CHICKEN ANTIBODIES TO A RECOMBINANT FRAGMENT OF THE EQUINE IMMUNOGLOBULIN-EPSILON HEAVY-CHAIN RECOGNIZING NATIVE HORSE IGE

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, n ucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His](6)-tag and purified over a Ni-NTA column . The recombinant protein was used to immunise hens. Testing of the ra ised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA reveale d high titres against the recombinant equine IgE fragment (reqIgEf). T he reqIgEf-specific IgG was successfully affinity-purified on an uncon ventional affinity matrix: the [His](6) -tagged recombinant IgE fragme nt was bound to Ni-NTA agarose and used to adsorb specific immunoglobu lins. In Western-blot of ammonium sulphate precipitated horse serum an d bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing -reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar f luid (BALF) was separated under non-reducing conditions. These reactio ns could be inhibited by preincubation of the immune IgG with reqIgEf while preincubation with horse IgG did not inhibit the reaction. Antib ody affinity chromatography of horse serum with the reqIgEf-specific c hicken IgG resulted in an enrichment of the 80 kDa protein in denaturi ng Western-blot. Determination of the amino acid composition of this p rotein and comparison with the equine IgE heavy-chain sequence strongl y indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-spccific chicken IgG was further characterised In an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG a nonrelevant [His](6)-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characte ristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases. (C) 1997 Els evier Science B.V.