Affinity purification of a major Alternaria allergen using a monoclonal antibody

An extract of Alternaria (ALT) was passed through an affinity column containing a monoclonal antibody directed to ALT. After washing the column, a single glycoprotein was eluted using 0.1 M glycine (pH 3.0). This glycoprotein accounted for 13% of the dry weight of our ALT preparation and had a carbohydrate to protein ratio of 0.35 as compared with 3.2 for the whole ALT extract. Its molecular weight was 70 kD by SDS-PAGE and isoelectric point was 3.5 by IEF. Though individual sensitivities varied, 14/16 patients skin reactive to ALT were also reactive to this glycoprotein. Quantitative skin tests of extracts adjusted to the same dry weight per volume showed that it required about a four times greater concentration of the purified glycoprotein to give a wheal size equal to whole Alternaria. The ability to purify large amounts of this allergen with affinity chromatography should make its complete characterization possible.