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.
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.
McElhiney
Jacqui
J
School of Life Sciences, The Robert Gordon University, St. Andrew Street, Aberdeen AB25 1HG, United Kingdom.
Drever
Mathew
M
Lawton
Linda A
LA
Porter
Andy J
AJ
D003429:Cross Reactions; D000458:Cyanobacteria / Q000737:chemistry*; D007118:Immunoassay; D008387:Marine Toxins; D052998:Microcystins; D019151:Peptide Library; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D010456:Peptides, Cyclic / Q000032:analysis* / Q000276:immunology*; D014867:Water / Q000032:analysis*
D008387:Marine Toxins; D052998:Microcystins; D019151:Peptide Library; D010455:Peptides; D010456:Peptides, Cyclic; D014867:Water; C078588:microcystin; C057862:cyanoginosin LR