title: Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach.
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.
authors: Apoptosis Biomarkers, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors: X
authors: X
authors: Ai
authors: B
authors: B
authors: Butts
authors: K
authors: K
authors: Vora
authors: W
authors: W
authors: Li
authors: C
authors: C
authors: Tache-Talmadge
authors: A
authors: A
authors: Fridman
authors: H
authors: H
authors: Mehmet
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology; D005472:Fluorouracil / Q000633:toxicity; D006801:Humans; D047468:Immunoprecipitation; D009842:Oligopeptides / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits
chemical_list: D000906:Antibodies; D000939:Epitopes; D009842:Oligopeptides; D020169:Caspases; D005472:Fluorouracil