Graduate School, Tianjin Medical University, Tianjin, 300070, China; Tianjin Chest Hospital, Tianjin, 300222, China.
Graduate School, Tianjin Medical University, Tianjin, 300070, China; School of Pharmacy, Tianjin Medical University, Tianjin, 300070, China; Sinopharm Group (Tianjin) East Bookcom Pharmaceutical Co., Ltd, Tianjin, 300051, China.
J Chromatogr A. 2020 May 24;1619:460974. doi: 10.1016/j.chroma.2020.460974. Epub 2020 Feb 14.
Human epidermal growth factor receptor 2 (HER2) gene expresses a transmembrane glycoprotein that is over-expressed in 15-30% breast, 3% lung, and other several digestive cancers. So HER2 is a good biomarker for tumor diagnostic and treatment monitoring. Clinically, detection of HER2 often employs invasive approaches with tissue samples, which at large extent limit its universal application. Shedding of the extracellular domain (ECD) of the HER2 (HER2-ECD) into the circulation has led to the development of a serum test of HER2-ECD as an additional approach to probe the HER2 overexpression. However, few methods were developed due to the high sensitivity required by the serum HER2-ECD determination. In this work, we prepared a novel immunoaffinity in-tube solid phase microextraction (IT-SPME) sorbent for selective enrichment of HER2-ECD. Two clinical available monoclonal antibodies against to HER2, trastuzumab and pertuzumab, were selected as immunoaffinity ligands. Porous layer open tubular capillary with oriented antibody immobilization were fabricated and systematically optimized to afford a higher extraction capacity. The capacity was reached to 120.4 μg/m, which is more than 1000 times higher than that obtained by a common method (directly antibody immobilization on a naked capillary). After sample extraction and enrichment by the IT-SPME, the eluent were determined by a particle-enhanced turbidimetric immunoassay (PETIA). Sensitive quantification of HER2-ECD by the PETIA was thereby accomplished. HER2-ECD concentrations in 82 clinical serum samples were determined by the developed IT-SPME/PETIA method, and the results were well-correlated with that by the clinical used chemiluminescence immunoassay (CLIA). Besides, the IT-SPME/PETIA method was found providing 5 times higher sensitivity than the CLIA, and 500 times higher than the PETIA without IT-SPME. The results indicate that the developed method is suitable for high-sensitive quantification of HER2-ECD in clinical samples.
人表皮生长因子受体 2（HER2）基因表达一种跨膜糖蛋白，在 15-30%的乳腺癌、3%的肺癌和其他几种消化道癌中过表达。因此，HER2 是肿瘤诊断和治疗监测的良好生物标志物。临床上，HER2 的检测通常采用组织样本的侵入性方法，在很大程度上限制了其广泛应用。HER2 的细胞外结构域（ECD）的脱落（HER2-ECD）进入循环系统，导致开发了一种血清 HER2-ECD 检测作为探测 HER2 过表达的附加方法。然而，由于血清 HER2-ECD 测定所需的高灵敏度，很少有方法得到发展。在这项工作中，我们制备了一种新型的免疫亲和管内固相微萃取（IT-SPME）吸附剂，用于选择性富集 HER2-ECD。选择了两种临床可用的针对 HER2 的单克隆抗体，曲妥珠单抗和帕妥珠单抗，作为免疫亲和配体。制备并系统优化了具有定向抗体固定化的多孔层开管毛细管，以提供更高的提取容量。容量达到 120.4μg/m，比常用方法（直接将抗体固定在裸毛细管上）高 1000 多倍。通过 IT-SPME 对样品进行提取和富集后，用颗粒增强浊度免疫分析（PETIA）测定洗脱液。从而通过 PETIA 实现了对 HER2-ECD 的灵敏定量。通过所开发的 IT-SPME/PETIA 方法测定了 82 份临床血清样本中的 HER2-ECD 浓度，结果与临床使用的化学发光免疫分析（CLIA）结果高度相关。此外，与 CLIA 相比，IT-SPME/PETIA 方法的灵敏度提高了 5 倍，与没有 IT-SPME 的 PETIA 相比提高了 500 倍。结果表明，所开发的方法适用于临床样本中 HER2-ECD 的高灵敏度定量。
