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 μg 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 ∼ 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 ∼ 30-fold, the eluted IgE was further purified by affinity chromatography using a panel of IUIS/WHO-documented mouse monoclonal anti-human immunoglobulin antibodies (αhIg-MAbs). First, the IgE-enriched DEAE-cellulose chromatography fraction was incubated in a batch mode with two αhIgE-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 αhIg-MAbs (αhIgA: HP6111 + HP6123; αhIgG: HP6017 + HP6046; αhIgM: HP6081 + HP6083) coupled to CNBr-Sepharose, CL-4B. IgE purity increased from 91% to > 99.9% with ∼ 70% recovery of IgE for this step. The ultrapurified IgE antibody was shown to be functionally reactive for allergen and Fc εRI receptors on human basophils. We conclude that αhIg-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.