Figure 2: MisTTR antibody selectively binds monomeric, misfolded conformations of TTR.

(A) Indirect ELISA using monomeric TTR (0.01 mg/mL TTR incubated at 25 °C , pH 3, 44 h), guanidine unfolded TTR (TTR in 6 M guanidine , incubated overnight at 25 °C), folded native TTR and BSA. (B) Competition ELISA using guanidine unfolded TTR as bound antigen, with monomeric and tetrameric TTR as competitors, and misTTR as the antibody. IC₅₀ = 63 ± 20 nM for monomeric TTR’s binding affinity for misTTR. (C) Native and SDS-PAGE analysis was performed on wild-type TTR purified from human plasma (purchased from Sigma-Aldrich). For native PAGE analysis, three pairs of sample lanes were run. The first lane consisted of molecular weight markers while the second lane consisted of 2 mg of wild-type TTR. The three pairs of lanes were excised from the gel. The first excised lane pair was stained with Coomassie Blue dye. The second pair was transferred to PDVF membrane and immunoblotted with misTTR antibody. The third pair was similarly transferred to PDVF membrane but immunoblotted with commercial anti-TTR antibody. The same procedure was repeated for an SDS-PAGE counterpart. In the native gels, misTTR did not recognize any species, while anti-TTR recognized putative dimers and higher molecular weight oligomers. In SDS gels misTTR only recognized monomers, while anti-TTR recognized monomers and putative dimers.