Subtractive immunization: a tool for the generation of discriminatory antibodies to proteins of similar sequence

Antibodies specific for a protein of interest are invaluable tools for monitoring the protein's structure, location and activity. Due to the tendency of an immune system to mount a response toward the abundant, immunodominant epitopes in a protein mixture, difficulties are inherent in the isolation of antibodies specific for proteins that are rare or poorly immunogenic. Likewise, isolation of antibodies specific for a protein with significant sequence similarity to other proteins, such as those derived from protein engineering, may be challenging. Subtractive immunization is a technique proven to facilitate efforts to produce monoclonal antibodies specific for antigens that are present in low abundance in a protein mixture, poorly immunogenic and/or similar in sequence or structure to other proteins. This protocol provides a detailed, stepwise procedure for the isolation of antibodies specific for a protein with sequence similarity to other proteins. As an example, we describe methods established to isolate antibodies specific to a methionine-enriched variant of soybean vegetative storage protein beta (VSPbeta-Met) that shares 91.8% amino acid sequence identity to the wild-type protein (VSPbeta-WT). These methods include cyclophosphamide-induced immunosuppression of mice for the wild-type protein followed by immunization with VSPbeta-Met. As a result of this procedure, mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPbeta-Met than VSPbeta-WT in an ELISA were generated. It is anticipated that this strategy will have utility for generating antibodies specific to protein variants derived from protein engineering.