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 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.

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Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.4.587-595.2003 ◽

2003 ◽

Vol 10 (4) ◽

pp. 587-595 ◽

Cited By ~ 21

Author(s):

Xiao K. Deng ◽

Lance A. Nesbit ◽

K. John Morrow

Keyword(s):

Monoclonal Antibody ◽

Clostridium Difficile ◽

Phage Display ◽

Recombinant Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Display System ◽

Single Chain ◽

Single Chain Antibody ◽

Toxin B ◽

Single Chain Antibodies

ABSTRACT Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10−8 M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

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