title: Extension of dynamic range of sensitive nanoparticle-based immunoassays.
abstract: Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07-0.11) and a y-intercept of -7.79 (-17.87-2.29)ng/L (n=65, r=0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.
authors.affiliation: Department of Biotechnology, University of Turku, 20520 Turku, Finland. Electronic address: heidi.hyytia@utu.fi.
authors.forename: Heidi
authors.initials: H
authors.lastname: Hyytiä
authors.affiliation: Department of Biotechnology, University of Turku, 20520 Turku, Finland.
authors.forename: Noora
authors.initials: N
authors.lastname: Ristiniemi
authors.affiliation: HUSLAB, Department of Clinical Chemistry, Helsinki University Hospital, 00029 HUS Helsinki, Finland.
authors.forename: Päivi
authors.initials: P
authors.lastname: Laitinen
authors.affiliation: Department of Biotechnology, University of Turku, 20520 Turku, Finland.
authors.forename: Timo
authors.initials: T
authors.lastname: Lövgren
authors.affiliation: Department of Biotechnology, University of Turku, 20520 Turku, Finland.
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053758:Nanoparticles; D019210:Troponin I / Q000097:blood / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; D019210:Troponin I; C036309:fibrin fragment D
keywords: Desensitization; Dynamic range; Immunoassay; Nanoparticle