Doucet, Julie, Fu, Jean, Zhao, An and Avrameas, Alexandre (2013) Development and validation of an ELISA at acidic pH for the quantitative determination of IL-13 in human plasma and serum. Journal of Pharmaceutical and Biomedical Analysis, 35. pp. 465-474. ISSN 0278-02401875-8630

Abstract

Interleukin-13 (IL-13) is an immunoregulatory cytokine secreted by T helper 2 lymphocytes and is a central mediator in the inflammation of airways and in the pathogenesis of asthma and allergy. The measurement of IL-13 in serum or plasma has been challenging using conventional immunoassays due to the low concentrations and to the presence of binding proteins. In this paper, we describe the development and validation of a highly sensitive enzyme immunoassay performed at acidic pH (pH 3.5) to remove any effect of IL-13-binding proteins. The lower limit of quantification (LLOQ) of the assay was validated at 0.59 pg/mL in human plasma. The intra- and inter-assay accuracy and precision were assessed using quality controls prepared with five different concentrations of endogenous and/or spiked IL-13 in plasma covering the entire working range. Intra-assay accuracy and precision were ranging from 84.6 to 116.2 % and from 1.1 to 11.2 %, respectively. Inter-assay accuracy and precision ranged from 98.2 to 105.5 % and 5.0 to 12.6 %, respectively. IL-13 was stable in plasma for at least one month when stored at -20°C or -80°C, and after three freeze and thaw cycles. The assay was also cross-validated in human serum with a LLOQ of 0.27 pg/mL. IL-13 was measured in plasma samples from atopic seasonal rhinitis patients and healthy controls. Concentrations ranged from < 0.59 – 23.2 pg/mL in atopic seasonal rhinitis patients (n=30) and from < 0.59 – 13 pg/mL in healthy controls. There were no statistical differences between the two groups. Taken together, these results showed that the assay was sensitive and reproducible quantification of total circulating IL-13 concentrations in human plasma and serum and can be used to support clinical studies.

Item Type:	Article
Additional Information:	Highlights: • 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 non-specific 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.
Keywords:	IL-13; binding proteins; ELISA; acidic incubation; immunoassay interference
Date Deposited:	10 May 2016 23:45
Last Modified:	10 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/8455