Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Pyo, Dongjin; Lee, Jung-Ae; Choi, Euiyule

doi:10.1016/j.microc.2004.07.015

Cited by 35 publications

(23 citation statements)

References 20 publications

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“…In most studies, carboxylic acids at position 3 or 6 of MC-LR were chosen for conjugation to the carrier proteins through the water-soluble carbodiimide method [18,36,37] or the active ester (AE) method [15,26,33]. Some other studies chose to introduce a free amino group at the double bond at position 7 and then use GA to combine H 2 N-MC-LR with carrier proteins [14,20,38].…”

Section: Mc-lr and Nod Conjugate Preparationmentioning

confidence: 99%

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

Yang

Dai²,

Zhang³

et al. 2016

Anal Bioanal Chem

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Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

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Section: Mc-lr and Nod Conjugate Preparationmentioning

confidence: 99%

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

Yang

Dai²,

Zhang³

et al. 2016

Anal Bioanal Chem

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“…EP022). This method is sensitive for low levels of microcystin (Pyo et al 2005;Carmichael and An 1999). The assays were run in 96-well plates containing 0.1 mU enzyme (recombinant protein phosphatase 1A, catalytic subunit, Roche Applied Science), 1.05 mg para-nitrophenyl phosphate (Sigma) and 10 μL of sample or microcystin-LR (Sigma Biochemical).…”

Section: Microcystin Extraction and Analysismentioning

confidence: 99%

Competition between toxic Microcystis aeruginosa and nontoxic Microcystis wesenbergii with Anabaena PCC7120

2011

J Appl Phycol

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To elucidate the changes in the proportions of microcystin (MC)-producing Microcystis, non-MCproducing Microcystis and Anabaena strains during cyanobacteria blooms, we compared their fitness under different initial biomass ratios. Culture experiments were carried out with three cyanobacterial strains: single-celled toxic Microcystis aeruginosa PCC7806 (Ma7806), singlecelled nontoxic Microcystis wesenbergii FACHB-929 (Mw929) and filamentous Anabaena PCC7120 (An7120). Growth curves expressed as biovolume, Ma7806 microcystin-LR (MC-LR) content (detected with HPLC and ELISA), and the culture medium dissolved total nitrogen and dissolved total phosphorous (DTP) were measured to monitor nutrient uptake. Results suggest that the dominant strain in competition experiments between Ma7806 and An7120 was mainly controlled by the initial biomass ratio of the two strains, but there was also evidence for allelopathic interactions, where MC-LR produced by Ma7806 played an important role in the competition process. However, Mw929 was always less competitive when co-cultured with An7120 regardless of initial biomass ratio. Culture medium DTP showed significant differences between competition experiments in all sets, suggesting that Mw929 could be more suited to low phosphorus environments than Ma7806 and An7120. Overall, the competitive ability of Ma7806 was stronger than Mw929 when co-cultured with An7120 in the case of excess nutrients and the results could well unravel the seasonal succession process of cyanobacteria blooms.

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“…LC-MS revealed 98% of 7 in the 40 and 45% fractions (together with 31% of 8), which were combined. This fraction was further purified by SPE, with a gradient of MeOH (25,30,35,40,45,50, and 90%) in water (5 mL per step). The 40% fraction contained 67% of the [Asp 3 ]MC-RY (7), essentially free of 8 or other peptides by LC-MS, and an aliquot containing 100 μg of 7 was evaporated to dryness under a stream of nitrogen.…”

Section: ■ Introductionmentioning

confidence: 99%

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Samdal

Ballot

Løvberg

et al. 2014

Environ. Sci. Technol.

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Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO's maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

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Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Cited by 35 publications

References 20 publications

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

Competition between toxic Microcystis aeruginosa and nontoxic Microcystis wesenbergii with Anabaena PCC7120

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

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