Abstract
A monoclonal antibody for microcystin–leucine–arginine (MC-LR) was produced by cell fusion. The immunogen was synthesized in two steps. First, ovalbumin/ bovine serum albumin was conjugated with 6-acetylthiohexanoic acid using a carbodiimide EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)/ NHS (N-hydroxysulfosuccinimide) reaction. After dialysis, the protein was reacted with MC-LR based on a free radical reaction under basic solution conditions. The protein conjugate was used for immunization based on low volume. The antibodies were identified by indirect competitive (ic)ELISA and were subjected to tap water and lake water analysis. The concentration causing 50% inhibition of binding of MC-LR (IC50) by the competitive indirect ELISA was 0.27 ng/mL. Cross-reactivity to the MC-RR, MC-YR and MC-WR was good. The tap water and lake water matrices had no effect on the detection limit. The analytical recovery of MC-LR in the water samples in the icELISA was 94%–110%. Based on this antibody, an immunochromatographic biosensor was developed with a cut-off value of 1 ng/mL, which could satisfy the requirement of the World Health Organization for MC-LR detection in drinking water. This biosensor could be therefore be used as a fast screening tool in the field detection of MC-LR.
Highlights
Microcystins (MCs) are a series of structurally related hepta- and pentacyclic peptide toxins with molecular weights between 900 and 1200 produced by certain freshwater cyanobacteria, includingOscillatoria agardhii, Nodularia spumigena, Microcystis aeruginosa and Anabaena flos-aquae [1].Contamination with cyanobacteria has a severe effect on drinking water safety and public health.As tumor promoters, MCs cause a range of adverse effects in animals and humans [2]
In monitoring the immune response and screening cell culture supernatants, we developed a highly sensitive indirect competitiveELISA against MC-LR
The protocol for the icELISA was the same as that described previously for screening of cell culture supernatants, except that the antibody was diluted in 0.01 M phosphate buffered saline tween-20 (PBST)-gelatin (0.1 μg/mL), and a series of MC-LR standard solutions (0, 0.02, 0.05, 0.1, 0.2 0.5, 1 and 2 ng/mL) was tested
Summary
Microcystins (MCs) are a series of structurally related hepta- and pentacyclic peptide toxins with molecular weights between 900 and 1200 produced by certain freshwater cyanobacteria, including. In order to monitor environmental contamination with MCs and reduce exposure to humans and animals, highly sensitive and specific methods have been developed for detection of MCs [8,9]. The most common analytical method for the detection of MCs is high-performance liquid chromatography (HPLC) [10,11]. This approach has been extensively applied to detection of toxins in cyanobacterial material. Capillary electrophoresis and related techniques have been considered for quantification of MCs. Capillary electrophoresis and related techniques have been considered for quantification of MCs These methods lack sensitivity compared with HPLC and are not suitable for routine and on-site monitoring of water samples. 10 min and could be used for field detection of MCs
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