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Dry-Reagent Double-Monoclonal Assay for Cystatin C

Ristiniemi, Noora ; Qin, Qiu-Ping ; Postnikov, Alexander ; Grubb, Anders LU orcid and Pettersson, Kim (2010) In Clinical Chemistry 56(9). p.1424-1431
Abstract
BACKGROUND: Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C. METHODS: We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one... (More)
BACKGROUND: Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C. METHODS: We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one immunoassay concept with time-resolved fluorometry. The assay was performed on an automated immunoanalyzer in single wells that contained all the required assay components. We used heparin-derived plasma samples for methodological evaluation of the assay. RESULTS: From a relative epitope map involving 7 cystatin C-specific antibodies, we selected a pair of antibodies for a 2-site sandwich-type dry-reagent assay. Total assay time was 15 min, and 10 mu L of a 100-fold diluted sample was used. The analytical detection limit (background + 3SD) and functional detection limit (CV 20%) were 0.01 mg/L and 0.02 mg/L, respectively. Within-run and total assay imprecision were < 4.7% and < 5.6% (at 0.84-3.2 mg/L), respectively, and plasma recoveries of added cystatinCwere 94%-110%. Regression analysis with the Roche particle-enhanced immunoturbidimetric method yielded the following (SD): slope, 1.391 (0.029); y-intercept, - 0.152 (0.045) mg/L; S-y vertical bar x = 0.294 mg/L (n = 31). CONCLUSIONS: The developed assay enables rapid and reliable measurement of cystatin C. (C) 2010 American Association for Clinical Chemistry (Less)
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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Clinical Chemistry
volume
56
issue
9
pages
1424 - 1431
publisher
Oxford University Press
external identifiers
  • wos:000281400600012
  • scopus:77956384500
  • pmid:20624851
ISSN
0009-9147
DOI
10.1373/clinchem.2009.141663
language
English
LU publication?
yes
id
8930cbe9-0a52-4e4e-87b6-1daba3f207e8 (old id 1672447)
date added to LUP
2016-04-01 10:15:26
date last changed
2025-01-14 10:09:17
@article{8930cbe9-0a52-4e4e-87b6-1daba3f207e8,
  abstract     = {{BACKGROUND: Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C. METHODS: We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one immunoassay concept with time-resolved fluorometry. The assay was performed on an automated immunoanalyzer in single wells that contained all the required assay components. We used heparin-derived plasma samples for methodological evaluation of the assay. RESULTS: From a relative epitope map involving 7 cystatin C-specific antibodies, we selected a pair of antibodies for a 2-site sandwich-type dry-reagent assay. Total assay time was 15 min, and 10 mu L of a 100-fold diluted sample was used. The analytical detection limit (background + 3SD) and functional detection limit (CV 20%) were 0.01 mg/L and 0.02 mg/L, respectively. Within-run and total assay imprecision were &lt; 4.7% and &lt; 5.6% (at 0.84-3.2 mg/L), respectively, and plasma recoveries of added cystatinCwere 94%-110%. Regression analysis with the Roche particle-enhanced immunoturbidimetric method yielded the following (SD): slope, 1.391 (0.029); y-intercept, - 0.152 (0.045) mg/L; S-y vertical bar x = 0.294 mg/L (n = 31). CONCLUSIONS: The developed assay enables rapid and reliable measurement of cystatin C. (C) 2010 American Association for Clinical Chemistry}},
  author       = {{Ristiniemi, Noora and Qin, Qiu-Ping and Postnikov, Alexander and Grubb, Anders and Pettersson, Kim}},
  issn         = {{0009-9147}},
  language     = {{eng}},
  number       = {{9}},
  pages        = {{1424--1431}},
  publisher    = {{Oxford University Press}},
  series       = {{Clinical Chemistry}},
  title        = {{Dry-Reagent Double-Monoclonal Assay for Cystatin C}},
  url          = {{http://dx.doi.org/10.1373/clinchem.2009.141663}},
  doi          = {{10.1373/clinchem.2009.141663}},
  volume       = {{56}},
  year         = {{2010}},
}