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

J. McElhiney, Matthew Ross Drever, L. A. Lawton, Andrew Justin Radcliffe Porter

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

A naive (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 mug 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 mug of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elation 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.

Original languageEnglish
Pages (from-to)5288-5295
Number of pages7
JournalApplied and Environmental Microbiology
Volume68
Issue number11
DOIs
Publication statusPublished - Nov 2002

Keywords

  • POLYCLONAL ANTIBODIES
  • ESCHERICHIA-COLI
  • PURIFICATION
  • FRAGMENTS
  • QUANTIFICATION
  • CHROMATOGRAPHY
  • PHOSPHATASE-1
  • MOUSE

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