Development and validation of a standardized ELISA for the detection of soluble Fc-epsilon-RI in human serum.

ABSTRACT: The aim of this study was to develop a standardized enzyme-linked immunosorbent assay (ELISA) for detection of human soluble Fc-epsilon-RI (sFc?RI), a serum isoform of the high affinity IgE receptor. A recombinant version of sFc?RI was produced in baculovirus and used as standard. ELISA plates were coated with anti-mouse IgG followed by incubation with the monoclonal capture antibody CRA1. This Fc?RI-alpha-specific antibody binds to the stalk region of the protein and does not inhibit IgE-binding. After incubation with standards or serum samples, plates were incubated with chimeric IgE followed by detection with horseradish peroxidase conjugated anti-human IgE. Enzymatic activity was visualized with (3,3',5,5')-tetramethylbenzidine. Specificity was demonstrated by omission of capture or detection reagents. Units (U) of detection were established and the dynamic range of the assay was defined as 10-640 U/ml for a 1/5 serum dilution. Parameters of linearity (R(2)>0.999), matrix interference test (recovery of 70-110%), intra-assay variability (coefficient of variation (CV) <20%) and inter-assay variability (CV <20%) met acceptance criteria for immunoassay validation. Correlation analysis of serum units of sFc?RI measured with the new ELISA and serum IgE levels confirmed earlier published data describing a weak correlation of the two parameters in patients with elevated serum IgE while no correlation in patients with normal serum IgE or the total patient group was found. In summary, we established and validated a standardized ELISA for the detection of sFc?RI. This novel method now allows for comparative analysis of sFc?RI levels in health and disease.

SUBMITTER: Lexmond W 

PROVIDER: S-EPMC3196264 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature