Abstract
Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.
Highlights
Organophosphorous pesticides (OPPs), which are known as acetylcholinesterase inhibitors, are widely used in pest control [1]
The results showed that this scFv antibody could be used as an effective reagent for triazophos monitoring because of its high affinity to the analyte
With the primer sets of specific isotypes, VH and VLλ genes were amplified from the cDNA of monoclonal antibody (mAb)-8C10 (Figure 1A)
Summary
Organophosphorous pesticides (OPPs), which are known as acetylcholinesterase inhibitors, are widely used in pest control [1]. The inappropriate and essentially unregulated use of these compounds has endangered human health because of occupational or environmental exposure [1,2]. Triazophos is a non-systemic broad spectrum OPP that negatively affects organisms. Female rats suffer from blood, kidney, and liver toxicities, as well as changes in hormone levels, after long-term exposure to low concentrations of triazophos [5]. Researchers found that chronic exposure to triazophos significantly impaired the learning and memory function of rats [6]. To minimize the danger of triazophos residues on human health, a strict limit of 0.01 mg/kg in food was set by the British Health and Safety Executive (https://secure.pesticides.gov.uk/MRLs/search.asp). Regular monitoring of triazophos residues in food and environmental samples is necessary
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