Generation of monospecific antibodies based on affinity capture of polyclonal antibodies

作者：Hjelm Barbara; Forsstrom Bjorn; Igel Ulrika; Johannesson Henrik; Stadler Charlotte; Lundberg Emma; Ponten Fredrik; Sjoberg Anna; Rockberg Johan; Schwenk Jochen M; Nilsson Peter; Johansson Christine; Uhlen Mathias^*

来源：Protein Science, 2011, 20(11): 1824-1835.

DOI：10.1002/pro.716

• epitope mapping
• monospecific antibody
• affinity chromatography

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.

• 出版日期2011-11