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

45 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

Cite this

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title = "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 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.",
keywords = "POLYCLONAL ANTIBODIES, ESCHERICHIA-COLI, PURIFICATION, FRAGMENTS, QUANTIFICATION, CHROMATOGRAPHY, PHOSPHATASE-1, MOUSE",
author = "J. McElhiney and Drever, {Matthew Ross} and Lawton, {L. A.} and Porter, {Andrew Justin Radcliffe}",
year = "2002",
month = "11",
doi = "10.1128/AEM.68.11.5288-5295.2002",
language = "English",
volume = "68",
pages = "5288--5295",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
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TY - JOUR

T1 - 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

AU - McElhiney, J.

AU - Drever, Matthew Ross

AU - Lawton, L. A.

AU - Porter, Andrew Justin Radcliffe

PY - 2002/11

Y1 - 2002/11

N2 - 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.

AB - 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.

KW - POLYCLONAL ANTIBODIES

KW - ESCHERICHIA-COLI

KW - PURIFICATION

KW - FRAGMENTS

KW - QUANTIFICATION

KW - CHROMATOGRAPHY

KW - PHOSPHATASE-1

KW - MOUSE

U2 - 10.1128/AEM.68.11.5288-5295.2002

DO - 10.1128/AEM.68.11.5288-5295.2002

M3 - Article

VL - 68

SP - 5288

EP - 5295

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 11

ER -