Abstract

Histone 3 isoform 3 (H3.3) is a specific histone isoform with hyperacetylation, methylation, and ubiquitination patterns. The hyperacetylated H3.3 is resistant to proteasomal degradation and present extracellularly both in the lumen of the airway, prominent in the bronchoalveolar lavage fluid and the plasma of subjects with chronic obstructive pulmonary disease (COPD). Plasma H3.3 is therefore a potential biomarker for histone hyperacetylation in COPD. The aim of this work was to develop an assay for H3.3 biomarker screening with sufficient throughput and sensitivity. Methods: We used Western blot and ELISA to identify a pair of highly specific antibodies to specifically detect total H3.3. After extensive optimisation, a highly sensitive sandwich ELISA assay in 384w format for the detection of total H3.3 was developed. Results: Antibodies for the sensitive and specific detection of H3.3 were identified. The composition of the blocking solution was optimized. The assay was established in 384 well format to allow for screening of large numbers of samples. The detection of total H3.3 is about 40 ng/mL. Conclusions: A novel high throughput H3.3 ELISA assay was developed for identification/stratification of COPD patients with increased histone 3.3 hyperacetylation.