There are three requirements for standardization of allergenic extracts: 1. identification of all important allergens; 2. development of reproducible assays for their measurement; and 3. a reliable source of antigen that can serve as a standard. To date, none of the above have been accomplished for the important allergenic fungus Alternaria. Some of its major allergens have been isolated, however, many more remain unidentified. In addition, biologic and immunologic methods currently in use for antigen measurement such as skin testing and RAST inhibition have limitations in that they provide information about total allergen content and not about individual antigens. Finally, preparation of an Alternaria standard has been difficult due to the extreme variability of antigens contained in source materials. We will address the first problem by initially separating extracts of Alternaria into individual allergenic fractions using affinity chromatography with antigen-specific human IgE in solid phase. This will be followed by preparative isoelectric focusing, PAGE, and gel filtration as necessary to obtain pure materials. Once these are available, the second problem can be addressed by producing fraction-specific antisera (mouse and human) which can be used for measurement of the individual fractions using a """"""""sandwich"""""""" ELISA. To further refine the assays and map the allergenic epitopes, Alternaria-specific mouse Mabs will then be produced and substituted for the polyclonal antibodies as they become available. These same Mabs will also permit purification of their recognized antigens by affinity chromatography which will help to solve the third problem. It should be possible to use the purified material as standards for calibration of the assays. Finally, on a more speculative note, production of anti-idiotype Mabs directed to one of the Alternaria Mabs will be attempted. If their idiotypes are similar enough to native antigens, such Mabs may substitute for standards in immunoassays.
