ISOELECTRIC FOCUSING PROCEDURES FOR ALLERGENIC EXTRACTS

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Isoelectric Focusing has proven to be a useful tool in the evaluation of allergenic extracts. This type of electrophoresis serves as a basis for examining the immunological activity of the extract, using a blotting procedure that passively transfers the focused extracts and identifies the IgE binding components in the products. The two companion methods have multiple uses. The identity of extracts submitted for standardization can be examined and compared to reference extracts with respect to IgE binding activity as well as stained IEF patterns. The optimal extraction method, in terms of IgE binding proteins generated, may be determined with these two methods. Differences in IgE binding in the sera of allergic individuals may be surveyed, and sera may be selected for testing pools with these methods, thereby assuring a fair representation of IgE binding activity in quantitative tests. A further adaptation, using select components to inhibit IgE binding in the immunoblotting, aids in the identification of cross reactive IgE binding components. Preparative IEF, using a new apparatus, has permitted the isolation of single components from allergenic extracts, separating non-IgE binding and IgE binding components in sufficient quantities for analysis and further testing. Following IEF, these components are being subjected to 2-D electrophoresis for further examination. IEF and immunoblotting has become a critical factor in the evaluation of stability studies for allergenic extracts. Using IEF as the preliminary screening method, the loss of well defined IEF banding patterns frequently indicates some loss of allergenic activity. Following evaluation of IEF, quantitative tests can be performed to confirm loss of allergenic activity. IEF has also proven to be useful in the evaluation of standardized cat extracts, with respect to demonstrating consistency between lots, and compositional differences related to allergenic source material. It has been proposed that IEF will become a release criteria for standardized cat extracts, in conjunction with Fel d I assays and protein assays that are already required.