Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

Abstract

Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

Clinical applications of capillary electrophoresis coupled to mass spectrometry in biomarker discovery: Focus on bladder cancer

“…. For the above issues, CIEF‐MS has not seen broader applications in the biomedical and biopharmaceutical fields yet, though applications have been reported for the characterization of monoclonal antibodies . For additional information, we refer to Haselberg et al.…”

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

“…Recently, various CE modes, e.g. CZE, CIEF and affinity CIEF, have been applied for the characterization of recombinant allergens 11–14. Although CE provides an outstanding selectivity, it becomes merely gradually implemented within the arsenal of analytical methods applied by the pharmaceutical industry 15.…”

Bioanalytical Methods for Food Allergy Diagnosis, Allergen Detection and New Allergen Discovery

“…Direct allergen detection and quantification are often realized by separation techniques such as LC [82,83] and CE [84] with UV [85][86][87][88] or fluorescence detection [89,90]. The utilization of such detectors suffers from false positive results due to the potential coelution of other compounds present in the sample matrix.…”