Heterologous antigen selection of chicken single-chain variable fragments against thiamethoxam.
Shaopeng GuYating WangDi WangYayun YangXianle XueTing XuShaopeng GuFang TangPublished in: Analytical methods : advancing methods and applications (2024)
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 (IC 50 ) 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.
Keyphrases
- high throughput
- high performance liquid chromatography
- label free
- loop mediated isothermal amplification
- real time pcr
- risk assessment
- mass spectrometry
- single cell
- tandem mass spectrometry
- climate change
- machine learning
- solid phase extraction
- big data
- saccharomyces cerevisiae
- monoclonal antibody
- bacillus subtilis