Chemical Tools for Studying Phosphohistidine: Generation of Selective -Phosphohistidine and -Phosphohistidine Antibodies

Nonhydrolysable stable analogues of & tau;-phosphohistidine (& tau;-pHis) and & pi;-pHis have been designed, aided by electrostatic surface potential calculations, and subsequently synthesized. The & tau;-pHis and & pi;-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both & tau;-pHis and & pi;-pHis conjugates in the form of BSA-glutaraldehyde-& tau;-pHis and BSA-glutaraldehyde-& pi;-pHis were synthesized and characterized by P-31 NMR spectroscopy. Commercially available & tau;-pHis (SC56-2) and & pi;-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-& tau;-pHis and BSA-G-& pi;-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the pHis antibodies generated by using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, & tau;-pHis and & pi;-pHis. Antibodies generated by using phosphopyrazole 8 as a hapten were found to be selective for & tau;-pHis, and antibodies generated by using pyridyl amino amide 13 were found to be selective for & pi;-pHis. Both & tau;- and & pi;-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The & tau;-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis.