title: Purification of immunoglobulin E (IgE) antibodies from sera with high IgE titers.
abstract: A three stage method for the ultrapurification of polyclonal IgE from human serum is reported using anion exchange chromatography followed by monoclonal antibody based positive and negative affinity chromatography. Following dialysis of 25-100 ml of serum (2.3-14 micrograms IgE/ml, n = 4) against 0.05 M Tris pH 8, each specimen was subjected to diethylaminoethyl (DEAE)-cellulose chromatography (serum/matrix = 1/4). IgE was eluted with 0.05 M Tris, 0.05 M NaCl pH 8, yielding an IgE recovery of 61-93%, with removal of approximately 90% of other serum proteins and an IgE purity ([IgE]/[Igs]) of 0.1-1.1%. After adjusting to 0.1 M NaCl and concentrating approximately 30-fold, the eluted IgE was further purified by affinity chromatography using a panel of IUIS/WHO-documented mouse monoclonal anti-human immunoglobulin antibodies (alpha hIg-MAbs). First, the IgE-enriched DEAE-cellulose chromatography fraction was incubated in a batch mode with two alpha hIgE-Fc MAbs (HP6029, HP6061) coupled to CNBr-Sepharose, CL-4B. IgE was eluted with 0.05 M glycine pH 2.8 and immediately neutralized. The IgE recovery was 32-52% and IgE purity was 72-97%. Silver-stained SDS-PAGE and noncompetitive solid-phase two-site immunoenzymetric assays for total human IgA, IgE, IgG and IgM indicated that IgA, IgG and IgM were the only contaminants. Next, the IgE was concentrated 10-30-fold in the presence of 0.1% HSA. One IgE specimen was ultrapurified in a batch mode by negative selection chromatography using three pairs of alpha hIg-MAbs (alpha hIgA: HP6111 + HP6123; alpha hIgG: HP6017 + HP6046; alpha hIgM: HP6081 + HP6083) coupled to CNBr-Sepharose, CL-4B. IgE purity increased from 91% to > 99.9% with approximately 70% recovery of IgE for this step. The ultrapurified IgE antibody was shown to be functionally reactive for allergen and Fc epsilon RI receptors on human basophils. We conclude that alpha hIg-MAbs are powerful tools to facilitate the affinity purification of functionally active human IgE from serum; however, when the analyte is present in low concentration, a carrier protein needs to be added to minimize non-specific loss of the material during this process.
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224-6801.
authors.forename: J
authors.initials: J
authors.lastname: Kleine-Tebbe
authors.forename: R G
authors.initials: RG
authors.lastname: Hamilton
authors.forename: M
authors.initials: M
authors.lastname: Roebber
authors.forename: L M
authors.initials: LM
authors.lastname: Lichtenstein
authors.forename: S M
authors.initials: SM
authors.lastname: MacDonald
mesh_terms: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006801:Humans; D007073:Immunoglobulin E / Q000302:isolation & purification*
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007073:Immunoglobulin E