Strategies\nfor the Immobilization and Signal Amplification\nof a Double Nanobody Sandwich ELISA for Human Microsomal Epoxide Hydrolase

The microsomal epoxide hydrolase (mEH) is important in\nthe detoxification\nof carcinogens in the liver and other tissues but is also a blood\nbiomarker of hepatitis and liver cancer. Improved analytical methods\nare needed for the study of its role in the metabolism of xenobiotics\nand endogenous roles as a blood biomarker of diseases. The development\nof a double nanobody sandwich ELISA offers significant improvements\nover traditional polyclonal or monoclonal antibody-based assays, enhancing\nboth the homogeneity and the stability of assay production. This study\nfocuses on selecting and optimizing nanobody pairs for detecting human\nmEH. Four high-affinity nanobodies were identified and tested for\nthermal stability. Combinations of these nanobodies were evaluated,\nrevealing that the MQ4–MQ30 pair achieved the best performance\nwith a limit of detection (LOD) of 1 ng/mL. Additionally, polyHRP\nwas also employed for signal amplification, enhancing detection capabilities\ndespite challenges related to the small size and single epitope recognition\nof the nanobodies. Comparative studies using microplates and NHS@MF\nmembranes were also performed. The superior performance of the NHS@MF\nmembranes highlighted their potential as a promising alternative for\npoint-of-care testing. The assay exhibited high specificity for human\nmEH and minimal cross-reactivity with related enzymes and effectively\naddressed matrix effects in plasma and tissue samples. These findings\nunderscore the potential of double nanobody sandwich ELISAs for reliable\nand sensitive biomarker detection.