Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
2011 (English)In: Protein Science, ISSN 0961-8368, Vol. 20, no 11, 1824-1835 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2011. Vol. 20, no 11, 1824-1835 p.
epitope mapping, monospecific antibody, affinity chromatography
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-47969DOI: 10.1002/pro.716ISI: 000296273700009ScopusID: 2-s2.0-80054717087OAI: oai:DiVA.org:kth-47969DiVA: diva2:457225
FunderKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 201111172011-11-172011-11-152014-11-11Bibliographically approved