Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov St, 119991 Moscow, Russia.
National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe Shosse, 115409 Moscow, Russia.
Int J Mol Sci. 2022 Apr 19;23(9):4474. doi: 10.3390/ijms23094474.
Express and highly sensitive immunoassays for the quantitative registration of cardiac troponin I (cTnI) are in high demand for early point-of-care differential diagnosis of acute myocardial infarction. The selection of antibodies that feature rapid and tight binding with antigens is crucial for immunoassay rate and sensitivity. A method is presented for the selection of the most promising clones for advanced immunoassays via simultaneous characterization of interaction kinetics of different monoclonal antibodies (mAb) using a direct label-free method of multiplex spectral correlation interferometry. mAb-cTnI interactions were real-time registered on an epoxy-modified microarray glass sensor chip that did not require activation. The covalent immobilization of mAb microdots on its surface provided versatility, convenience, and virtually unlimited multiplexing potential. The kinetics of tracer antibody interaction with the “cTnI—capture antibody” complex was characterized. Algorithms are shown for excluding mutual competition of the tracer/capture antibodies and selecting the optimal pairs for different assay formats. Using the selected mAbs, a lateral flow assay was developed for rapid quantitative cTnI determination based on electronic detection of functionalized magnetic nanoparticles applied as labels (detection limit—0.08 ng/mL, dynamic range > 3 orders). The method can be extended to other molecular biomarkers for high-throughput screening of mAbs and rational development of immunoassays.
对于急性心肌梗死的早期床边快速鉴别诊断，人们迫切需要能够定量检测心肌肌钙蛋白 I（cTnI）的表达和高灵敏度的免疫分析方法。选择具有快速和紧密结合抗原能力的抗体对于免疫分析的速度和灵敏度至关重要。本研究提出了一种通过使用直接无标记的多重光谱相关干涉测量法同时对不同单克隆抗体（mAb）的相互作用动力学进行特征描述，从而筛选出最有前途的克隆，以用于高级免疫分析的方法。在未进行活化处理的情况下，将 mAb 与 cTnI 的相互作用实时登记在经过环氧化修饰的微阵列玻璃传感器芯片上。在其表面共价固定 mAb 微球提供了多功能性、便利性和几乎无限的多重化潜力。对示踪抗体与“cTnI-捕获抗体”复合物相互作用的动力学进行了特征描述。展示了用于排除示踪/捕获抗体相互竞争并为不同分析形式选择最佳配对的算法。使用所选 mAbs，开发了一种基于功能化磁性纳米颗粒作为标记物（检测限为 0.08 ng/mL，动态范围>3 个数量级）的电子检测的快速定量 cTnI 检测的侧向流动测定法。该方法可扩展到其他分子生物标志物，用于高通量筛选 mAbs 和合理开发免疫分析。
