Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.