Wang Yufeng, Gulati Nitya, Regeenes Romario, Migliorini Adriana, Oakie Amanda, Nostro Maria Cristina, Rocheleau Jonathan V
Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto M5G 1L7, Canada.
Institute of Biomedical Engineering, University of Toronto, Toronto M5S 3G9, Canada.
ACS Omega. 2025 Mar 13;10(11):11595-11606. doi: 10.1021/acsomega.5c00761. eCollection 2025 Mar 25.
Fluorescence anisotropy immunoassays (FAIAs) are widely used to quantify the concentration of target proteins based on competitive binding to a monoclonal antibody with a tracer. We recently designed an FAIA to measure mouse C-peptide secretion from living islets in a continuous-flow microfluidic device (InsC-chip). To develop a similar assay for human C-peptide, we selected two monoclonal antibodies (Ab1 and Ab2) that initially showed a low dynamic range and slow kinetics. One option to measure this assay on-chip was to extend the length of the mixing channels. However, this strategy would increase dispersion and ultimately lower the temporal resolution of secreted C-peptide. To shorten the time-to-reach equilibrium for Ab1, we reengineered the tracer based on a comparison between the human and mouse C-peptide sequences, resulting in >30-fold shorter time-to-reach equilibrium. To increase the relatively small dynamic range for Ab2, we used partial epitope mapping and targeted point mutations to increase the dynamic range by 45%. Finally, we validated both FAIAs by measuring depolarization-induced secretion from individual human stem cell-derived islets in our InsC-chip. These data demonstrate a strategy to optimize FAIA kinetics to be measured in continuous-flow microfluidic devices.
荧光偏振免疫分析（FAIA）基于与带有示踪剂的单克隆抗体的竞争性结合，被广泛用于定量目标蛋白的浓度。我们最近设计了一种FAIA，用于在连续流动微流控装置（胰岛芯片）中测量活胰岛分泌的小鼠C肽。为开发一种类似的人C肽检测方法，我们选择了两种单克隆抗体（Ab1和Ab2），它们最初显示出较低的动态范围和较慢的动力学。在芯片上测量该检测方法的一种选择是延长混合通道的长度。然而，这种策略会增加扩散并最终降低分泌C肽的时间分辨率。为缩短Ab1达到平衡的时间，我们基于人C肽和小鼠C肽序列的比较对示踪剂进行了重新设计，使达到平衡的时间缩短了30倍以上。为增加Ab2相对较小的动态范围，我们使用部分表位作图和靶向点突变将动态范围提高了45%。最后，我们通过在我们的胰岛芯片中测量来自单个源自人干细胞的胰岛的去极化诱导分泌，验证了这两种FAIA。这些数据证明了一种优化FAIA动力学以在连续流动微流控装置中进行测量的策略。
