An ultrasensitive assay format for detecting ULK1 inhibition by monitoring the phosphorylation status of Atg13

详细信息    查看全文

• 作者：Zhong-Hua Yan ; Wenhai Zhang ; Neil Rollins ; Olga Tayber ; Jiejin Chen ; Dongyun Wu ; Pam Brauer ; Johara Chouitar ; Robin Bosse ; Jie Yu ; Michael E. Bembenek ; ^{michael.bembenek@takeda.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Sandwich ELISA ; Immunoassay ; ULK1 inhibition ; Quanterix ; SiMoA ; Endogenous Atg13
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 September 2016
• 年：2016
• 卷：509
• 期：Complete
• 页码：73-78
• 全文大小：798 K

文摘

A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer³¹⁸) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer³¹⁸-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer³¹⁸-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer³¹⁸-Atg13 upon ULK1 inhibition.