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Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay.
Anal Biochem. 1997 Jul 01; 249(2):219-27.AB

Abstract

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques.

Authors+Show Affiliations

Institute of Applied Microbiology, University of Agriculture, Vienna, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9212874

Citation

Kerschbaumer, R J., et al. "Single-chain Fv Fusion Proteins Suitable as Coating and Detecting Reagents in a Double Antibody Sandwich Enzyme-linked Immunosorbent Assay." Analytical Biochemistry, vol. 249, no. 2, 1997, pp. 219-27.
Kerschbaumer RJ, Hirschl S, Kaufmann A, et al. Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Anal Biochem. 1997;249(2):219-27.
Kerschbaumer, R. J., Hirschl, S., Kaufmann, A., Ibl, M., Koenig, R., & Himmler, G. (1997). Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Analytical Biochemistry, 249(2), 219-27.
Kerschbaumer RJ, et al. Single-chain Fv Fusion Proteins Suitable as Coating and Detecting Reagents in a Double Antibody Sandwich Enzyme-linked Immunosorbent Assay. Anal Biochem. 1997 Jul 1;249(2):219-27. PubMed PMID: 9212874.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. AU - Kerschbaumer,R J, AU - Hirschl,S, AU - Kaufmann,A, AU - Ibl,M, AU - Koenig,R, AU - Himmler,G, PY - 1997/7/1/pubmed PY - 1997/7/1/medline PY - 1997/7/1/entrez SP - 219 EP - 27 JF - Analytical biochemistry JO - Anal. Biochem. VL - 249 IS - 2 N2 - A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/9212874/Single_chain_Fv_fusion_proteins_suitable_as_coating_and_detecting_reagents_in_a_double_antibody_sandwich_enzyme_linked_immunosorbent_assay_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(97)92171-0 DB - PRIME DP - Unbound Medicine ER -