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

• Published in: Journal of immunological methods 2002-03, Vol.261 (1), p.213-220
• Main Authors: Sleister, Heidi Major, Rao, A.Gururaj
• Format: Article
• Language: English
• Subjects: Animals; Antibody; Antibody Formation; Antibody Specificity; Bacteriology; Biological and medical sciences; Cyclophosphamide; Cyclophosphamide - administration & dosage; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Genetics; Glycine max; Glycine max - genetics; Glycine max - immunology; Immunization - methods; Immunodominant Epitopes - genetics; Immunosuppression; Immunosuppressive Agents - administration & dosage; Mice; Mice, Inbred BALB C; Microbiology; Molecular immunology; Plant Proteins - genetics; Plant Proteins - immunology; Proteins - genetics; Proteins - immunology; Subtractive immunization; Techniques; Tolerization; Vegetative storage protein; vegetative storage protein-^b
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00567-1

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 β (VSPβ-Met) that shares 91.8% amino acid sequence identity to the wild-type protein (VSPβ-WT). These methods include cyclophosphamide-induced immunosuppression of mice for the wild-type protein followed by immunization with VSPβ-Met. As a result of this procedure, mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPβ-Met than VSPβ-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.