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Applied and Environmental Microbiology, November 2002, p. 5288-5295, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5288-5295.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Jacqui McElhiney,1 Mathew Drever,2 Linda A. Lawton,1* and Andy J. Porter2*

School of Life Sciences, The Robert Gordon University, St. Andrew Street, Aberdeen AB25 1HG,1 Department of Molecular and Cell Biology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom2

Received 24 May 2002/ Accepted 15 August 2002

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 µg 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 µg 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.

* Corresponding author. Mailing address for L. A. Lawton: School of Life Sciences, The Robert Gordon University, St. Andrew St., Aberdeen AB25 1HG, United Kingdom. Phone: 01224 262823. Fax: 01224 262828. E-mail: l.lawton{at}rgu.ac.uk Mailing address for A. J. Porter: Department of Molecular and Cell Biology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Phone: 01224 555870. Fax: 01224 273144. E-mail: a.porter{at}abdn.ac.uk.

Applied and Environmental Microbiology, November 2002, p. 5288-5295, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5288-5295.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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