Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.

中文翻译：

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开发检测细胞和组织中内源性cGAMP的新型高灵敏度方法。

细胞内DNA在先天免疫应答过程中触发干扰素释放。环状GMP-AMP合酶（cGAS）不仅可以响应病毒感染，而且可以在自身免疫条件下感应细胞内双链DNA。测量循环GMP-AMP（cGAMP）作为cGAS激活的第二个信使的水平对于阐明cGAS的生理和病理作用很重要。因此，我们使用杂交瘤技术生成了针对cGAMP的单克隆抗体，以测试抗体的特异性并建立检测细胞内cGAMP的方法。所得的cGAMP特异性抗体能够开发时间分辨的荧光能量转移测定法，其定量范围为0.1 nM至100 nM cGAMP。使用该测定法，我们检测到细胞和组织cGAMP。我们证实，cGAMP抗体通过免疫细胞化学分析成功靶向细胞内cGAMP。这些结果表明，cGAMP抗体是一种功能强大的工具，可以确定cGAS在细胞和组织水平上参与自身免疫和疾病病理的程度。