Serum Histone H3 Quantification

Serum histone H3 levels were measured as recently described [9 (link)]. Briefly, polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μL of 1 mg/L anti-histone H3 antibody (Shino-Test Corporation, Sagamihara, Japan). After washing and blocking, the plates were incubated with 100 μL of diluted calibrator and serum samples for 24 h at room temperature. After washing, the plates were incubated with 100 μL of anti-histone H3 peroxidase-conjugated antibody (Shino-Test Corporation) for 2 h at room temperature. The plates were washed again, and incubated with the chromogenic substrate 3,3′,5,5′-tetra-methylbenzidine (Dojindo Laboratories, Kumamoto, Japan). The reaction was terminated with 0.35 mol/L Na₂SO₄, and the absorbance at 450 nm was measured with a microplate reader (Model 680; Bio-Rad, Hercules, CA). A standard curve was obtained with purified calf thymus histone H3 (Roche, Stockholm, Sweden). The lower detection limit of the ELISA was 2 ng/mL, and linearity was observed in the range up to 250 ng/mL. The ELISA specifically detected histone H3, and did not react with other histone family proteins, including histone H2A, H2B and H4 [9 (link)].