Strategies to generate antibodies capable of distinguishing between proteins with >90% amino acid identity.

Protein engineering is a common strategy for the generation of protein variants with new properties. The engineered variants often have a high degree of similarity with the wild-type progenitor protein, necessitating a tool (e.g., antibody) to distinguish the wild-type and variant protein forms. As part of an overall effort to understand the process of incorporation of amino acids into storage proteins during seed fill in soybean, we have engineered a variant of soybean vegetative storage protein beta (VSPbeta) that is 91.8% identical in amino acid sequence to the wild-type protein, but contains 10% methionine (VSPbeta-Met, unpublished results). Thus, it would be desirable to have antibodies that specifically recognize VSPbeta-Met over the endogenously expressed wild-type protein in transgenic plants. To this end, we compared three strategies for the isolation of VSPbeta-Met-specific antibodies: (1) hybridoma production using VSPbeta-Met protein as the antigen, (2) polyclonal antibody production in rabbits using a peptide antigen corresponding to a methionine-rich region of VSPbeta-Met, and (3) subtractive immunization in mice using VSPbeta-WT as the tolerogen, cyclophosphamide for immunosuppression and VSPbeta-Met as the immunogen. While the first strategy generated antibodies cross-reactive to both antigens, the second strategy generated polyclonal antibodies that preferentially recognized the variant protein in immunoblots. However, using subtractive immunization, we were able to generate mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPbeta-Met than VSPbeta-WT in an ELISA.