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.