Nanopore detection of antibody prion interactions

Binding of bovine T194A PrPCby PrPSc-specific antibodies

“…It was previously shown that the antibody M2188 binds wildtype bPrP 35 and confirmed that pre-treatment with 1 M GdnHCl does not interfere with this interaction ( Fig. 3A; Table 3).…”

“…As shown previously, the events centered between these type-I and type-II may represent peptide/protein molecules that interact with the pore either by orientation through N-or C-terminal entry or by different conformations. 35 At present these possibilities cannot be distinguished. Previously, our group has used nanopore analysis to probe various aspects of PrP C behavior, including metal binding 37 and the interaction with PrP C specific-antibodies.…”

“…Previously, our group has used nanopore analysis to probe various aspects of PrP C behavior, including metal binding 37 and the interaction with PrP C specific-antibodies. 35 As well, it was demonstrated that the event profiles after pre-incubation in 1.5 M guanidinium hydrochloride (Gdn-HCl) of wild-type bPrP and mutant bPrP(T194A) were different as were those of full length human PrP(23-231) and truncated PrP(90-231). 38 Thus the electronic signal at the instant that the protein interacts with the pore is very sensitive to the protein's structure and conformation.…”

“…Antibody prion interactions were analyzed using an α-hemolysin pore as described previously. 34,35 In nanopore analysis, generally, type-I events have small blockade currents and short blockade times and correspond to individual molecules bumping into the pore before rapidly diffusing away. 36 Type-II events have larger blockade currents and longer blockade times and correspond to either translocation of a molecule through the pore after unfolding or transient insertion of a loop or strand of the protein into the pore before diffusing back to the cis side.…”

“…9B). 35 In the case of the bovine wildtype PrP C the epitope for SN6b is buried in the globular domain of the protein and will not be affected by the addition of antibody. 19,20,45 Previously it was shown that wild-type bPrP generated about 35%−40% of type-II events.…”