Establishment of a Novel Quantum Dots-Encoded Microbead-Based Flow Cytometric Method for Quantification of Soluble Fc epsilon RI alpha in Serum

摘要

The soluble form of the transmembrane glycoprotein, Fc epsilon RI alpha which corresponds to the high-affinity receptor for IgE, is found in serum. Growing evidence suggests the pathogenic role of IgE and Fc epsilon RI in systemic lupus erythematosus (SLE). The goal of this study is to develop a sensitive and standardized cytometric assay for quantification of sFc epsilon RI alpha. A membrane emulsification technique was utilized to incorporate CuInS2/ZnS quantum dots and Fe3O4 nanoparticles into poly (styrene-co-maleic anhydride) microbeads. The beads were then carboxylated and coated with capture antibody monoclonal anti-human Fc epsilon RI alpha. This antibody binds to Fc epsilon RI alpha but does not block the binding of Fc epsilon RI alpha to IgE. After incubation with standards or serum samples, the microbeads were incubated with excessive native human IgE, followed by incubation with Phycoerythrin (PE) conjugated anti-human IgE. The resulting quantum dot microbeads were gated, and sFc epsilon RI alpha quantification was analyzed based on PE fluorescence intensity. The method exhibited good linearity (R-2 > 0.99), and the limit of detection was established at 0.29 ng/mL with the dynamic range of up to 200 ng/mL. The precision of the assay validated by intra- and inter-assay variability met the acceptance criteria with the mean recovery falling within 80-110% of the theoretical concentration and a corresponding CV < 20%. We tested 149 serum samples which 89 were from SLE patients and 60 were from healthy volunteers. For the first time, we detected an increased sFc epsilon RI alpha level in the serum of SLE patients, which was confirmed by a commercial ELISA kit. Compared to ELISA, this novel method is more sensitive and efficient. It allows for the simple comparative analysis of sFc epsilon RI levels in health and disease. (C) 2017 International Society for Advancement of Cytometry