Isolation by mAb based affinity chromatography of two Par j I isoallergens. Comparison of their physicochemical, immunochemical and allergenic properties.
We report the identification and separation of two isoallergen components of Par j I, the major allergen from Parietaria judaica pollen. First, electrophoretic conditions for consistently separating both isoforms in an SDS-PAGE system were established, and mol. wt values of 13,000 (isoallergen IA) and 10,500 (isoallergen IB) were estimated. Immunoblot, 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 immunized with a purified Par j I preparation comprising IA and IB isoallergens. Three mAbs were directed to an epitope shared by both isoallergens, 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 molecules. 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-terminal sequencing demonstrated an extensive homology and also the existence of some structural differences between Par j I isoallergens, which is in agreement with the high, but not complete, cross-reactivity observed in competition ELISA experiments. Finally, skin prick tests performed on 28 P. judaica-sensitive patients showed that all of them recognized both isoforms and that allergenic epitopes present in Par j IA and IB are responsible for most of the allergenic activity of the whole extract.
Ayuso
R
R
Departamento Investigación, Alergia e Immunología Abelló S.A., Madrid, Spain.
Carreira
J
J
Lombardero
M
M
Duffort
O
O
Peris
A
A
Basomba
A
A
Polo
F
F
D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011058:Pollen / Q000737:chemistry* / Q000276:immunology; D012882:Skin Tests
D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; C083860:Par j I protein, Parietaria judaica; D010940:Plant Proteins