ELISA-Based Enzyme Kinetics Assay for Measuring cGAS Activity.

Cyclic GMP-AMP synthase (cGAS) is a key cytosolic double-stranded DNA sensor that activates innate immune responses. Upon binding double-stranded DNA, cGAS undergoes conformational activation and catalyzes the synthesis of the second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) from ATP and GTP. 2'3'-cGAMP then triggers a downstream signaling cascade that induces type-I interferon and inflammatory gene expression and has been shown to exert antitumor effects in the context of cancer. Accurate measurement of this enzymatic activity is therefore important for mechanistic studies. Traditional kinetic methods such as radiolabeling, HPLC, or mass spectrometry provide precise results but require specialized equipment and expertise. Here, we describe a rapid and accessible ELISA-based protocol to quantify 2'3'-cGAMP product formation and derive cGAS enzymatic parameters. Reactions are initiated with defined DNA ligands and quenched at multiple time points, and product accumulation is quantified by a commercially available 2'3'-cGAMP ELISA. Time course measurements are used to calculate initial velocities, which can be plotted against substrate concentration to obtain Michaelis-Menten parameters. This approach enables direct, product-specific quantification of 2'3'-cGAMP formation using only an absorbance plate reader. The protocol provides a sensitive and broadly applicable alternative to traditional methods, allowing laboratories without advanced instrumentation to perform reliable cGAS enzyme kinetics. Key features • Developed to evaluate how cofactors or regulatory proteins modulate cGAS activity in vitro. • Suitable for assessing cGAS kinetics with diverse nucleic acid substrates under defined reaction conditions. • Determination of initial (V