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Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA.
J Immunoassay Immunochem. 2002; 23(2):129-43.JI

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+Show Affiliations

Technische Universität München, Center of Life Sciences Weihenstephan, Department of Plant Sciences, Freising-Weihenstephan, Germany. rau@weihenstephan.deNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

12033639

Citation

Rau, Doris, et al. "Single-chain Fv Antibody-alkaline Phosphatase Fusion Proteins Produced By One-step Cloning as Rapid Detection Tools for ELISA." Journal of Immunoassay & Immunochemistry, vol. 23, no. 2, 2002, pp. 129-43.
Rau D, Kramer K, Hock B. Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA. J Immunoassay Immunochem. 2002;23(2):129-43.
Rau, D., Kramer, K., & Hock, B. (2002). Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA. Journal of Immunoassay & Immunochemistry, 23(2), 129-43.
Rau D, Kramer K, Hock B. Single-chain Fv Antibody-alkaline Phosphatase Fusion Proteins Produced By One-step Cloning as Rapid Detection Tools for ELISA. J Immunoassay Immunochem. 2002;23(2):129-43. PubMed PMID: 12033639.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA. AU - Rau,Doris, AU - Kramer,Karl, AU - Hock,Bertold, PY - 2002/5/30/pubmed PY - 2002/11/30/medline PY - 2002/5/30/entrez SP - 129 EP - 43 JF - Journal of immunoassay & immunochemistry JO - J Immunoassay Immunochem VL - 23 IS - 2 N2 - 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. SN - 1532-1819 UR - https://www.unboundmedicine.com/medline/citation/12033639/Single_chain_Fv_antibody_alkaline_phosphatase_fusion_proteins_produced_by_one_step_cloning_as_rapid_detection_tools_for_ELISA_ L2 - http://www.tandfonline.com/doi/full/10.1081/IAS-120003657 DB - PRIME DP - Unbound Medicine ER -