Heterologous antigen selection of chicken single-chain variable fragments against thiamethoxam.

Single-chain variable fragments (scFvs) are valuable in the development of immunoassays for pesticide detection. In this study, scFvs specific to thiamethoxam (Thi) were successfully isolated from a library generated by chicken immunization through heterologous coating selection. These scFvs were subsequently expressed with fusion with an Avi tag and alkaline phosphatase. After combination and optimization, a scFv-biotin based enzyme linked immunosorbent assay (ELISA) was developed for the detection of Thi, demonstrating an impressive half-maximum signal inhibition concentration (IC50) of 30 ng mL-1 and a limit of detection (LOD) of 1.8 ng mL-1. The immunoassay exhibited minimal cross-reactivity with other neonicotinoid insecticides, except for 7.5% for imidacloprid and 6.7% for imidaclothiz. The accuracy of the assay was confirmed by testing spiked samples of apple, pear, cabbage, and cucumber, which resulted in average recoveries ranging between 82% and 119%, closely aligning with the results obtained through high-performance liquid chromatography. Therefore, the chicken scFv-biotin based assay showed promise as a high-throughput screening tool for Thi in agricultural samples.

Biosynthesis of magnetosome-nanobody complex in Magnetospirillum gryphiswaldense MSR-1 and a magnetosome-nanobody-based enzyme-linked immunosorbent assay for the detection of tetrabromobisphenol A in water.

In this study, two mutant strains, TBC and TBC+, able to biosynthesize a novel functional magnetosome-nanobody (Nb), were derived from the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1. The magnetosome-Nbs biosynthesized by TBC+ containing multi-copies of the Nb gene had a higher binding ability to an environmental pollutant, tetrabromobisphenol A (TBBPA), than those biosynthesized by TBC containing only one copy of the Nb gene. The magnetosome-Nbs from TBC+ can effectively bind to TBBPA in solutions with high capacity without being affected by a broad range of NaCl and methanol concentrations as well as pH. Therefore, a magnetosome-Nb-based enzyme-linked immunosorbent assay (ELISA) was developed and optimized for the detection of TBBPA, yielding a half-maximum signal inhibition concentration of 0.23 ng/mL and a limit of detection of 0.025 ng/mL. The assay was used to detect TBBPA in spiked river water samples, giving average recoveries between 90 and 120% and coefficients of variation of 2.5-6.3%. The magnetosome-Nb complex could be reused 4 times in ELISA without affecting the performance of the assay. Our results demonstrate the potential of magnetosome-Nbs produced by TBC+ as cost-effective and environment-friendly reagents for immunoassays to detect small molecules in environmental waters.

Proteomic analysis reveals oxidative stress-induced activation of Hippo signaling in thiamethoxam-exposed Drosophila.

Thiamethoxam (THIA) is a widely used neonicotinoid insecticide. However, the toxicity and defense mechanisms activated in THIA-exposed insects are unclear. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology to identify changes in protein expression in THIA-exposed Drosophila. We found that the antioxidant proteins Cyp6a23 and Dys were upregulated, whereas vir-1 was downregulated, which may have been detoxification in response to THIA exposure. Prx5 downregulation promoted the generation of reactive oxygen species. Furthermore, the accumulation of reactive oxygen species led to the induction of antioxidant defenses in THIA-exposed Drosophila, thereby enhancing the levels of oxidative stress markers (e.g., superoxide dismutase, glutathione S-transferase, and glutathione) and reducing catalase expression. Furthermore, the Hippo signaling transcription coactivator Yki was inactivated by THIA. Our results suggesting that Hippo signaling may be necessary to promote insect survival in response to neonicotinoid insecticide toxicity.

Development of a Genetically Encoded Magnetic Platform in Magnetospirillum gryphiswaldense MSR-1 for Downstream Processing of Protein Expression System.

Background: Protein downstream processing remains a challenge in protein production, especially in low yields of products, in spite of ensuring effective disruption of cell and separation of target proteins. It is complicated, expensive and time-consuming. Here, we report a novel nano-bio-purification system for producing recombinant proteins of interest with automatic purification from engineered bacteria. Results: This system employed a complete genetic engineering downstream processing platform for proteins at low expression levels, referred to as a genetically encoded magnetic platform (GEMP). GEMP consists of four elements as follows. (1) A truncated phage lambda lysis cassette (RRz/Rz1) is controllable for lysis of Magnetospirillum gryphiswaldense MSR-1 (host cell). (2) A surface-expressed nuclease (NucA) is to reduce viscosity of homogenate by hydrolyzing long chain nucleic acids. (3) A bacteriogenic magnetic nanoparticle, known as magnetosome, allows an easy separation system in a magnetic field. (4) An intein realizes abscission of products (nanobodies against tetrabromobisphenol A) from magnetosome. Conclusions: In this work, removal of most impurities greatly simplified the subsequent purification procedure. The system also facilitated the bioproduction of nanomaterials. The developed platform can substantially simplify industrial protein production and reduce its cost.

Development of a nanobody-based ELISA for the detection of the insecticides cyantraniliprole and chlorantraniliprole in soil and the vegetable bok choy.

Cyantraniliprole and chlorantraniliprole are anthranilic diamide insecticides acting on ryanodine receptors. In this study, two camel-derived nanobodies (Nbs, named C1 and C2) recognizing cyantraniliprole as well as chlorantraniliprole were generated. C1-based enzyme-linked immunosorbent assays (ELISAs) for the detection of the two insecticides were developed. The half-maximum signal inhibition concentrations (IC50) of cyantraniliprole and chlorantraniliprole by ELISA were 1.2 and 1.5 ng mL-1, respectively. This assay was employed to detect these two insecticides in soil and vegetables. The average recoveries of cyantraniliprole from both bok choy (Brassica chinensis L.) and soil samples were 90-129%, while those of chlorantraniliprole were in a range of 89-120%. The insecticide residues in soil and bok choy, which were collected from plots sprayed with cyantraniliprole and chlorantraniliprole, were simultaneously detected by the resulting ELISA and a high-performance liquid chromatography (HPLC) method, showing a satisfactory correlation. Higher concentrations of chlorantraniliprole than cyantraniliprole were detected in soil and vegetables, which indicates the longer persistence of chlorantraniliprole in the environment.