Comparative Studies on Tree Pollen Allergens

Abstract

The major allergen of birch pollen (BV45) was conjugated to 2–0-methoxy polyethylene glycol-4,6-dichloro-5-triazine (mPEG). Three molecular weight variants of 4,000, 6,000 and 20,000 daltons, respectively, of the activated PEG were used. The modified preparations were labelled by ¹²⁵I and both the native and the radiolabelled protein conjugates were purified by gel filtration chromatography. The relative molecular weights of the purified two peaks were preliminarily estimated by high performance liquid chromatography (two populations of ≥ 100,000 or 20,000 daltons). The amino acid composition of the acid hydrolysates of the three conjugated proteins indicated that 5 residues of lysine were modified by mPEG. Other charged amino acid side chains could also be bound to the activated PEG. The immunochemical properties of the copolymers were studied. The immunogenicity and antigenicity were examined by immunizing rabbits with ¹²⁵I-mPEG 4,000 daltons BV45 and ¹²⁵I-mPEG 20,000 daltons BV45 and subsequently crossed immunoelectrophoresis. The clearence of the radiolabelled protein showed normal pattern. A sharp decline in the radioactivity could be measured. At days 10–12, the remaining radioactivity was below 2%. Preliminary studies showed that the modified proteins were immunogenic in rabbits. The findings were demonstrated by crossed immunoelectrophoresis of the ¹²⁵I-mPEG 4,000, 6,000 or 20,000 daltons BV45 and the corresponding autoradiographs. Apparently, the immunogenicity and antigenicity of the preparations were qualitatively unaltered. The allergenicity of the modified preparation was measured in vitro by RAST and RAST inhibition. The results indicated that the allergenic reactivity of the preparation was reduced by at least 25%. Similar results were shown in vivo by skin prick test (SPT) and PK technique. The reduced allergenic reactivity is likely to be attributed to masking of the immunoreactive determinants of the allergen by attachment to an inert net of polyethylene glycol. These studies showed that conjugation of the major allergen of birch pollen (BV45) resulted in a reduction of the allergenicity. Although the immunogenic and antigenic reactivities seemed qualitatively unaltered, a similar reduction could not be excluded. The data provide an experimental model for the modification of tree pollen allergens, suitable for further application on the manipulation of antibody synthesis in animals.