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

le was homogenized by vortexing, diluted 1:4 with incubation buffer and placed in the microtiter plate [16].

Results

The original version of the CDDE showed deficits with respect to the intra- and inter-assay comparability of the results because of the lack of a standard curve, of a standardized time for ABTS incubation or a stop reagent, and because the results were interpreted by use of an enrichment factor.

Modifications of Cell Death Detection ELISAplus

In a first step, we fixed the ABTS incubation time at 30 minutes which corrected the intra-assay error caused by delays as the procedure is carried out by hand. In addition, we introduced a standard curve: nucleosome-rich standard material was produced by mixing and incubating blood of several individuals, inducing massive apoptosis by mixed lymphocyte reaction. Dilution of the material obtained with incubation buffer (IB) (1:24, 1:32, 1:48, 1:64, 1:96 and only IB) resulted in a linear curve which passed through the origin. Scaling was performed in arbitrary units (AU): After 30 minutes of ABTS incubation, 1,000AU corresponded to 2,500mU (OD), 500AU to 1,250 mU etc. Being related to the standard curve, the AUs were less dependent on the period of color development [16]. Corresponding to the standard curve, dilution of serum by incubation buffer (1:2, 1:4, 1:8, 1:16 and only IB) resulted in a linear and proportional curve. To prevent values exceeding the measurable range, all serum-samples were diluted 1:4 with IB (Figure 1). The lowest detection dosage (LDD) was calculated at 16AU=38 mU [16].



Analytical specificity

The analytic specificity was investigated by titra
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