High-Throughput Epigenetic Profiling Immunoassays for Accelerated Disease Research and Clinical Development

Abstract

Epigenetics, which examines the regulation of genes without modification of the DNA sequence, plays a crucial role in various biological processes and disease mechanisms. Among the different forms of epigenetic modifications, histone post-translational modifications (PTMs) are important for modulating chromatin structure and gene expression. Aberrant levels of histone PTMs are implicated in a wide range of diseases, including cancer, making them promising targets for biomarker discovery and therapeutic intervention. In this context, blood, tissues, or cells serve as valuable resources for epigenetic research and analysis. Traditional methods such as mass spectrometry and western blotting are widely used to study histone PTMs, providing qualitative and (semi)quantitative information. However, these techniques often face limitations that could include throughput and scalability, particularly when applied to clinical samples. To overcome these challenges, we developed and validated 13 Nu.Q(R) immunoassays to detect and quantify specific histone PTM-nucleosomes from K2EDTA plasma samples. Then, we tested these assays on other types of samples, including chromatin extracts from frozen tissues, as well as cell lines and white blood cells Our findings demonstrate that the Nu.Q(R) assays offer high specificity, sensitivity, precision and linearity, making them effective tools for epigenetic profiling. A comparative analysis of HeLa cells using mass spectrometry, Western blot, and Nu.Q(R) immunoassays revealed a consistent histone PTMs signature, further validating the effectiveness of these assays. Additionally, we successfully applied Nu.Q(R) assays across various biological samples, including human tissues from different organs and specific white blood cell subtypes, highlighting their versatility and applicability in diverse biological contexts.