College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, 510642, China.
Anal Chim Acta. 2024 Feb 8;1289:342209. doi: 10.1016/j.aca.2024.342209. Epub 2024 Jan 4.
Nanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation.
Here, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM K, as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 10 CFU mL for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %.
In this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens.
纳米抗体（Nbs）仅由重链抗体的抗原结合结构域组成，由于其优异的特性，已被广泛应用于各种领域。然而，Nbs 的体积非常小，其抗原结合位点在随机固定为捕获抗体后可能会受到空间位阻，从而导致免疫分析中的检测性能不佳。为了解决这个问题，我们专注于 Nbs 的多价修饰，希望通过将 Nbs 的尺寸在一定程度上扩大来保留其良好稳定性的优势，同时提高其亲和力并减少空间取向的影响。
在这里，我们基于识别不同沙门氏菌表位的 Nb413 和 Nb422 设计了同源和异源二聚体 Nb。与单价 Nb 相比，工程化的二价纳米抗体对肠炎沙门氏菌的亲和力高 2 个数量级，亲和常数（K）低至亚纳摩尔级，根据 Scatchard 分析计算得出。为了进一步探索二价 Nb 在沙门氏菌检测中的潜力，我们基于二价和噬菌体展示的 Nb（BNb-ELISA）建立了用于多重沙门氏菌检测的三明治 ELISA。与基于单价 Nb 的 ELISA 相比，BNb-ELISA 的检测限（LOD）提高了 7.5 倍，达到 2.364×10 CFU mL-1 肠炎沙门氏菌。此外，还评估了该方法用于实际样品中肠炎沙门氏菌检测的可行性，回收率在 73.0%至 125.6%之间，变异系数（CV）低于 7.68%。
在这项研究中，我们首次开发了针对沙门氏菌的二价 Nb，并研究了它们提高的亲和力和对 ELISA 检测性能的影响。它证实了二聚体 Nb 的高结合亲和力和减少固定化抗体结合位点占有率的良好能力。因此，Nbs 的多价修饰被证明是增强基于 Nbs 的食源性病原体免疫分析性能的一种有前途的方法。
