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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.


Related in: MedlinePlus

Sensitive readout after 700 s with 1 mW excitation. (a) Photobleaching decays of the SAF intensity. (b) Plot of the photobleaching amplitudes after 11 s, minus the background (zero concentration decay) against IFN-γ concentration. A straight line through the origin was fitted through the data points (Adjusted R-squared = 0.980). The dashed line corresponds to the 3σ value of the zero concentration measurement.
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biosensors-03-00108-f004: Sensitive readout after 700 s with 1 mW excitation. (a) Photobleaching decays of the SAF intensity. (b) Plot of the photobleaching amplitudes after 11 s, minus the background (zero concentration decay) against IFN-γ concentration. A straight line through the origin was fitted through the data points (Adjusted R-squared = 0.980). The dashed line corresponds to the 3σ value of the zero concentration measurement.

Mentions: For the measurement of low rmIFN-γ concentrations, the excitation intensity was increased to 1 mW after 700 s, enhancing the SAF intensity by three orders of magnitude and causing the surface-bound fluorophores to photobleach within a few seconds. Figure 4(A) shows the intensity decays during photobleaching for selected rmIFN-γ concentrations. The intensity decay obtained in the absence of rmIFN-γ was caused by non-specific adsorption of detection antibodies at the surface. This background was subtracted from the rmIFN-γ concentration-dependent decay amplitudes. The plot of the background-corrected decay amplitude versus rmIFN-γ concentration is shown in Figure 4(B). The data were fitted by a straight line through the origin, and the limit of detection was calculated by its intersection with three-times the standard deviation (3σ value) of the zero concentration measurements, to 1.9 pM. Accordingly, the concentration range of the measurement was 30–32,000 pg/mL, with an assay time of only 12 min. For comparison, the supplier of the employed antibodies (eBioscience) specifies the recombinant standard range for the ELISA of 15–2,000 pg/mL, with an assay time of 4 h. In the one-step sandwich assay performed with the SAF immunodiagnostic system, there is a trade-off between sensitivity and dynamic range. The detection of high rmIFN-γ concentration requires the use of high detection antibody concentrations, leading to an elevated background. The use of a lower detection antibody concentration shifts the dynamic range towards even lower rmIFN-γ concentrations. The mean coefficient of variation of 14.8% of the assay was larger compared to commercial ELISA kits, which is around 10%. This comparably large variation can mainly be ascribed to variations in the capture antibody density from tube to tube, as a result of the manual immobilization procedure.


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

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

Sensitive readout after 700 s with 1 mW excitation. (a) Photobleaching decays of the SAF intensity. (b) Plot of the photobleaching amplitudes after 11 s, minus the background (zero concentration decay) against IFN-γ concentration. A straight line through the origin was fitted through the data points (Adjusted R-squared = 0.980). The dashed line corresponds to the 3σ value of the zero concentration measurement.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-03-00108-f004: Sensitive readout after 700 s with 1 mW excitation. (a) Photobleaching decays of the SAF intensity. (b) Plot of the photobleaching amplitudes after 11 s, minus the background (zero concentration decay) against IFN-γ concentration. A straight line through the origin was fitted through the data points (Adjusted R-squared = 0.980). The dashed line corresponds to the 3σ value of the zero concentration measurement.
Mentions: For the measurement of low rmIFN-γ concentrations, the excitation intensity was increased to 1 mW after 700 s, enhancing the SAF intensity by three orders of magnitude and causing the surface-bound fluorophores to photobleach within a few seconds. Figure 4(A) shows the intensity decays during photobleaching for selected rmIFN-γ concentrations. The intensity decay obtained in the absence of rmIFN-γ was caused by non-specific adsorption of detection antibodies at the surface. This background was subtracted from the rmIFN-γ concentration-dependent decay amplitudes. The plot of the background-corrected decay amplitude versus rmIFN-γ concentration is shown in Figure 4(B). The data were fitted by a straight line through the origin, and the limit of detection was calculated by its intersection with three-times the standard deviation (3σ value) of the zero concentration measurements, to 1.9 pM. Accordingly, the concentration range of the measurement was 30–32,000 pg/mL, with an assay time of only 12 min. For comparison, the supplier of the employed antibodies (eBioscience) specifies the recombinant standard range for the ELISA of 15–2,000 pg/mL, with an assay time of 4 h. In the one-step sandwich assay performed with the SAF immunodiagnostic system, there is a trade-off between sensitivity and dynamic range. The detection of high rmIFN-γ concentration requires the use of high detection antibody concentrations, leading to an elevated background. The use of a lower detection antibody concentration shifts the dynamic range towards even lower rmIFN-γ concentrations. The mean coefficient of variation of 14.8% of the assay was larger compared to commercial ELISA kits, which is around 10%. This comparably large variation can mainly be ascribed to variations in the capture antibody density from tube to tube, as a result of the manual immobilization procedure.

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.


Related in: MedlinePlus