Antibodies are very sensitive to protein conformation, and monoclonal antibodies are therefore well-suited to probe conformational changes in proteins. This is illustrated in the case of monoclonal antibody 4H4, which was raised against a putative 'EF Hand' calcium-binding region of the PC-1 antigen (Belli et al., 1994). The EF Hand region from human PC-1 was subcloned into the bacterial expression vector pGEX-KT (Hakes and Dixon, 1992), and used to immunize mice. Monoclonal antibodies were screened on methanol-fixed mouse L cells that had been transfected with human PC-1. A monoclonal antibody (4H4) which reacted with methanol-fixed cells did not stain intact unfixed cells, suggesting that it recognized only an unfolded conformation of PC-1. However, the 4H4 antibody did stain PC-1 on intact living cells in the presence of EDTA. This result suggests that the release of calcium from the EF hand caused a conformational change that allowed the antibody to bind
(Belli et al., 1994). In contrast, polyclonal antibodies to human PC-1, raised by immunizing mice against syngeneic mouse fibroblasts transfected with human PC-1, showed no difference in binding to PC-1 in the presence of calcium or EDTA. Taken together, these results suggest that the conformational change associated with calcium binding was restricted to the C-terminal portion of the protein (Belli et al., 1994).
Calmodulin is also known to undergo large conformational changes upon binding of calcium. Wolf et al. (1995) have shown that monoclonal antibodies can be generated which detect these changes.
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