Research and development scientists at allervet® have produced an oligoclonal assay based on canine recombinant IgE (rIgE), see fig 3. This incorporates 3 monoclonal antibodies raised against known epitopes on the heavy chain domains (2, 3 and 4) of the rIgE molecule. Oligoclonal technology combines the advantages of monoclonal specificity with polyclonal sensitivity resulting in enhanced performance of the assay (Alvarez et Zalve 2010).
The test is an indirect ELISA; the enzymatic reaction produces a colour change measured as the optical densitiy using a densitometer. The optical density reading is directly proportional to the patient’s serum concentration of allergen specific IgE. Results are reported as negative, borderline, positive or high positive, rather than numerically – whilst the OD reflects the serum concentration of IgE the presence and quantity of allergen specific serum IgE does not necessarily correlate with the severity of clinical signs (Lee et al 2009).
Figure 3: Production of a Recombinant Canine IgE Molecule
Immunoglobulin E (IgE) is a key molecule in immediate allergic reactions in dogs, and it is used in-vitro for detection of allergen-specific IgE employing anti-canine IgE antibodies. Usually, these reagents are developed against IgE purified from sera that frequently are contaminated with other immunoglobulins, cross-reaction with which could produce false positives. Molecular biology techniques now allow the production of immunoglobulins free of contamination with other isotypes. The aim of this study was to produce recombinant canine IgE (rIgE) using the known sequence of its gene that comprises the domains CH2-CH3-CH4 of the constant heavy chain region. A mammalian cell system was employed in order to maintain the glycosylation motifs of the molecule. rIgE was affinity purified and characterized in order to check its molecular weight and structure in SDS-PAGE and immunoblotting assays. The rIgE represented a minority protein in the supernatant of transfected cells. The purification needed several washing steps, and the elution required a high concentration of imidazole (500mM). rIgE was specifically recognized by an anti-canine IgE-peroxidase polyclonal antibody, showing an apparent molecular weight of 50 kDa in the presence of beta-2-mercaptoethanol and a molecular weight of 100 kDa in non- reducing conditions consistent with the predicted molecular weight for mono and dimeric forms. The rIgE maintains native IgE epitopes and can be used to produce new monoclonal antibodies against the Fc region of canine IgE that are free of contamination and cross- reaction with other immunoglobulins.
Cloning of CH2-CH3-CH4 region of heavy chain of canine IgE.
Detection of rIgE in supernatants of transfected cells.
Purification of canine rIgE by affinity.
Structure of purified recombinant canine IgE.