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: Barbara Hjelm, Björn Forsström, Ulrika Igel, Henrik Johannesson, Charlotte Stadler, Emma Lundberg, Fredrik Ponten, Anna Sjöberg, Johan Rockberg, Jochen M Schwenk, Peter Nilsson, Christine Johansson, Mathias Uhlén
• Journal: Protein science : a publication of the Protein Society (Protein Sci) Vol. 20 Issue 11 Pg. 1824-35 (Nov 2011) ISSN: 1469-896X [Electronic] United States
• PMID: 21898641 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 The Protein Society.

Chemical References

• Antibodies
• Antibodies, Monoclonal
• Biomarkers, Tumor
• Epitopes
• Matrix Attachment Region Binding Proteins
• Microfilament Proteins
• RBM3 protein, human
• RNA, Small Interfering
• RNA-Binding Proteins
• SATB2 protein, human
• Transcription Factors
• CNDP1 protein, human
• Dipeptidases

Topics

• Antibodies (immunology)
• Antibodies, Monoclonal (immunology)
• Antibody Formation
• Antibody Specificity
• Biomarkers, Tumor (immunology)
• Cell Line
• Dipeptidases (immunology)
• Epitope Mapping (methods)
• Epitopes (chemistry, immunology)
• Fluorescent Antibody Technique
• Humans
• Matrix Attachment Region Binding Proteins (immunology)
• Microfilament Proteins (immunology)
• Neoplasms
• RNA Interference
• RNA, Small Interfering
• RNA-Binding Proteins (immunology)
• Transcription Factors (immunology)