该研究将通过反向杨树菇凝集素（Agrocybe aegerita lectin 2，AAL2）的酶联免疫吸附测定法（Enzyme-Linked Immunosorbent Assay，ELISA）检测血浆中末端β-N-乙酰葡糖胺（β-N-acetylglucosamine，GlcNAc）修饰蛋白的水平。分别通过免疫印迹和反向AAL2的ELISA法对血浆中三种候选肿瘤标志物血浆蛋白酶C1抑制剂（plasma protease C1 inhibitor，C1Inh）、Serpin B4和血清转铁蛋白（serotransferrin，TF）的蛋白表达和糖蛋白表达水平进行检测，发现相比健康对照组（Healthy volunteers，HE），乳腺癌组（Breast Cancer，BC）血浆中C1Inh和TF的蛋白水平显著降低而serpin B4蛋白水平显著升高，而其GlcNAc修饰水平均呈相反趋势（p<0.05）。使用接收者操作特征曲线（receiver operating characteristic，ROC）分析指标在健康对照组和早期乳腺癌组的诊断价值，发现蛋白C1Inh、serpin B4和TF的ROC线下面积分别为0.66、0.62和0.73，而GlcNAc修饰的C1Inh、serpin B4和TF的ROC线下面积分别为0.69、0.76和0.83。联合检测GlcNAc修饰的serpin B4和TF可将线下面积在健康组/早期乳腺癌组、健康组/局限性疾病组、健康组/扩散性疾病组中分别提升至0.89、0.89和0.94。该研究为反向AAL2的ELISA法检测血浆中GlcNAc修饰蛋白的应用提供理论依据，且GlcNAc修饰的serpin B4和TF可作为乳腺癌潜在的诊断标志物。

The level of terminal β-N-acetylglucosamine (GlcNAc) - modified protein in plasma was detected using enzyme-linked immunosorbent assay (ELISA) with reverse Agrocybe aegerita lectin2 agglutinin (AAL2). The protein and GlcNAcylated protein levels of three candidate tumor biomarkers - plasma protease C1 inhibitor (C1Inh), serpin B4, and serotransferrin (TF) - in plasma were determined by western blotting and reverse AAL2-based ELISA. When compared to healthy volunteers (HE),in the breast cancer (BC) group, the protein levels of C1Inh and TF in plasma decreased significantly, whereas the level of serpin B4 protein increased significantly, and the level of GlcNAcylated protein showed the opposite trend (p<0.05). Additionally, between the HE group and early-stage BC samples, the area under the receiver operating characteristic (ROC) curve (AUC) for C1Inh, serpin B4, and TF proteins was 0.66, 0.62, and 0.73, respectively, whereas that for GlcNAcylated C1Inh, serpin B4, and TF was 0.69, 0.76, and 0.83, respectively. The combined detection of GlcNAcylated serpin B4 and TF showed improved performance for differentiating BC from HE, with the AUC for HE/early-stage BC samples, HE/limited-stage disease, and HE/extensive-stage disease increasing to 0.89, 0.89, and 0.94, respectively. This study provides a theoretical basis for the detection of GlcNAcylated protein in plasma using reverse AAL2-based ELISA, and GlcNAcylated serpinB4 and TF can serve as potential diagnostic markers of BC.

国家自然科学基金项目（81102850）；广东医科大学大学生创新创业训练计划项目（S202010571060）