title: Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay.
abstract: Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s.
authors.affiliation: Ultrasensitive Protein Detection Unit, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Dobslaff
authors.affiliation: Ultrasensitive Protein Detection Unit, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Zscharnack
authors.affiliation: Ultrasensitive Protein Detection Unit, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany. thomas.kreisig@bbz.uni-leipzig.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kreisig
authors.affiliation: Ultrasensitive Protein Detection Unit, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.|Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, 69120, Heidelberg, Germany.
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D055316:Cystatin C / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007118:Immunoassay / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011933:Reagent Kits, Diagnostic
chemical_list: D000911:Antibodies, Monoclonal; D055316:Cystatin C; D010455:Peptides; D011933:Reagent Kits, Diagnostic
keywords: Affinity; Antibody generation; FRET; Homogeneous immunoassays; Peptide design