title: Generation of monospecific antibodies based on affinity capture of polyclonal antibodies.
abstract: A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.
authors: Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, 10691 Stockholm, Sweden.
authors: Barbara
authors: B
authors: Hjelm
authors: Björn
authors: B
authors: Forsström
authors: Ulrika
authors: U
authors: Igel
authors: Henrik
authors: H
authors: Johannesson
authors: Charlotte
authors: C
authors: Stadler
authors: Emma
authors: E
authors: Lundberg
authors: Fredrik
authors: F
authors: Ponten
authors: Anna
authors: A
authors: Sjöberg
authors: Johan
authors: J
authors: Rockberg
authors: Jochen M
authors: JM
authors: Schwenk
authors: Peter
authors: P
authors: Nilsson
authors: Christine
authors: C
authors: Johansson
authors: Mathias
authors: M
authors: Uhlén
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000917:Antibody Formation; D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000276:immunology*; D002460:Cell Line; D004150:Dipeptidases / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D036961:Matrix Attachment Region Binding Proteins / Q000276:immunology; D008840:Microfilament Proteins / Q000276:immunology; D009369:Neoplasms; D034622:RNA Interference; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000276:immunology; D014157:Transcription Factors / Q000276:immunology
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D000939:Epitopes; D036961:Matrix Attachment Region Binding Proteins; D008840:Microfilament Proteins; C099506:RBM3 protein, human; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C521297:SATB2 protein, human; D014157:Transcription Factors; C522928:CNDP1 protein, human; D004150:Dipeptidases