Purification, characterization and immunological studies of rat urinary proteins causing allergy in humans

Sammanfattning: Rats are among the most frequently used laboratory animals and allergy to them constitutes a common occupational problem. Approximately 20-30% of the persons engaged in work with laboratory animals acquire symptoms of allergy. These include rhinitis, conjunctivitis, urticaria and sometimes asthma, symptoms which usually develop during the first three years of exposure. The allergic reaction arises from direct contact with rat urine, or from airborne dusts originating from dried animal urine. Rat urine contains a complex mixture of proteins some of which are known to be allergenic, for example a2u-globulin. In urine from fertile male rats a2u-globulin is one of the most abundant proteins.The primary aim of this thesis was to purify and identify the most potent allergens in rat urine. Optimization of various methods for evaluation of the allergenicity of the identified allergens was the second aim. The third aim was to examine in some detail the IgE binding regions of the major allergen, Rat n 1.02, a2u-globulin.Rat urinary proteins were separated and purified by ultrafiltration and high resolution chromatographic methods. The two dominating proteins were identified in this study as different forms of the same parent protein, i.e. a2u-globulin. It was also proved that the urinary protein previously named pre-albumin had no amino acid sequence resemblance to prealbumin (transthyretin) present in rat serum.Rat urinary proteins separated by SDS-PAGE were used as antigens when IgE antibodies in sera from allergic patients were studied with immunoblotting. A detection system utilizing chemiluminescence and a luminometer apparatus gave scanning curves of the IgE-binding reactivities and allowed arbitrary measurement of the amount of IgE bound to certain urine proteins. Specific immunoblotting patterns of IgE binding to the electrophoretically separated proteins were shown for each individual. Differences were seen in regard of the distribution among the recognized antigens and the amount of IgE bound. All rat allergic subjects studied had IgE antibodies binding to the major urinary protein, a2u-globulin. However, this protein was not always the most dominating allergen.Overlapping octapeptides corresponding to the amino acid sequence of the major allergen Rat n 1.02 were synthesized on solid support and screened in parallel to pinpoint the IgE binding regions of the protein using a modified ELISA procedure. Our results indicate the existence of linear IgE-binding epitopes, mainly located towards the N-terminal and C-terminal parts of the protein, as recognized by IgE antibodies in the studied sera. The role of these short amino acid sequences in the allergic reaction and their appropriateness for immunotherapy calls for further investigation.In conclusion, this thesis has contributed to the isolation, identification and characterization of the allergens involved in a major occupational disease among people who work with laboratory animals, namely rat allergy.