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Quantification of cystatin C by time-resolved fluorometry-based immunoassays

Ristiniemi, Noora; Qin, Qiu-Ping; Lindström, Veronica LU ; Grubb, Anders LU and Pettersson, Kim (2012) In Journal of Immunological Methods 378(1-2). p.56-61
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... (More)
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 mu L of sample, while methods 1 and 2 had washing steps, took 40 min and 15 min at 37 degrees C, respectively, but used only 10 mu L 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 0.25-20 mg/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. (C) 2012 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cystatin C, Immunoassay, Heterogeneous assay, Homogeneous assay, Time-resolved fluorescence
in
Journal of Immunological Methods
volume
378
issue
1-2
pages
56 - 61
publisher
Elsevier
external identifiers
  • wos:000304285900007
  • scopus:84859425466
ISSN
1872-7905
DOI
10.1016/j.jim.2012.02.004
language
English
LU publication?
yes
id
4155d9c5-cea1-4663-aaa6-96f6fb8f9a1a (old id 2809703)
date added to LUP
2012-07-03 10:24:25
date last changed
2017-01-01 06:25:36
@article{4155d9c5-cea1-4663-aaa6-96f6fb8f9a1a,
  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 mu L of sample, while methods 1 and 2 had washing steps, took 40 min and 15 min at 37 degrees C, respectively, but used only 10 mu L 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 0.25-20 mg/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. (C) 2012 Elsevier B.V. All rights reserved.},
  author       = {Ristiniemi, Noora and Qin, Qiu-Ping and Lindström, Veronica and Grubb, Anders and Pettersson, Kim},
  issn         = {1872-7905},
  keyword      = {Cystatin C,Immunoassay,Heterogeneous assay,Homogeneous assay,Time-resolved fluorescence},
  language     = {eng},
  number       = {1-2},
  pages        = {56--61},
  publisher    = {Elsevier},
  series       = {Journal of Immunological Methods},
  title        = {Quantification of cystatin C by time-resolved fluorometry-based immunoassays},
  url          = {http://dx.doi.org/10.1016/j.jim.2012.02.004},
  volume       = {378},
  year         = {2012},
}