Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach |
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Authors: | X Ai B Butts K Vora W Li C Tache-Talmadge A Fridman H Mehmet |
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Affiliation: | 1Apoptosis Biomarkers, Merck Research Laboratories, Rahway, NJ 07065, USA;2Immunology Franchise, Merck Research Laboratories, Rahway, NJ 07065, USA;3Applied Computer Science & Mathematics, Merck Research Laboratories, Rahway, NJ 07065, USA |
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Abstract: | Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity. |
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Keywords: | Biomarker apoptosis neo-epitope antibodies |
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