Inactivation of Allergenic Proteins by Compressed Carbon Dioxide

Dust mite allergen Der p 1 and cat allergen Fel d 1 are two major indoor allergens that triggering allergenic disease risks by activating human immune system. These allergens are known indoor risk factors for some sensitive individuals, especially for children under age 13. Avoidance and effective protecting protocols from Der p 1 and Fel d 1 call for serious attention. It has been shown that supercritical carbon dioxide, a benign solvent, can kill a wide spectrum of living organisms, from easy-to-kill mammalian cells to hard-to-kill bacterial spores. Recently, carbon dioxide was proven to achieve more than 90% reduction in enzyme activity of trypsin, a model protein, with similar molecular weight and structure as that of Der p 1. Objectives of this thesis are to demonstrate whether and how carbon dioxide, at either the liquid state (25, 68bar) or the supercritical state (60, 25bar), with selected additives can inactivate Der p 1 and Fel d 1. The reduction level of allergenic activity (inactivation) was measured by ELISA (Enzyme-linked immunosorbent assay). The work was done with commercial high pressure apparatus, inactivation of allergens were compared to that by simple dry heat treatment (60). Conclusively, the highest inactivation resulted at supercritical conditions, where we observed 80% inactivation for Der p 1 and 37% for Fel d 1. This compares to deactivation with dry heat treatment where only 2.6% Der p 1 was measured and no deactivation of Fel d 1was seen. We also explored the possible inactivation mechanisms, using SDS-PAGE and Circular Dichroism. Gel electrophoresis indicated supercritical treatment was irrelevant to molecules cleavage of Der p 1 or Fel d 1, because no molecular weight changes were observed. Circular Dichroism indicated some changes in secondary structure and tertiary structure, one possible mechanism explanation