Development of an scFv-based Q-body fluorescence immunoassay for detection of hexachlorobenzene in soil and water samples.

Hexachlorobenzene (HCB) is a persistent organic pollutant that remains a concern because of its environmental persistence, bioaccumulation potential, and toxicity to ecosystems and human health. In this study, we developed an anti-HCB single-chain variable fragment (scFv)-based Q-body (quenchbody) fluorescence immunoassay for the rapid detection of HCB in environmental samples. Six scFvs differing in N-terminal cysteine-containing tags and VH/VL orientation were designed, expressed in HEK293 cells, purified, and site-specifically labeled with 5-TAMRA C6 maleimide. Among the tested constructs, the VL-VH format carrying the longer cysteine-containing tag AQIEVNCSNETG showed the most favorable antigen-dependent fluorescence response and was selected for further assay development. After optimization of the working concentration, a homogeneous photoinduced electron transfer (PET) immunoassay was established. Under the optimized conditions, the assay showed a concentration-dependent fluorescence response to HCB, with an EC50 of 584.62 ng mL-1 and a linear working range of 180.39-1894.68 ng mL-1. In spike-and-recovery experiments, recoveries ranged from 85.8 to 107.7% for soil and from 93.2 to 106.4% for water, with relative standard deviations below 4.54%. The developed assay offered the advantages of simple operation, rapid response, and a wash-free format. These results demonstrate that the proposed Q-body-based PET immunoassay is a feasible approach for rapid HCB screening in environmental samples and provides a useful reference for the development of homogeneous fluorescence immunoassays for small-molecule contaminants.