Figure 2. TnI and TnT constructs and site-specific mAbs.

Intact TnI and TnT and their fragments were prepared to map the location of epitopes recognized by the mAbs used in our study and for conformational analysis. (A) Mouse cardiac TnI fragments were constructed with a deletion of the cardiac TnI-specific N-terminal 28 amino acids (cTnI-ND) or deletions of the N-terminal 28 amino acids and the C-terminal 19 amino acids (cTnI-ND-CD). The Western blots showed that mAb 4H6 recognizes both fragments as well as intact cardiac TnI, indicating specificity to a middle epitope. In contrast, mAbs TnI-1 and 4D12 recognize intact cardiac TnI and cTnI-ND but not cTnI-ND-CD, indicating specificity to the C-terminus. (B) Mouse cardiac, fast and slow TnT fragments with N-terminal deletions were constructed by selectively deletion of the N-terminal variable region (TnT-ND), or extended deletion into the conserved middle region (cardiac TnT-ND₉₂ and cardiac TnT-ND₁₃₀), or deletion of both N-terminal and middle regions (TnT-T2, Heeley et al., 1987). The Western blot analysis localized the epitopes of mAbs 2C8, T12 and 1G9 in the middle or C-terminal regions of the TnT polypeptide.