Tips on using Global DNA Methylation ELISAs

DNA methylation ELISAs are a simple and convenient way to rapidly assess global levels of a specific methylated DNA modification, before pursuing more in-depth and costly applications such as qPCR and NGS. Compared to traditional ELISAs, DNA methylation ELISAs are extremely sensitive with much lower detection limits, allowing for the assessment of a wider range of sample types. Whether your target is 5-mC, 5-hmC, or 5-fC, DNA methylation ELISAs provide a high-throughput, cost-effective means of analyzing a variety of modified DNA forms.

While similar to conventional ELISAs in terms of assay workflow (e.g., sample binding to assay wells, target capture by primary antibody, signal detection with enzyme-conjugated secondary antibody), DNA methylation ELISAs require additional considerations that should be taken into account to ensure reproducibility. Below are some benchtop tips for improving the reliability and consistency of your assay results.

Quality of input DNA

Methylation from contaminating RNA can also be detected by these assays, as the capture antibody does not discriminate between nucleic acid species. Your DNA sample should be relatively pure, with a 260/280 ratio of ~1.8. Use fluorometric-based quantification methods such as Qubit, PicoGreen, and SYBR Green, which are more reliable, sensitive, and accurate than photometric approaches like NanoDrop that can overestimate sample concentrations due to salts, solvents, detergents, proteins, and other contaminants.




Choice of primary antibody

There are a number of different methylated DNA forms. It is essential to choose a primary antibody that is validated for use in ELISA, highly specific for your target methylation of interest, and not cross-reactive with other modifications.




Importance of washing

Residual wash buffer can increase background during signal detection. Use manual pipetting during wash steps instead of the common flicking or dumping techniques. Avoid scraping the bottom well surface by guiding the pipette tip down along the sides of the wells, and completely remove the wash buffer from the wells.







Closely monitor color development

DNA methylation ELISAs typically have a greater sensitivity and faster color development time than traditional protein ELISAs. To reduce cross-variation between sample replicates, consider placing the replicates vertically within a single column instead of horizontally in the plate, and add color developer to all wells in each column at the same time using a multichannel pipette.




Know what you are calculating

Confusion often arises when comparing results obtained with DNA methylation ELISAs and those reported in the literature. For example, some ELISA kits calculate 5-mC levels as a percentage of methylated C per total DNA, and these values will differ from reported percentages for the same sample type that are instead expressed as %5-mC per total number of cytosines. Be mindful of this discrepancy, and make literature comparisons accordingly.

For quick and accurate quantitation of global DNA methylation via a simplified “one-step” ELISA method, EpiGentek offers highly cited MethylFlash Global DNA Methylation (5-mC) ELISA Kits. With these easy-to-use kits, 5-mC methylation levels as low as 0.05% from 100 ng of fully intact input DNA can be directly measured in just 2 h, without the need for additional sample processing like DNA denaturation and fragmentation.

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