Combining Histone ELISA and Quantitative PCR Assays to Extract and Quantify Histone Methylation-related Circulating DNA

Abstract

Aim: Liquid biopsy-based measurement of post-translational histone modifications (PTHMs) in bodily fluids of patients with cancer represents a novel area of biomarker research. Here, we tested the applicability of an approach combining enzyme-linked immunosorbent assays (ELISA)- like measurement of histone methylation and quantitative polymerase chain reaction to extract and quantify pericentric histone 4 lysine 20 trimethylation (H4K20me3)-related circulating DNA. Materials and Methods: After adding plasma into ELISA wells and letting H4K20me3-related nucleosomes bind to the antibody, nucleosomal DNA was detached from immune complexes using a buffer including NaHCO3 , SDS, and proteinase K and purified. A single copy- and a multiple copysequence from pericentric regions of chromosomes 16 and 1 were amplified to quantify H4K20me3-related DNA from patients with colon cancer or colonic polyps. Results: Relative plasma level of H4K20me3-related nucleosomal DNA was lower in the patients with colon cancer than in controls for both target sequences, with a statistically significant difference at the chr16-specific target (relative values were 0.037 vs. 0.073, respectively; p=0.008). Conclusion: That H4K20me3-related nucleosomal DNA is present in decreased levels in colon cancer confirms our previous findings attained using other techniques. In conclusion, our findings indicate the applicability of direct extraction of protein-bound DNA from ELISA immune complexes and it could provide a surrogate means in the assessment of PTHMs or other protein-DNA interactions.