title: Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA.
abstract: A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.
authors.affiliation: Technische Universität München, Center of Life Sciences Weihenstephan, Department of Plant Sciences, Freising-Weihenstephan, Germany. rau@weihenstephan.de
authors.forename: Doris
authors.initials: D
authors.lastname: Rau
authors.forename: Karl
authors.initials: K
authors.lastname: Kramer
authors.forename: Bertold
authors.initials: B
authors.lastname: Hock
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001280:Atrazine / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013997:Time Factors
chemical_list: D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D001280:Atrazine