Recombinant single chain antibodies in bioelectrochemical sensors

Benhar, Itai; Eshkenazi, Inna; Neufeld, Tova; Opatowsky, J; Shaky, Shelly; Rishpon, Judith

doi:10.1016/s0039-9140(01)00497-0

Cited by 39 publications

(8 citation statements)

References 30 publications

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“…This methodology enabled the isolation of four unique scFv antibodies which were highly reactive against B. anthracis spores. Solution-based screening was applied in the past by several groups for the successful isolation of scFv antibodies (2,23). Previous observations indicate that solution-based screening has some advantages over solid-based screening, particularly for the selection of monomeric over dimeric scFv antibodies (15) and for better recognition of the native antigen (16 6 CFU/ml) were challenged with the purified scFv antibody (1 nM).…”

Section: Discussionmentioning

confidence: 99%

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Mechaly

Zahavy

Fisher

2008

Appl Environ Microbiol

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A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 ؋ 10 6 PFU) was biopanned against live, native B. anthracis ATCC ⌬14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 ؋ 10 8 ؎ 1 ؋ 10 8 M ؊1 ) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.Bacillus anthracis spores, the primary infectious agents causing anthrax, are probably the most likely candidates for a biological terrorist assault. Therefore, rapid detection of spores is critical for a timely response and successful treatment of the disease. Various immunoassays based on the high specificity of antibodies have been developed for the rapid detection of several pathogens. Over the past decade, recombinant technology enabled the production of engineered antibody fragments such as Fabs or single-chain Fv (scFv) antibodies. An scFv antibody is a small engineered antibody in which the variable heavy chain and light chain of the antibody molecule are connected by a short, flexible polypeptide linker. Phage display technology enables the presentation of scFv antibody on the phage surface and has been used successfully for the isolation of specific scFv antibodies from animal repertoire libraries via several enrichment cycles.Using scFv antibodies for antigen detection has several advantages. scFv antibodies can be produced in large quantities in bacterial expression systems, with high reproducibility at low cost, and can be manipulated genetically for improved specificity and affinity (5,15,17). The recombinant antibody can also be fused to marker molecules for detection purposes (25). Recombinant antibodies have been developed for treatment of anthrax infection (26) and disease detection in clinical applications (25). However, the use of recombinant antibodies for detection of B. anthracis spores has not been described. In this study, we describe the construction of an scFv antibody and its successful application for B. anthracis spore detection. Bacillus cultures and sporulation. Spores of all strains were produced in SSM sporulation medium, as previously described (6). MATERIALS AND METHODS Bacteria. B. anthracis ⌬14185 is a nontoxinogenic, nonencapsulated (ToxImmunization. Female BALB/c mice were immunized subcutaneously with 1 ϫ 10 7 CFU of irradiated (15 min at maximum intensity in a microwave oven) B. ...

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Section: Discussionmentioning

confidence: 99%

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Mechaly

Zahavy

Fisher

2008

Appl Environ Microbiol

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“…Industries such as Caliper Technologies, Cepheid, Nanogen, ACLARA BioSciences, MICROGEN Systems, and Lawrence Livermore Laboratories are developing microfabricated systems for detection and identification of specific microbial agents [105]. Advances in antibody production and the recent emergence of phage-displayed peptide biosensors offer increased possibilities for the rapid detection of pathogens [110,111], but still require time-consuming preenrichment in order to detect low numbers of pathogens in water [112].…”

Section: Microorganismsmentioning

confidence: 99%

“…Current research includes combinatorial screening or design of the amino acid sequence of the binding region of antibody. In this sense, recombinant antibodies provide an emerging strategy in the development of new immunosensors [110]. A recently developed method that generates antibody-like molecules that bind specifically to target molecules is "phage display".…”

Section: Bioengineeringmentioning

confidence: 99%

“…Benhar et al [110], for example, presented single-chain phage-displayed antibodies combined with amperometric detection and its application as an immunosensor for the detection of three different analytes. Goldman et al [111] demonstrated the potential of a phagebased biosensor for the determination of a staphylococcal enterotoxin B (SEB).…”

Section: Bioengineeringmentioning

confidence: 99%

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Biosensors for environmental applications: Future development trends

Rodríguez-Mozaz

Marco

Alda

et al. 2004

Pure and Applied Chemistry

199

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Biosensors can be excellent analytical tools for monitoring programs working to implement legislation. In this article, biosensors for environmental analysis and monitoring are extensively reviewed. Examples of biosensors for the most important families of environmental pollutants, including some commercial devices, are presented. Finally, future trends in biosensor development are discussed. In this context, bioelectronics, nanotechnology, miniaturization, and especially biotechnology seem to be growing areas that will have a marked influence on the development of new biosensing strategies in the next future.

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“…It involves the display of a library of single‐chain antibody fragments (scFv) on the surface of filamentous phage followed by selection of the desired recombinant phage by means of specific binding to an antigen of interest. Although phage display has advantages over conventional polyclonal and monoclonal antibody production, few reports (Benhar et al. 2001) have employed this technique for the selection of reagents for the detection of foodborne pathogens.…”

Section: Introductionmentioning

confidence: 99%

A LISTERIA MONOCYTOGENES‐SPECIFIC PHAGE‐DISPLAYED ANTIBODY FRAGMENT RECOGNIZES A CELL SURFACE PROTEIN WHOSE EXPRESSION IS REGULATED BY PHYSIOLOGICAL CONDITIONS¹

Paoli¹,

Brewster

2007

Rapid Methods Automation Mic

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We recently selected a phage-displayed single-chain antibody that detects several strains of Listeria monocytogenes and does not cross-react with any of the other five species of Listeria. The efficacy of a phage-displayed anti-L. monocytogenes single-chain antibody as a detection reagent was examined by enzyme-linked immunosorbent assay using L. monocytogenes grown at different temperatures and in a variety of media commonly used for the isolation of L. monocytogenes from food. The best results were observed when cells were grown in supplemented Fraser enrichment broth. The antigen detected by the phage-displayed antibody was present on the surface of the cells grown between 20 and 42C, but was not present on the cell surface when cells were grown at or below 15C. As determined by Western blot analysis, the antibody bound to a protein with apparent molecular mass of approximately 90 kD. Identification of the protein antigen would allow a more rational approach to the development of an antibody-based method for the specific detection of L. monocytogenes. PRACTICAL APPLICATIONSAntibody phage display is an alternative approach to the selection of immunoreagents for the detection of food-borne pathogens. Recently, a phage-1 Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. 2 Corresponding 77 displayed antibody that detected several strains of Listeria monocytogenes, and did not cross-react with any of the other five species of Listeria, was selected from an antibody phage display library. In this work, the efficacy of this phage-displayed antibody for the detection of L. monocytogenes was examined using cells grown under a variety of conditions. In addition, the target that is detected by the phage-displayed antibody was characterized. Characterization of the L. monocytogenes-specific phage-displayed antibody fragment will allow a more rational approach toward the development of methods for the rapid and specific detection of L. monocytogenes from food.

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Recombinant single chain antibodies in bioelectrochemical sensors

Cited by 39 publications

References 30 publications

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Biosensors for environmental applications: Future development trends

A LISTERIA MONOCYTOGENES‐SPECIFIC PHAGE‐DISPLAYED ANTIBODY FRAGMENT RECOGNIZES A CELL SURFACE PROTEIN WHOSE EXPRESSION IS REGULATED BY PHYSIOLOGICAL CONDITIONS¹

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Recombinant single chain antibodies in bioelectrochemical sensors

Cited by 39 publications

References 30 publications

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

Biosensors for environmental applications: Future development trends

A LISTERIA MONOCYTOGENES‐SPECIFIC PHAGE‐DISPLAYED ANTIBODY FRAGMENT RECOGNIZES A CELL SURFACE PROTEIN WHOSE EXPRESSION IS REGULATED BY PHYSIOLOGICAL CONDITIONS1

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A LISTERIA MONOCYTOGENES‐SPECIFIC PHAGE‐DISPLAYED ANTIBODY FRAGMENT RECOGNIZES A CELL SURFACE PROTEIN WHOSE EXPRESSION IS REGULATED BY PHYSIOLOGICAL CONDITIONS¹