NDLI: Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

Content Provider	Royal Society of Chemistry (RSC)
Author	Ding, Shuangyang Wen, Kai Pei, Xingyao Xie, Jie Wang, Qi Jiang, Haiyang Chen, Min Xie, Sanlei Wang, Zhanhui
Copyright Year	2015
Abstract	A fluorescent single-domain antibody (fluobody), consisting of a single-chain variable fragment antibody (scFv) and a green fluorescent protein extracted from aequorea coerulescens (AcGFP), was produced and used to develop a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) for the detection of avermectins (AVMs). The scFv gene was prepared by cloning VH and VL genes from a hybridoma cell line (2C11) and then fused to the C-terminus of AcGFP (fluobody) with a flexible peptide linker (Gly4Ser)2 between the two domains. After expression and purification, the functional fluobody was used to develop a one-step FLISA protocol for the determination of AVMs in milk samples. The 50% inhibition concentration (IC50) value and the limit of detection (LOD) of the optimized immunoassay for abamectin (ABM) were 2.13 and 1.07 ng mL−1, respectively. Cross-reactivity studies showed that the fluobody exhibited high affinity for the other four AVMs. The recoveries from the spiked milk samples ranged between 86.8 and 125.0%, with relative standard deviation lower than 10.2%. Moreover, the developed FLISA was applied to field samples, followed by confirmation with liquid chromatography-fluorescence detection (LC-FLD) analysis. The consistency of results between the immunoassay and instrumental techniques in detecting the presence of AVMs near the detection limit of the FLISA indicated that the newly developed method is suitable for rapid screening of AVM contamination in food samples prior to chromatographic analysis.
Starting Page	3728
Ending Page	3734
Page Count	7
File Format	HTM / HTML PDF
ISSN	17599660
Volume Number	7
Issue Number	9
Journal	Analytical Methods
DOI	10.1039/c5ay00305a
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Abamectin Immunoassay Cross-reactivity Coefficient of variation C-terminus Chromatography Hybridoma technology Gene Green fluorescent protein IC50 AVM Single-chain variable fragment Flisa Peptide
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Engineering Chemical Engineering

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