It is long-cherished to develop rapid approaches for the identification and quantification of pesticide and its metabolites. Carbofuran (CBF) and its metabolite 3-hydroxy-carbofuran (3-OH-CBF) are highly toxic to humans and non-target organisms, which makes it mandatory for the detection of their residues in foods and the environment in many countries. Herein, a colloidal goldstrip assay based on a broad-specific monoclonal antibody (mAb) for simultaneous determination of CBF and 3-OH-CBF was developed. The colloidal gold-strips have a runtime within 5 min without complex sample pretreatment and perform a cut-off limit of detection (LOD) of 7-10 ng/mL for both carbofuran and 3-OH-CBF. The developed portable strip assay is a versatile and robust tool for the rapid and simultaneous detection of CBF and 3-OH-CBF in water samples as well as for the determination of illegal addition of CBF in other pesticide preparations.
Carbofuran is a highly toxic insecticide and has been banned or restricted for pest control in many countries. Being a potent acetylcholinesterase inhibitor, carbofuran and its metabolite 3hydroxycarbofuran (3-OH-CBF) are required for the analysis of their residues in food. However, an immunochemical method for simultaneous analysis of carbofuran and 3-OH-CBF is still not available. Herein, we report an enzyme-linked immunosorbent assay (ELISA) based on a broad-specific monoclonal antibody (mAb) for simultaneous detection of carbofuran and 3-OH-CBF. The mAb, designated as 2E3, against both carbofuran and 3-OH-CBF was used to develop an indirect competitive ELISA (icELISA) with 50% inhibition concentrations of 0.76 and 0.69 ng/mL, respectively. The developed icELISA was validated by UPLC-MS/MS and is suitable for the rapid and simultaneous detection of carbofuran and 3-OH-CBF in fruits and vegetables.
BackgroundKynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals.MethodsKAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively.ResultsThis study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1.ConclusionsMouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase.General significanceOur data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked.
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