title: 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 (sFcεRI), a serum isoform of the high affinity IgE receptor. A recombinant version of sFcεRI 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 FcεRI-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 sFcεRI 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 sFcεRI. This novel method now allows for comparative analysis of sFcεRI levels in health and disease.
authors: Division of Gastroenterology and Nutrition, Children's Hospital Boston, Boston, MA, USA.
authors: Willem
authors: W
authors: Lexmond
authors: John van
authors: Jv
authors: der Mee
authors: Floortje
authors: F
authors: Ruiter
authors: Barbara
authors: B
authors: Platzer
authors: Georg
authors: G
authors: Stary
authors: Elizabeth H
authors: EH
authors: Yen
authors: Eleonora
authors: E
authors: Dehlink
authors: Samuel
authors: S
authors: Nurko
authors: Edda
authors: E
authors: Fiebiger
mesh_terms: D000293:Adolescent; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007223:Infant; D017455:Receptors, IgE / Q000097:blood* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000592:standards; D012015:Reference Standards; D012016:Reference Values; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E