title: Quantification of cystatin C by time-resolved fluorometry-based immunoassays.
abstract: Plasma cystatin C is increasingly used as a marker of glomerular filtration rate. Most assays for cystatin C are based on turbidimetric or nephelometric detection and studies of other rapid methods are limited. This study aimed to develop and compare differently configured immunoassays for quantification of plasma cystatin C, using recombinant cystatin C and two cystatin C-specific antibodies. Method 1 was a two-step sandwich assay with polyclonal antibody as capture and europium chelate-labeled monoclonal antibody as tracer. Method 2 was a one-step heterogeneous competitive assay using immobilized polyclonal antibody and europium-labeled cystatin C. Method 3 was a one-step homogeneous competitive assay with europium-labeled polyclonal antibody as donor and cyanine 5-labeled cystatin C as acceptor. All three assays were evaluated with plasma samples and their performance was compared to a conventional particle-enhanced turbidimetric immunoassay (PETIA). Method 3 was the easiest to perform, with incubation at ambient temperature for 10 min and 20 μL of sample, while methods 1 and 2 had washing steps, took 40 min and 15 min at 37°C, respectively, but used only 10 μL of 100- or 10-fold diluted sample, respectively. The working ranges for methods 1, 2 and 3 were 0.0005-0.2, 0.05-1.0 and 0.25-20mg/L, respectively. Kinetics for method 3 was the fastest with >95% binding completion and for method 2 the slowest with 60% binding completion. All three methods showed good correlation to PETIA, but produced higher cystatin C levels than PETIA. Methods 1 and 3 offered the most favorable performance characteristics and especially method 3 enabled rapid and simple measurement of circulating cystatin C.
authors.affiliation: Department of Biotechnology, University of Turku, Tykistökatu 6A, 20520 Turku, Finland. noora.ristiniemi@utu.fi
authors.forename: Noora
authors.initials: N
authors.lastname: Ristiniemi
authors.forename: Qiu-Ping
authors.initials: QP
authors.lastname: Qin
authors.forename: Veronica
authors.initials: V
authors.lastname: Lindström
authors.forename: Anders
authors.initials: A
authors.lastname: Grubb
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
mesh_terms: D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D055316:Cystatin C / Q000097:blood* / Q000276:immunology*; D005063:Europium / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D005919:Glomerular Filtration Rate; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007677:Kidney Function Tests / Q000379:methods*; D007700:Kinetics; D009391:Nephelometry and Turbidimetry / Q000379:methods; D011994:Recombinant Proteins / Q000097:blood / Q000276:immunology
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D055316:Cystatin C; D011994:Recombinant Proteins; D005063:Europium