This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.
Copyright (c) 2007 John Wiley & Sons, Ltd.