Characterization and Chemical Modification of Isolated Allergens from Horse Hair and Dandruff

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Abstract
Three previously isolated major allergens of horse hair and dandruff, antigens No. 6, 9 and 11 (Ag 6, 9 and 11) have been further characterized and chemically modified. Carbohydrate analyses revealed that Ag 9 and 11 contained 12 and 17 hexoses, amino sugars, sialic acids as well as low amounts of unidentified sugar molecules. The carbohydrate part of the glycoproteins constituted 5 and 10% (w/w) of dry-weight material. Addition of the non-ionic detergent Berol did not change the molecular structure of the three allergens. Denaturation using 6 M guanidinium hydrochloride was reversible for Ag 6 and 11, whereas Ag 9 separated into two non-identical molecules of almost equal size. Several derivatives of the three allergens coupled to ethylene maleic anhydride copolymer (EMA) were prepared by coupling the amino groups of the allergens (molecular weight EMA: 20,000 daltons). The recovery of the prepared water-soluble complexes was greater than 85% in all experimental cases studied and the complexes obtained were from one to several EMA and allergen molecules; both intra- and intermolecular bridges were established. Immunochemical analyses revealed that through attachment of EMA chains, antigenic sites were masked. The preserved activity of modified relative to native allergens was from 1 to 31% as measured by means of single radial immunodiffusion (on the basis of polyspecific rabbit antibodies against the allergens), RAST inhibition (on the basis of human IgE antibodies from 22 horse-allergic patients), and prick tests (on the basis of the individual reactions of 4–6 horse-allergic patients).