Hazardous small molecules in food and environment seriously threatens human health, which requires sensitive and rapid tools for monitoring. Using a previously identified nanobody against ochratoxin A (OTA), we herein proposed a homogeneous sensing platform \"nanobody/NanoLuc Binary Technology (NanoBiT) system\" and developed a nanobody/NanoBiT system-mediated bioluminescence immunosensor (NBL-Immunosens) for OTA using LgBiT (Lg) and SmBiT (Sm), two subunits of the split nanoluciferase (NanoLuc). The core elements of NBL-Immunosens include Lg-nanobody fusion (NLg) and Sm-labeled OTA-bovine serum albumin conjugate (OSm). The antigen-antibody interaction between NLg and OSm triggers the reconstitution of NanoLuc for generating luminescence signals. Moreover, free OTA can compete with OSm for binding to NLg, resulting the decrease of dose-dependent signals. NBL-Immunosens can detect OTA in a one-step assay of 5 min without washing and exhibit a limit of detection of 0.01 ng/mL with a linear range of 0.04-2.23 ng/mL. It shows high selectivity for OTA and has good accuracy and precision in the spiking-and-recovery experiments. Furthermore, its effectiveness was evaluated with real cereal samples and confirmed by liquid chromatography tandem mass spectrometry and commercial ELISA kits. Hence, the NBL-Immunosens is a very promising tool for rapid, accurate, and selective detection of trace OTA in food.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently emerged picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. A recombinant reporter virus (rSVA-Nluc) stably expressing the nanoluciferase (Nluc) gene between SVA 2A and 2B was developed to rapidly detect anti-SVA neutralizing antibodies and establish a high-throughput screen for antiviral agents. This recombinant virus displayed similar growth kinetics as the parental virus and remained stable for more than 10 passages in BHK-21 cells. As a proof-of-concept for its utility for rapid antiviral screening, this reporter virus was used to rapidly quantify anti-SVA neutralizing antibodies in 13 swine sera samples and screen for antiviral agents, including interferons ribavirin and interferon-stimulated genes (ISGs). Subsequently, interfering RNAs targeting different regions of the SVA genome were screened using the reporter virus. This reporter virus (rSVA-Nluc) represents a useful tool for rapid and quantitative screening and evaluation of antivirals against SVA.

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.

Insulin-like peptide 3 (INSL3) is a reproduction-related peptide hormone belonging to the insulin/relaxin superfamily, which mediates testicular descent in the male fetus, suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the relaxin family peptide receptor 2 (RXFP2). To establish an ultrasensitive receptor-binding assay for INSL3-RXFP2 interaction studies, in the present work we labeled a recombinant INSL3 peptide with a newly developed nanoluciferase (NanoLuc) reporter through a convenient chemical conjugation approach, including the introduction of an active disulfide bond to INSL3 by chemical modification and engineering of a 6× His-Cys-NanoLuc carrying a unique exposed cysteine at the N-terminus. The bioluminescent NanoLuc-conjugated INSL3 retained high binding affinity with the target receptor RXFP2 (Kd = 2.0 ± 0.1 nM, n = 3) and was able to sensitively monitor the receptor-binding of a variety of ligands, representing a novel ultrasensitive tracer for non-radioactive receptor-binding assays. Our present chemical conjugation approach could readily be adapted for conjugation of NanoLuc with other proteins, even other macrobiomolecules, for various highly sensitive bioluminescent assays.