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. 2013;35(5):465-74.
doi: 10.1155/2013/290670. Epub 2013 Oct 2.

Development and validation of an ELISA at acidic pH for the quantitative determination of IL-13 in human plasma and serum

Julie Doucet  1 , An Zhao, Jean Fu, Alexandre Avrameas
Affiliations

Development and validation of an ELISA at acidic pH for the quantitative determination of IL-13 in human plasma and serum

Julie Doucet et al. Dis Markers. 2013.

Abstract

A novel sandwich ELISA for the quantitative and sensitive determination of IL-13 in human serum and plasma was established. The assay employs an incubation step at acidic pH, which was shown to decrease nonspecific binding and interference from IL-13 binding proteins. The assay was validated and was shown to be accurate and precise over the entire quantification range (0.59 to 68.4 pg/mL in human EDTA plasma). The validated assay was successfully applied to samples from healthy volunteers and patients with atopic seasonal rhinitis. The assay is suitable for use in clinical trials to monitor efficacy or pharmacodynamic effects of drug candidates.

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Figures

Figure 1
Figure 1
Titration curves of rhIL-13 incubated at pH 7.4, pH 5, pH 3.5, and pH 3.25 on immobilized capture antibody.
Figure 2
Figure 2
NSB obtained in plasma samples at pH 7.4, pH 5, and pH 3.5. NSB is the signal obtained by the detection (biotinylated anti-IL13 antibody and enzyme linked streptavidin) of plasma components nonspecifically bound to the plate surface in the absence of capture antibody.
Figure 3
Figure 3
Interrun precision of endogenous IL-13 measurement in two human plasma samples at pH 5 and pH 3.5. The endogenous IL-13 concentration was measured at pH 5 and pH 3.5 in three independent runs. Precision (% CV) of the three measures was calculated for the two plasma samples.
Figure 4
Figure 4
Recovery of rhIL-13 concentration in the presence of its receptor rhIL-13Rα2 as a function of incubation pH. rhIL-13 (10 pg/mL) and its receptor were spiked in a human serum. IL-13 concentration was then measured by ELISA at pH 5 and pH 3.5.
Figure 5
Figure 5
Recovery of endogenous IL-13 concentration in the presence of rhIL-13Rα2 as a function of incubation pH. IL-13 was measured in two human serums spiked with rhIL-13Rα2. The IL-13 concentration measured in the absence of spiked rhIL-13Rα2 was taken as reference for the calculation of IL-13 recovery.
Figure 6
Figure 6
IL-13 concentration in EDTA plasma of healthy donors and atopic seasonal rhinitis patients.
See this image and copyright information in PMC

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