Phosphorylation-specific antibodies for human cardiac troponin-I

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin-I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac-specific but cross-species reactive [Cummins, B., Aukland, M. L. & Cummins, P. (1987) Amer. Heart J. 113, 1333−1344], were used in this study. These mAbs were tested against recombinant wild-type and mutant human CdTnI proteins to assess their value as probes for the phosphorylation status of CdTnI. Four mAbs were found to react positively with the recombinant wild-type protein and their epitopes were contained in residues 31−210 of the human cardiac protein. Two of these mAbs appeared to be directed against the same epitope site within this region. The remaining three mAbs only reacted against the recombinant wild-type protein when it was phosphorylated, showing that these three antibodies were directed against the phosphate group(s) on Ser23 and/or Ser24. In order to investigate this further, a series of single and double mutants of CdTnI were used in which either Ala (to direct the enzymatic phosphorylation) or Asp (to mimic the phosphate group) replaced the Ser23 and/or Ser24. It was found the all three mAbs were able to react with the mono-phosphorylated form of the [Ala23]CdTnI single mutant but not the mono-phosphorylated form of the [Ala24]CdTnI single mutant, showing that they specifically required phosphorylation at Ser24. Experiments with a synthetic peptide composed of residues 1−29 of human CdTnI confirmed these data. Two of the three phosphorylation-specific mAbs were able to react with mutants containing either two Asp residues replacing Ser23 and Ser24 or one Asp residue instead of Ser24, indicating that a negative charge at position Ser24 is sufficient to invoke a reaction. The other mAb was more specific in that it would only react with CdTnI species with a phosphate group on Ser24.