title: Improvement of an antibody-enzyme coupling yield by enzyme surface supercharging.
abstract: BACKGROUND
  Protein cross-coupling reactions demand high yields, especially if the products are intended for bioanalytics, like enzyme-linked immunosorbent assays. Amongst other factors, the coupling yield depends on the concentration of the proteins being used for coupling. Protein supercharging of enzymes can increase the solubility dramatically, which could promote enzyme-antibody coupling reactions. A highly soluble, supercharged variant of the enzyme human enteropeptidase light chain was created by a site-directed mutagenesis of surface amino acids, used for the production of an antibody-enzyme conjugate and compared to the wild type enzyme.
  
  
  RESULTS
  Wild type and mutant enzyme could successfully be cross-coupled to an antibody to give antibody-enzyme conjugates suitable for ELISA. Their assay performances and the analysis of the enzyme activities in solution demonstrate that the supercharged version could be coupled to a higher extent, which resulted in better assay sensitivities. The generated conjugate, based on the supercharged enzyme, was feasible as a reporter molecule in a sandwich ELISA and allowed the detection of epidermal growth factor with a detection limit of 15.63 pg (25 pmol/L).
  
  
  CONCLUSIONS
  The highly soluble, surface supercharged, human enteropeptidase light chain mutant provided better yields in coupling the enzyme to an antibody than the wild type. This is most likely related to the higher protein concentration during the coupling. The data suggest that supercharging can be applied favourably to other proteins which have to be covalently linked to other polymers or surfaces with high yields without losses in enzyme activity or specificity.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Centre of Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. agneta.prasse@uni-leipzig.de.
authors.forename: Agneta A
authors.initials: AA
authors.lastname: Prasse
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Centre of Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. T.Zauner@wolfener-analytik.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Zauner
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Centre of Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. karin.buettner@uni-leipzig.de.
authors.forename: Karin
authors.initials: K
authors.lastname: Büttner
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Centre of Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. hoffmann@chemie.uni-leipzig.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Centre of Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. Thole.Zuechner@octapharma.com.|Current address: Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, 69120, Heidelberg, Germany. Thole.Zuechner@octapharma.com.
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D004765:Enteropeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed
chemical_list: D000906:Antibodies; D004765:Enteropeptidase