Department of Chemistry, The University of Sheffield, Brook Hill, Sheffield, S3 7HF, UK.
Department of Infection Immunity and Cardiovascular Disease, Medical School, The University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK.
Chembiochem. 2023 Aug 15;24(16):e202300182. doi: 10.1002/cbic.202300182. Epub 2023 Jul 19.
Nonhydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed, aided by 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 BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by P 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 pHis antibodies generated by 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 by using phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated by using 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.
已设计并随后合成了τ-磷酸组氨酸（τ-pHis）和π-pHis 的不可水解稳定类似物，这得益于静电表面电势计算。τ-pHis 和 π-pHis 类似物（分别为磷酸吡唑 8 和吡啶基氨基酰胺 13）被用作半抗原来产生 pHis 多克隆抗体。BSA-戊二醛-τ-pHis 和 BSA-戊二醛-π-pHis 形式的 τ-pHis 和 π-pHis 缀合物均通过 31P NMR 光谱进行了合成和表征。使用市售的 τ-pHis（SC56-2）和 π-pHis（SC1-1；SC50-3）单克隆抗体表明，BSA-G-τ-pHis 和 BSA-G-π-pHis 缀合物可用于竞争性 ELISA 中评估 pHis 抗体的选择性。随后，通过竞争性 ELISA 评估了使用磷酸吡唑 8 和吡啶基氨基酰胺 13 作为半抗原产生的 pHis 抗体对 His、pSer、pThr、pTyr、τ-pHis 和 π-pHis 的选择性。发现使用磷酸吡唑 8 作为半抗原产生的抗体对 τ-pHis 具有选择性，而使用吡啶基氨基酰胺 13 产生的抗体对 π-pHis 具有选择性。两种 τ-和 π-pHis 抗体均被证明在免疫实验中有效，包括 ELISA、western blot 和免疫荧光。τ-pHis 抗体还被证明在含有 pHis 的蛋白质的免疫沉淀中很有用。
