Yu Fei, Xiong Ya-Min, Yu Song-Cheng, He Lei-Liang, Niu Shan-Shan, Wu Yu-Ming, Liu Jie, Qu Ling-Bo, Liu Li-E, Wu Yong-Jun
College of Public Health.
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Int J Nanomedicine. 2018 Jan 17;13:429-437. doi: 10.2147/IJN.S152618. eCollection 2018.
DNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity.
A magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.
Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1-1,500 ng/mL, the recovery was 91.67%-106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%-11.29% and 7.03%-11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient =0.866, =0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits.
These indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples.
DNA甲基转移酶1（DNMT1）是一种主要的酶，负责将甲基从通用甲基供体转移到DNA中胞嘧啶残基的5位，对哺乳动物发育至关重要，且与癌症及多种与年龄相关的慢性疾病密切相关。DNMT1已成为疾病早期诊断中的有用生物标志物以及癌症治疗和药物开发中的潜在治疗靶点。然而，迄今为止，大多数关于DNA甲基转移酶（MTase）检测的研究都集中在原核生物MTase及其活性上。
本研究提出了一种以CdSe/ZnS量子点为荧光探针的磁荧光联免疫吸附测定法（FLISA），用于快速灵敏地检测DNMT1水平。对影响DNMT1测定精密度和准确度的关键因素进行了优化。
在最佳条件下，检测限为0.1 ng/mL，线性范围为0.1 - 1500 ng/mL，回收率为91.67% - 106.50%，批内和批间相对标准偏差分别为5.45% - 11.29%和7.03% - 11.25%。与DNA甲基转移酶3a和3b的交叉反应率分别仅为4.0%和9.4%。此外，FLISA成功用于检测人血清样本中DNMT1的水平，并与商业酶联免疫吸附测定（ELISA）试剂盒进行比较。结果显示，FLISA与商业ELISA试剂盒之间具有良好的相关性（相关系数 = 0.866，P = 0.001）。FLISA的线性范围比ELISA更宽，测量时间比ELISA试剂盒短得多。
这些表明所提出的FLISA方法灵敏且高通量，能够快速筛查血清样本中DNMT1的水平。
