Engineering antibody conjugation and valency for optimized nanoparticle-based immunoassays

The introduction of nanoparticles (NPs) has led to substantial improvements in the performance of immunoassays. However, the further development of NP-based immunoassays is hampered by the lack of precise conjugation strategies and a systematic investigation of how antibody valency affects assay performance.

Using homogeneous chemiluminescence immunoassays (HCLIA) as a model system, we present a novel site-specific coupling strategy. This strategy integrates cysteine-engineered single-chain variable fragment (scFv), click chemistry, and biotin-streptavidin interactions to precisely control scFv loading density on NPs. Compared to the traditional chemical conjugation and secondary antibody capture methods, this strategy shown significantly higher conjugates structural uniformity, superior antibody functionality retention, and enhanced assay sensitivity and reproducibility. Then, we present a quantitative study elucidating how antibody valency governs the sensitivity and robustness of HCLIA. Results show that low valency (coupling proportion <10 %) compromised sensitivity and robustness, medium antibody valency (10 % < coupling proportion <50 %) maximized sensitivity, while high valency (coupling proportion >50 %) significantly enhanced robustness at the expense of sensitivity. These insights enabled development of an optimized HCLIA for detecting 17 quinolones (QNs) in milk, which exhibited exceptional sensitivity with limits of detection ranging from 0.010 to 0.131 μg/L.

This study presents a precisely controllable antibody conjugation strategy and advances our understanding of the effect of antibody valency, offering key guidelines for the rational design of future NP-based immunoassays. The established HCLIA method can be used as a practical tool for the detection of QNs residues in milk.