Quantification and Clinical Relevance of Cystatin C

An aging population and increasing rates of diabetes mellitus contribute to a high prevalence of kidney dysfunction – approximately 10 percent of adults in developed countries have chronic kidney disease (CKD). CKD is a progressive loss of kidney function and this remains permanent. Early recognition of this condition is important for prevention or impeding severe adverse cardiac and renal outcomes. Cystatin C is a low molecular weight cysteine protease inhibitor that has emerged as a biomarker of kidney function. The special potential of plasma cystatin C in this setting is related to its independency of muscle mass, which is a remarkable limitation of the traditional marker creatinine. Cystatin C is a sensitive marker in diagnosing mild and moderate CKD, especially in small children, in the elderly and in conditions where muscle mass is affected. Cystatin C is quantified with immunoassays, mainly based on particle-enhanced nephelometry (PENIA) or turbidimetry (PETIA). The aim of this study was to develop a rapid and reliable assay for quantification of human cystatin C in plasma or serum by utilizing time-resolved fluorescence-based immunoassay methods. This was accomplished by utilizing different antibodies, including polyclonal and 7 monoclonal antibodies against cystatin C. Different assay designs were tested and the best assay was further modified to a dry-reagent double monoclonal assay run on an automated immunonalyzer. This assay was evaluated for clinical performance in estimating reduced kidney function and in predicting risk of adverse outcomes in patients with non-ST elevation acute coronary syndrome. Of the tested assay designs, heterogeneous non-competitive assay had the best performace and was chosen to be developed further. As an automated double monoclonal assay, this assay enabled a reliable measurement of clinically relevant cystatin C concentrations. It also showed a stronger concordance with the reference clearance method than the conventional PETIA method in patients with reduced kidney function. Risk of all-cause mortality and combined events, defined by death and myocardial infarction, increased with higher cystatin C and cystatin C remained an independent predictor of death and combined events after adjustment to nonbiochemical baseline factors. In conclusion, the developed dry-reagent double monoclonal assay allows rapid and reliable quantitative measurement of cystatin C. As measured with the developed assay, cystatin C is a potential predictor of adverse outcomes in cardiac patients.Väestön ikääntyminen ja diabeteksen yleistyminen ovat johtaneet munuaisten vajaatoiminnan esiintyvyyden kasvuun. Nykyään arviolta kymmenyksellä kehittyneiden maiden aikuisväestöstä on krooninen munuaisten vajaatoiminta, joka tarkoittaa etenevää ja pysyvää munuaistoiminnan menetystä. Jos munuaisten vajaatoiminta havaitaan ajoissa, vakavia munuaisiin ja sydämeen liittyviä seurauksia on mahdollista estää tai hidastaa. Kystatiini C on pienimolekyylinen kysteiiniproteaasien inhibiittori, joka on lupaava uusi munuaistoiminnan merkkiaine. Kystatiini C:n suurin tunnettu etu perinteiseen munuaismerkkiaineeseen, kreatiniiniin, nähden on, ettei lihasmassan määrä vaikuta sen pitoisuuteen veressä. Kystatiini C:n on osoitettu olevan herkkä merkkiaine lievän ja kohtalaisen munuaisten vajaatoiminnan diagnosoinnissa, erityisesti pikkulapsilla, ikääntyneillä sekä erilaisissa tiloissa, joissa lihasmassan määrä on normaalista poikkeava. Kystatiini C -pitoisuutta mitataan immunomäärityksillä, pääasiallisesti turbidimetrisilla tai nefelometrisilla menetelmillä. Tutkimuksen tavoitteena oli kehittää nopea ja luotettava aikaerotteiseen fluoresenssiin perustuva immunomääritys ihmisen kystatiini C -pitoisuuden mittaamiseen veriplasmasta tai -seerumista. Määrityksen kehittämisessä käytettiin polyklonaalista ja seitsemää eri monoklonaalista vasta-ainetta. Erilaisten määritystyyppien, kuten kilpailevan ja ei-kilpailevan määrityksen, soveltuvuus kystatiini C:n mittaamiseen selvitettiin. Parhainta määritystä kehitettiin edelleen ja se muokattiin kahta monoklonaalista vasta-ainetta käyttäväksi kuivakemiaan perustuvaksi automatisoiduksi määritykseksi. Lopuksi selvitettiin kehitetyn määrityksen kliininen suorituskyky munuaisten vajaatoiminnan arvioinnissa sekä sepelvaltimotautipotilaiden kuoleman ja sydänkohtauksen riskin ennustajana. Testatuista määritystyypeistä ei-kilpaileva määritys toimi parhaiten ja valittiin jatkokehitykseen. Kehitetty automatisoitu kahta monoklonaalista vasta-ainetta käyttävä määritys soveltui kliinisesti merkittävien kystatiini C -pitoisuuksien luotettavaan mittaukseen. Sillä mitatut pitoisuudet vastasivat paremmin munuaistoiminnan tarkimpana pidetyn mittarin eli puhdistumamittauksen tuloksia kuin turbidimetrisella menetelmällä mitatut kystatiini C -pitoisuudet, erityisesti potilailla, joiden munuaistoiminta oli heikentynyt. Kuoleman ja sydänkohtauksen riski oli suurempi korkeammilla kystatiini C -pitoisuuksilla ja kystatiini C oli näiden tapahtumien itsenäinen ennustaja myös sen jälkeen kun ei-biokemiallisten taustatekijöiden vaikutus oli huomioitu riskianalyysissä.Siirretty Doriast

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Peer reviewe

Novel sensitive cardiac troponin I immunoassay free from troponin I-specific autoantibody interference

BACKGROUND\nCardiac troponins (cTnI and cTnT) are the recommended biomarkers of myocardial infarction. As cTn-specific autoantibodies (cTnAAb) can interfere with the cTn detection by state-of-the-art cTnI assays, our objective was to develop a sensitive cTnI immunoassay free from this analytical interference.\nMETHODS\nThe assay used antibody-coated spots containing three capture Mabs/Fabs directed against the N-terminus, midfragment and C-terminus of cTnI and a europium chelate-labeled tracer Mab against the C-terminus. Following a 3-h sample incubation and washing, cTnI was quantified by time-resolved fluorometry.\nRESULTS\nThe limit of detection (LoD) was 2.9 ng/L and the assay was linear up to 50,000 ng/L. The total precision of 10% CV was not reached, but 20% CV was reached at 10 ng/L. Mean cTnI (10-50,000 ng/L) recoveries were 100% and 119% in three cTnAAb-positive and two cTnAAb-negative individuals, respectively, verifying the interference resistance of the antibody design used. On average, Architect hs-cTnI assay gave seven-fold higher cTnI concentrations than the new assay but the correlation between the assays was good (r=0.958). Of apparently healthy individuals (n=159), 18% had measurable cTnI values (&gt;LoD) and 10% were cTnAAb-positive. The proportion of measurable cTnI values, however, was significantly higher in cTnAAb-positive individuals (13/16, median cTnI 8.5 ng/L) than in cTnAAb-negative individuals (15/143, median cTnI</p

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

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

Dry-Reagent Double-Monoclonal Assay for Cystatin C

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 Chemistr

Cystatin C as a predictor of all-cause mortality and myocardial infarction in patients with non-ST-elevation acute coronary syndrome

Objectives: To investigate the predictive value of cystatin C among patients diagnosed with non-ST-elevation acute coronary syndrome (nSTE-ACS). Design and methods: Admission serum samples from 245 nSTE-ACS patients were measured with a novel cystatin C immunoassay based on a dry-reagent, double monoclonal design. Creatinine concentrations, estimated glomerular filtration rates (eGFR) and one-year follow-up data were available for these patients. Results: During the follow-up period, 34 (14%) of patients had myocardial infarction (MI) and 25 (11%) died. Increased serum cystatin C was an independent predictor of all-cause mortality and combined events (all-cause mortality and MI) after adjustment to non-biomarker baseline factors, hazard ratio (HR) 2.19 (per increase of 1 tertile; 95% CI 1.28-3.78, p = 0.0046) and 1.75 (1.22-2.51, p = 0.0024), respectively. Corresponding values for eGFR were 2.56 (1.43-4.59, p = 0.0016) and 1.76 (1.23-2.53, p = 0.0022), respectively. Creatinine was not an independent predictor of endpoints (p > 0.05). Conclusions: Cystatin C was associated with an increased risk of death and combined events in patients with nSTE-ACS. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved