Isolation and characterization of two IgE-reactive proteins from Azadirachta indica pollen

Two allergenically active components present in the Azadirachta indica whole pollen extract have been isolated by sequential ammonium sulfate precipitation (0-90%), DEAE-Sephadex A-50 ion-exchange chromatography followed by gel filtration through Sephadex G-200. The allergenicity of fractionated materials has been tested by skin prick test and ELISA inhibition which reveal that AIaI and AIaIVb are the major allergens. Immunoblot confirms the IgE-binding activity of the proteins. Although both fractions are found to be homogeneous by SDS-PAGE, isoelectric focusing produces more than one isoelectric point in AIaI (pI = 3.15, 3.3 and 3.5) and AIaIVb (pI = 6.0 and 6.2). Amino acid analyses of the two allergens, the effect of pH on them and cross-reactivity between them have been discussed.