A1 Refereed original research article in a scientific journal
Extension of dynamic range of sensitive nanoparticle-based immunoassays
Authors: Heidi Hyytiä, Noora Ristiniemi, Päivi Laitinen, Timo Lövgren, Kim Pettersson
Publisher: Elsevier
Publication year: 2014
Journal: Analytical Biochemistry
Journal acronym: anal biochem
Volume: 446
First page : 82
Last page: 86
Number of pages: 5
ISSN: 0003-2697
DOI: https://doi.org/10.1016/j.ab.2013.10.034
Abstract
Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000ng/ml, and with cTnI from 25 to 1000ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07-0.11) and a y-intercept of -7.79 (-17.87-2.29)ng/L (n=65, r=0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.
Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000ng/ml, and with cTnI from 25 to 1000ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07-0.11) and a y-intercept of -7.79 (-17.87-2.29)ng/L (n=65, r=0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.
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