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Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water.

ABSTRACT: A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

SUBMITTER: McElhiney J

PROVIDER: S-EPMC129936 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water.

McElhiney Jacqui J Drever Mathew M Lawton Linda A LA Porter Andy J AJ

Applied and environmental microbiology 20021101 11

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels ...[more]

PMID: 12406716

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Project description:BackgroundCyanobacterial harmful algal blooms (CyanoHABs) originate from the excessive growth or bloom of cyanobacteria often referred to as blue-green algae. They have been on the rise globally in both marine and freshwaters in recently years with increasing frequency and severity owing to the rising temperature associated with climate change and increasing anthropogenic eutrophication from agricultural runoff and urbanization. Humans are at a great risk of exposure to toxins released from CyanoHABs through drinking water, food, and recreational activities, making CyanoHAB toxins a new class of contaminants of emerging concern.ObjectivesWe investigated the toxic effects and mechanisms of microcystin-LR (MC-LR), the most prevalent CyanoHAB toxin, on the ovary and associated reproductive functions.MethodsMouse models with either chronic daily oral or acute intraperitoneal exposure, an engineered three-dimensional ovarian follicle culture system, and human primary ovarian granulosa cells were tested with MC-LR of various dose levels. Single-follicle RNA sequencing, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, immunohistochemistry (IHC), and benchmark dose modeling were used to examine the effects of MC-LR on follicle maturation, hormone secretion, ovulation, and luteinization.ResultsMice exposed long term to low-dose MC-LR did not exhibit any differences in the kinetics of folliculogenesis, but they had significantly fewer corpora lutea compared with control mice. Superovulation models further showed that mice exposed to MC-LR during the follicle maturation window had significantly fewer ovulated oocytes. IHC results revealed ovarian distribution of MC-LR, and mice exposed to MC-LR had significantly lower expression of key follicle maturation mediators. Mechanistically, in both murine and human granulosa cells exposed to MC-LR, there was reduced protein phosphatase 1 (PP1) activity, disrupted PP1-mediated PI3K/AKT/FOXO1 signaling, and less expression of follicle maturation-related genes.DiscussionUsing both in vivo and in vitro murine and human model systems, we provide data suggesting that environmentally relevant exposure to the CyanoHAB toxin MC-LR interfered with gonadotropin-dependent follicle maturation and ovulation. We conclude that MC-LR may pose a nonnegligible risk to women's reproductive health by heightening the probability of irregular menstrual cycles and infertility related to ovulatory disorders. https://doi.org/10.1289/EHP12034.

Dataset Information

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water.

Publications

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water.

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