Cayman Chemical, in collaboration with the nucleotide experts at Biolog Life Science Institute, has developed a new 3'2'-cGAMP ELISA Kit (Item No. 502340) to measure 3'2'-cyclic GMP-AMP (3'2'-cGAMP) levels in fruit fly (Drosophila) homogenates and bacterial lysates. This new assay kit is sensitive, selective, and thoroughly validated to ensure accurate, reliable results and provide a straightforward and inexpensive alternative to mass spectrometry.

Assay Kit At-A-Glance

About 3'2'-cGAMP

3'2'-cGAMP is a cyclic dinucleotide (CDN) second messenger found in Drosophila and bacteria that functions in antiviral immunity.^1-3

Infection of mammalian cells by DNA viruses leads to detection of cytosolic DNA by cyclic GMP-AMP synthase (cGAS), which in turn produces the second messenger 2'3'-cGAMP (Figure 1).^1,2 2'3'-cGAMP then activates stimulator of interferon genes (STING), triggering an innate immune response.

In Drosophila, however, 3'2'-cGAMP is the primary CDN produced in response to infection by RNA and DNA viruses.^1,2 3'2'-cGAMP is synthesized by the innate immune sensor cGAS-like receptor 1 (cGLR1), which is activated by foreign double-stranded RNA, or by cGLR2, which also synthesizes 2'3'-cGAMP and whose ligand is yet to be determined (Figure 1). 3'2'-cGAMP binds to Drosophila STING (dSTING) and induces the expression of Sting and several Sting-regulated genes (Srg), which is dependent on dSTING and the NF-κB homolog Relish.¹ In flies infected with Drosophila C virus (DCV), an RNA virus, 3'2'-cGAMP reduces viral load and increases survival.¹ Additionally, cGLR1 and cGLR2 are required for the survival of flies infected with DCV or Kallithea virus, a DNA virus.²

Figure 1. Infection of mammalsby DNA viruses induces cGAS-mediated production of 2'3'-cGAMP, activation of STING signaling, and innate immunity. Infection of Drosophila by RNA or DNA viruses induces cGLR-mediated production of 3'2'-cGAMP, as well as 2'3'-cGAMP, which activates dSTING signaling and antiviral immunity.

In bacteria, 3'2'-cGAMP is a second messenger in certain cyclic-oligonucleotide-based antiphage signaling systems (CBASS).³ The cGAS/DncV-like nucleotidyltransferase (CD-NTase) CdnG from Asticcacaulis sp. (AsCdnG) synthesizes 3'2'-cGAMP, which then activates the CBASS effector protein AsCap5 to degrade DNA.

About the Assay

Cayman's 3'2'-cGAMP ELISA Kit is an easy-to-use competitive ELISA for the quantification of 3'2'-cGAMP in fruit fly homogenates and bacterial lysates. This kit includes all necessary reagents and uses a straightforward protocol that provides rapid results in under three hours.

The assay is based on the competition between free 3'2'-cGAMP in samples (or standards) and a 3'2'-cGAMP-HRP conjugate (3'2'-cGAMP-HRP Tracer) for binding to a limited amount of 3'2'-cGAMP polyclonal antibody binding sites. In one single-step incubation, samples are added to IgG-coated wells along with the specific 3'2'-cGAMP polyclonal antiserum and the 3'2'-cGAMP-HRP Tracer (Figure 2). After two hours of incubation, plates are washed to remove unbound reagents and developed with a TMB substrate solution containing an HRP substrate. This results in the formation of a colorimetric product that can be detected by a plate reader at 450 nm. The absorbance intensity is proportional to the amount of 3'2'-cGAMP-HRP Tracer bound to the well, and therefore inversely proportional to the concentration of 3'2'-cGAMP in the tested sample.

Figure 2. Schematic of the competitive ELISA. For the complete assay protocol, please refer to the kit booklet (PDF).

High Sensitivity and Specificity

Cayman's 3'2'-cGAMP ELISA Kit uses a 3'2'-cGAMP polyclonal antiserum that offers excellent sensitivity and low cross-reactivity to structurally related molecules. The assay has a range of 7.8-1,000 pM, with a midpoint (50% B/B₀) of 72 pM, a sensitivity (80% B/B₀) of 15 pM, and a lower limit of detection (LLOD) of 4.0 pM (Figure 3). The dynamic range of this assay allows for dilution to eliminate interference commonly seen with whole fly homogenates and crude bacterial lysates.

Figure 3. Typical standard curve.

This kit demonstrates a high degree of specificity, with minimal detection of 2’2’-cGAMP, 3’3’-cGAMP, 2’3’-cGAMP, cyclic di-GMP, and cyclic di-AMP.

Cross reactivity of the 3'2'-cGAMP ELISA Kit