Assay Principle: 

The 3′,3′-Cyclic GAMP ELISA Kit quantitatively measures 3’,3’-cGAMP present in cell lysates, tissue extracts, and tissue culture media. This competitive ELISA has a run time of 1.5 hours. Please read the complete kit insert before performing this assay. Use our provided 3’,3’-Cyclic GAMP (cGAMP) standard to generate a standard curve for the assay.

Protocol Summary:

• Add standards or diluted samples to the transparent microtiter plate, pre-coated with goat anti-rabbit IgG antibody.
• Introduce the 3’,3’-cGAMP-peroxidase conjugate and 3’,3’-cGAMP polyclonal rabbit antibody into each well.
• Incubate the plate covered, shaking at room temperature, for 1 hour to facilitate the immunological reaction.
• Wash the plate to remove excess conjugate and then add the TMB substrate. This substrate reacts with the bound conjugate to generate a detectable signal.
• Measure the absorbance at 450nm using a plate reader and calculate the 3’,3’-cGAMP concentration using the standard curve.

Background:

3′,3′-Cyclic guanosine monophosphate–adenosine monophosphate (3’,3’-cGAMP) is an essential messenger molecule in both bacterial and mammalian systems. In bacteria, it serves as a mediator in signal transduction and regulation, affecting a diverse range of targets involved in transcription and enzyme activity. Notably, bacterial riboswitches, which are gene-regulatory RNA elements, bind cGAMP with high specificity, influencing genes related to motility, biofilm formation, colonization, and virulence. This functionality makes cGAMP signaling a promising target for antimicrobial therapies.

In mammalian cells, 3′,3′-cGAMP, along with its analog 2′,3′-cGAMP produced by cGAS, plays a critical role in the innate immune response. It binds to STING (stimulator of interferon genes), inducing a cascade leading to the production of IFN-β. The cGAS-cGAMP-STING pathway enables antiviral and antitumor immunity and mediates autoimmune responses. Dysregulation of this pathway, particularly through the aberrant activation by self-DNA, is linked to tumorigenesis and autoimmune disorders.

The DetectX® 3′,3′-Cyclic GAMP ELISA Kit is an important tool for researchers in immunology and microbiology, providing precise quantification of 3′,3′-cGAMP levels and aiding in the study of its diverse biological roles.