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Chemical Tools for Study of Phosphohistidine: Generation of Selective τ-Phosphohistidine and π-Phosphohistidine Antibodies

preprint
submitted on 23.09.2020 and posted on 23.09.2020 by Mehul Makwana, Cleide Dos Santos Souza, Barry T. Pickup, Mark J. Thompson, Santosh Kumar Lomada, Yushi Feng, Thomas Wieland, Richard F. W. Jackson, Richmond MUIMO
Non-hydrolysable stable analogues of τ-pHis and π-pHis have been designed using electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the generated pHis antibodies generated using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated using the pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis

History

Email Address of Submitting Author

r.muimo@sheffield.ac.uk

Institution

University of Sheffield, Infection, Immunity & Cardiovascular Disease The Medical School

Country

United Kingdom

ORCID For Submitting Author

0000-0003-4242-0188

Declaration of Conflict of Interest

Two of the authors are named on relevant patents (WO/2015/033120; EU - 20160207948 and USA – US 10,053,476).

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