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

Cell death as a counterpart to cell proliferation plays an essential role in the homeostasis of cell numbers in adult organisms, furthermore in eliminating cells damaged by irradiation, chemotherapeutic drugs, hyperthermia and other influences [1-3]. There are two distinct ways of eukaryotic cell death: oncosis and apoptosis. How cells die does not so much depend on the type of the stimulus, as on dose and severity of a lesion, as well as on cell type [4-6]. During apoptosis, various endonucleases are activated which cleave the chromatin into nucleosomesized fragments of 180 bp and multiples thereof, generating the typical ladder pattern observed with agarose gel electrophoresis [1-8].
Nucleosomes are elementary units of chromatin formed by an octamer of histones and 146 bp DNA wrapped around it. The nucleosomes are connected by linker DNA of about 15 -100bp which are the preferential binding sites of the endonucleases [9-11]. Under physiological conditions, nucleosomes are packed into apoptotic bodies and phagocytized by macrophages or neighboring cells [1-4]. In situations of enhanced cell death these mechanisms are overloaded and nucleosomes are also released into circulation [12-15]. Quantifying these circulating nucleosomes may offer the fascinating possibility of estimating the extent of cell death wherever it happens. This could be especially useful for follow up studies and for monitoring individuals during therapy as the material is obtained easily by venipuncture.

The Cell Death Detection ELISAplus (CDDE) was developed as a cellular test to detect nucleosomes in cytoplasm prior to disintegration of the plasma membrane which is a well-known hallmark of apoptosis [1-3, 7, 8]. We adapted the CDDE for its use in serum samples and im 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 le was homogenized by vortexing, diluted 1:4 with incubation buffer and placed in the microtiter plate [16].


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 ting native anti-histone antibodies to the normal immunoreagent containing biotinylated anti-histone antibodies. With increasing concentrations of the native anti-histone antibodies, the absorbance values declined in a dosedependent manner indicating that only complexes of DNA and histones bound to peroxidase-labeled anti- DNA antibodies and biotinylated anti-histone antibodies were detected by the CDDE. DNA, histones, nucleosomes, anti-histone antibodies or anti-DNA antibodies alone do not alter the measured values (16) (Figure 2).


The imprecision, as a criterion for the reliability of the ELISA, ranged in the intra-assay comparison (n=10) between 3.0% and 4.1 %, in the inter-assay comparison (n=14) between 8.6% and 13.5 % [16].


In the serum and plasma of ten individuals, which were stored under varying conditions (storage time, temperature, addition of a stabilizer), the best stability was obtained for serum which has been treated with 10mM EDTA (pH 8) and stored at -20 C or -80 C [16].

Long-term stability

Serum samples which were treated with EDTA and stored at -20 C showed a good long-term stability at low and high absorbance levels, even after six months. This was indicated by a coefficient of variation between 4.2% and 9.2 % [16] (Figure 3).

Preanalytic handling of the samples

The standardization of the preanalytic handling of the samples was essential for the reliable quantification of nucleosomes in serum. Whereas hemolysis and prolongation of the period between ven 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].


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 the acidic DNase II [18]. Summarizing our studies, we recommend the preanalytic handling of the blood samples as indicated in Table 1.

Our first findings seem to indicate that the quantification of the concentration of nucleosomes in serum is a sensitive and fast method useful for monitoring the efficacy of antitumor therapy and estimating the individual sensitivity to therapy. Further studies with more samples, defined tumors and therapeutic regimens will have to prove its significance in clinical practice.



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