandwich. Quantification of the nucleosomes was performed by photometrical determination of the absorbance at 405 nm against substrate solution as blank (reference wavelength 492 nm). Serum was used as matrix. One to two hours after venipuncture, the sample was centrifuged, treated with 10mM EDTA (pH 8) and stored at -20 C. After thawing, the samp
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