Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation.
In Alzheimer's disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231+pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.
Singer
D
D
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.
Herth
N
N
Kuhlmann
J
J
Holland-Nell
K
K
Beck-Sickinger
A G
AG
Hoffmann
R
R
D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016875:tau Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D016875:tau Proteins