Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
J Pharm Biomed Anal. 2022 Jul 15;216:114804. doi: 10.1016/j.jpba.2022.114804. Epub 2022 Apr 27.
Enzyme-labeled secondary antibody is often used to amplify the output signal in the process of antibody detection. However, its preparation process is complex and time-consuming. Herein, we fabricated an innovative hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide (HRBGO) nanocomposite, used as a substitute of enzyme-labeled second antibody. The synthetic HRBGO was loaded with generous rhodamine B and modified with boronic acid. Therefore, the HRBGO could selectively label the carbohydrate chains of Fc fragment of primary antibody through specific boronate affinity recognition, and then perform signal output and amplification by releasing rhodamine B. To verify the practicability of HRBGO, trastuzumab as a humanized monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2) was selected as model antibody. A glycosylation site-blocked / HER2-immobilized magnetic nanoparticles (GHMN) was also prepared for selectively capturing trastuzumab from complex samples via specific immunoaffinity. Because the glycosylation sites of HER2 can also be labeled with the HRBGO by boronate affinity recognition, these sites were blocked by a masking agent to minimize the background signal. For specific and ultrasensitive detection of trastuzumab, the integration of GHMN and HRBGO was proposed and optimized in detail. Trastuzumab detection based on HRBGO consisted of three steps: specific capture, selective labeling, and output signal. The proposed strategy provided ultrahigh sensitivity with limit of detection of 0.35 fg mL and was successfully applied in the detection of trastuzumab in spiked serum sample with recovery and relative standard deviation in the range of 98.7-103.8% and 3.8-6.0%, respectively. To assess universal applicability, the HRBGO was also successfully used for the determination of anti-SARS-COV2 RBD antibody in human serum sample.
酶标二级抗体常用于抗体检测过程中放大输出信号。然而，其制备过程复杂且耗时。在此，我们制备了一种创新的亲水型负载罗丹明 B/硼酸基修饰氧化石墨烯（HRBGO）纳米复合材料，用作酶标二级抗体的替代品。该合成的 HRBGO 负载有大量罗丹明 B，并修饰有硼酸。因此，HRBGO 可以通过特异性硼酸亲和识别选择性标记抗体 Fc 片段的糖链，然后通过释放罗丹明 B 进行信号输出和放大。为了验证 HRBGO 的实用性，选择曲妥珠单抗作为针对人表皮生长因子受体 2（HER2）的人源化单克隆抗体作为模型抗体。还制备了糖基化位点封闭/HER2 固定化磁性纳米颗粒（GHMN），用于通过特异性免疫亲和作用从复杂样品中选择性捕获曲妥珠单抗。由于 HER2 的糖基化位点也可以通过硼酸亲和识别标记 HRBGO，因此使用掩蔽剂封闭这些位点以最小化背景信号。为了特异性和超灵敏检测曲妥珠单抗，详细提出并优化了 GHMN 和 HRBGO 的整合。基于 HRBGO 的曲妥珠单抗检测由三个步骤组成：特异性捕获、选择性标记和输出信号。所提出的策略提供了超高的灵敏度，检测限为 0.35 fg mL，成功应用于加标血清样品中曲妥珠单抗的检测，回收率和相对标准偏差分别在 98.7-103.8%和 3.8-6.0%范围内。为了评估通用适用性，还成功地将 HRBGO 用于人血清样本中抗 SARS-COV2 RBD 抗体的测定。
