Enhanced sandwich immunoassay based on bivalent nanobody as an efficient immobilization approach for foodborne pathogens detection

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 KD, 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 × 103 CFU mL−1 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.