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Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence.

Winterflood CM, Ruckstuhl T, Seeger S - Biosensors (Basel) (2013)

Bottom Line: We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude.The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader.The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

View Article: PubMed Central - PubMed

Affiliation: Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; E-Mails: c.winterflood@pci.uzh.ch (C.M.W.); t.ruckstuhl@pci.uzh.ch (T.R.).

ABSTRACT
We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

No MeSH data available.


(a) Real-time measurement of selected concentrations of Interferon-γ (IFN-γ) . (b) SAF intensity increase after 700 s plotted against IFN-γ concentration. A straight line through the origin was fitted to the data for IFN-γ concentrations up to 2 nM (Adjusted R-squared = 0.995). The linear relationship is lost at higher concentrations due to depletion of the free detection antibody.
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biosensors-03-00108-f003: (a) Real-time measurement of selected concentrations of Interferon-γ (IFN-γ) . (b) SAF intensity increase after 700 s plotted against IFN-γ concentration. A straight line through the origin was fitted to the data for IFN-γ concentrations up to 2 nM (Adjusted R-squared = 0.995). The linear relationship is lost at higher concentrations due to depletion of the free detection antibody.

Mentions: Figure 3(A) shows SAF intensity curves for the measurement of selected rmIFN-γ concentrations. Due to the excellent sensitivity of the system, fairly smooth binding curves were obtained for picomolar analyte concentrations. The binding followed a rather complex kinetics, as the sandwich formation at the surface proceeded through two pathways, with rmIFN-γ molecules binding to either the detection antibody in solution or to the capture antibody on the surface first. The calibration curve shown in Figure 3(B) was obtained by plotting the rmIFN-γ concentrations from 10 pM to 5 nM versus the SAF intensity measured after 700 s. The saturation of the signal for rmIFN-γ concentrations above 2 nM was caused by the depletion of the detection antibodies.


Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence.

Winterflood CM, Ruckstuhl T, Seeger S - Biosensors (Basel) (2013)

(a) Real-time measurement of selected concentrations of Interferon-γ (IFN-γ) . (b) SAF intensity increase after 700 s plotted against IFN-γ concentration. A straight line through the origin was fitted to the data for IFN-γ concentrations up to 2 nM (Adjusted R-squared = 0.995). The linear relationship is lost at higher concentrations due to depletion of the free detection antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263590&req=5

biosensors-03-00108-f003: (a) Real-time measurement of selected concentrations of Interferon-γ (IFN-γ) . (b) SAF intensity increase after 700 s plotted against IFN-γ concentration. A straight line through the origin was fitted to the data for IFN-γ concentrations up to 2 nM (Adjusted R-squared = 0.995). The linear relationship is lost at higher concentrations due to depletion of the free detection antibody.
Mentions: Figure 3(A) shows SAF intensity curves for the measurement of selected rmIFN-γ concentrations. Due to the excellent sensitivity of the system, fairly smooth binding curves were obtained for picomolar analyte concentrations. The binding followed a rather complex kinetics, as the sandwich formation at the surface proceeded through two pathways, with rmIFN-γ molecules binding to either the detection antibody in solution or to the capture antibody on the surface first. The calibration curve shown in Figure 3(B) was obtained by plotting the rmIFN-γ concentrations from 10 pM to 5 nM versus the SAF intensity measured after 700 s. The saturation of the signal for rmIFN-γ concentrations above 2 nM was caused by the depletion of the detection antibodies.

Bottom Line: We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude.The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader.The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

View Article: PubMed Central - PubMed

Affiliation: Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; E-Mails: c.winterflood@pci.uzh.ch (C.M.W.); t.ruckstuhl@pci.uzh.ch (T.R.).

ABSTRACT
We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

No MeSH data available.