State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210093, China.
School of Medicine, Nankai University, Tianjin, 300350, China.
Biosens Bioelectron. 2024 Nov 1;263:116598. doi: 10.1016/j.bios.2024.116598. Epub 2024 Jul 22.
Antibody pairs-based immunoassay platforms served as essential and effective tools in the field of pathogen detection. However, the cumbersome preparation and limited detection sensitivity of antibody pairs challenge in establishment of a highly sensitive detection platform. In this study, using COVID-19 testing as a case, we utilized readily accessible nanobodies as detection antibodies and further proposed an accurate design concept with a more scientific and efficient screening strategy to obtain ultrasensitive antibody pairs. We employed nanobodies capable of binding different antigenic epitopes of the nucleocapsid (NP) or receptor-binding domain (RBD) antigens sandwich as substitutes for monoclonal antibodies (mAbs) sandwich in fast detection formats and utilized time-resolved fluorescence (TRF) microspheres as the signal probe. Consequently, we developed a multi-epitope nanobody sandwich-based fluorescence lateral flow immunoassay (FLFA) strip. Our results suggest that the NP antigen had a detection limit of 12.01pg/mL, while the RBD antigen had a limit of 6.51 pg/mL using our FLFA strip. Based on double mAb sandwiches, the values presented herein demonstrated 4 to 32-fold enhancements in sensitivity, and 32 to 256-fold enhancements compared to commercially available antigen lateral flow assay kits. Furthermore, we demonstrated the excellent characteristics of the proposed test strip, including its specificity, stability, accuracy, and repeatability, which underscores its the prospective utility. Indeed, these findings indicate that our established screening strategy along with the multi-epitope nanobody sandwich mode provides an optimized strategy in the field of pathogen detection.
基于抗体对的免疫分析平台在病原体检测领域是必不可少且有效的工具。然而，抗体对的制备繁琐和检测灵敏度有限，这给建立高灵敏度的检测平台带来了挑战。在本研究中，以 COVID-19 检测为例，我们利用易于获得的纳米抗体作为检测抗体，并进一步提出了一种准确的设计概念和更科学、高效的筛选策略，以获得超灵敏的抗体对。我们采用能够结合核衣壳（NP）或受体结合域（RBD）抗原不同抗原表位的纳米抗体作为快速检测模式中单克隆抗体（mAb）夹心的替代品，并利用时间分辨荧光（TRF）微球作为信号探针。因此，我们开发了一种基于多表位纳米抗体夹心的荧光侧向流动免疫分析（FLFA）条。我们的结果表明，使用我们的 FLFA 条，NP 抗原的检测限为 12.01pg/mL，而 RBD 抗原的检测限为 6.51pg/mL。与双 mAb 夹心相比，本文所呈现的值在灵敏度上提高了 4 到 32 倍，与市售的抗原侧向流动检测试剂盒相比，提高了 32 到 256 倍。此外，我们还展示了所提出的测试条的优异特性，包括特异性、稳定性、准确性和重复性，这突出了其潜在的应用价值。事实上，这些发现表明，我们建立的筛选策略以及多表位纳米抗体夹心模式为病原体检测领域提供了一种优化的策略。
