DNA甲基转移酶1（DNA methyltransferases1, DNMT1）负责维持DNA甲基化遗传稳定性, 其表达量与多种肿瘤的发生发展密切相关, 是一种重要的肿瘤分子标志物。 DNA甲基化转移酶（DNA methyltransferases, DNMTs）的现有检测方法大多基于原核生物酶建立, 且局限于实验室研究, 因此建立一种高灵敏、 高通量测定人血清中DNMT1含量的荧光免疫分析方法（FLISA）, 以期为癌症早期筛查及临床应用提供新思路。 以Fe3O4磁珠为固相载体, 采用碳二亚胺/N-羟基硫代琥珀酰亚胺(EDC/Sulfo-NHS)键合法分别制备磁性免疫捕获探针Fe3O4@DNMT1小鼠单克隆抗体（Fe3O4@McAbDNMT1）及荧光免疫检测探针5-羧基四甲基罗丹明@DNMT1兔多克隆抗体（5-TAMRA@PcAbDNMT1）。 用红外光谱、 紫外-可见（UV-Vis）光谱、 Zeta电位、 免疫活性分别表征偶联产物的结构与活性。 在此基础上, 采用双抗体夹心法, 检测黑色96微孔板中反应产物的荧光强度, 根据荧光强度与DNMT1浓度的关系进行定量分析, 并进行方法学评价与比较。 实验结果显示, 双探针偶联成功, 并保持了原抗体的免疫活性。 该方法的线性方程y=222.046+48.323x, 线性范围0.05～80 ng·mL-1, 相关系数为0.991 4, 检出限为0.005 ng·mL-1, 板内、 板间的RSD分别为4.7%～8.8%, 1.6%～10.0%（n=6）； 板内、 板间的回收率分别为91.3%～102.4%, 88.0%～98.8%（n=6）； 方法特异性良好。 与酶联免疫吸附法（ELISA）和磁酶免疫吸附法（MELISA）相比, FLISA法的检出限最低, 灵敏度最高, 所需分析时间最短； FLISA法与商品化ELISA试剂盒对15例人血清样品进行分析, 差异无统计学意义（p＞0.05）。 表明本研究所建立的FLISA方法灵敏快速, 适用于大批量人血清样品中DNMT1的快速测定, 在临床诊断方面具有重要的应用价值和广阔的应用前景。

DNA methyltransferases1 (DNMT1), a dominant enzyme responsible for maintaining the genetic stability of DNA methylation, is closely related to the occurrence and development of a variety of tumors. However, till now, most of the studies on DNA methyltransferases (DNMTs) detection have focused on prokaryote methyltransferases and been limited to laboratory studies. Therefore, a fluorescence immunoassay (FLISA) for the high sensitivity and high throughput detection of DNMT1 level in human serum samples was established to provide new ideas for early cancer diagnosis and clinical cancer therapy. The functional carboxyl Fe3O4 magnetic beads were used as solid phase carriers. 1-chloride-3-dimethylamino-propyl-3-ethylcarbodiimide hydrochloric acid (EDC) and sulfo-N-hydroxysuccinimide (Sulfo-NHS) were used as coupling agents to prepare immune-magnetic capture probe Fe3O4@ McAbDNMT1 (monoclonal antibody DNMT1). After that, the immune-fluorescent detective probe 5-TAMRA@PcAbDNMT1（5-carboxytetramethylrhodamine@polyclonal antibody DNMT1）was also made. Their structure and activity were characterized by infrared absorption, UV-Vis spectra, Zeta potential and immunocompetence. Then, the content of DNMT1 in human serum samples was detected based on the double-anti-sandwich method by FLISA with high sensitivity. Finally, methodology evaluation and comparison were conducted. The results showed that the probes conjugated successfully and maintained the immunocompetence of the original antibody. The linear equation was y=222.046+48.323x, the linear range was 0.05～80 ng·mL-1, and the correlation coefficient was 0.991 4, the detection limit was 0.005 ng·mL-1, the intra-and inter-panel RSD was 4.7%～8.8% and 1.6%～10.0% (n=6), respectively, intra- and inter-panel recoveries were 91.3%～102.4% and 88.0%～98.8% (n=6), respectively. The cross-reactivity rates with other two DNA methyltransferase 3a/3b were lower, and the specificity of FLISA was well. Compared with ELISA and MELISA, FLISA has the lowest detection limit and shortest analysis time. Compared with ELISA kits, the result displayed a high correlation between two methods, and the difference of them was not statistically significant (p＞0.05). The result suggests that the FLISA system would be used to detect multiple samples at the same time for the rapid analysis of DNMT1 in human serum samples.