ISOLATION BY MAB BASED AFFINITY-CHROMATOGRAPHY OF 2 PAR J I ISOALLERGENS - COMPARISON OF THEIR PHYSICOCHEMICAL, IMMUNOCHEMICAL AND ALLERGENIC PROPERTIES

We report the identification and separation of two isoallergen compone nts of Par j I, the major allergen from Parietaria judaica pollen. Fir st, electrophoretic conditions for consistently separating both isofor ms in an SDS-PAGE system were established, and mol. wt values of 13,00 0 (isoallergen IA) and 10,500 (isoallergen IB) were estimated. Immunob lot, after SDS-PAGE experiments, with individual P. judaica-sensitive human sera revealed a slightly different IgE-binding pattern for each isoallergen. Four anti-Par j I mAbs were obtained from BALB/c mice imm unized with a purified Par j I preparation comprising IA and IB isoall ergens. Three mAbs were directed to an epitope shared by both isoaller gens, and the fourth one recognized specifically one epitope on Par j IB. Dot-blot experiments with the deglycosylated allergen showed that the mAbs did not recognize the carbohydrate prosthetic group of the mo lecules. Affinity chromatography using the mAbs allowed the separation of the isoallergens that retained their IgE-binding ability after the purification process. Amino acid composition analyses and partial N-t erminal sequencing demonstrated an extensive homology and also the exi stence of some structural differences between Par j I isoallergens, wh ich is in agreement with the high, but not complete, cross-reactivity observed in competition ELISA experiments. Finally, skin prick tests p erformed on 28 P. judaica-sensitive patients showed that all of them r ecognized both isoforms and that allergenic epitopes present in Par j IA and IB are responsible for most of the allergenic activity of the w hole extract.