Simultaneous detection of tau phospho-epitopes with haptenylated antibodies Wolfgang HÌrtig a, * , J˛rg Lehmann d, * , Jens Stieler a , David Singer b , Jens Grosche a,c , Thomas Arendt a and Ralf Ho¡mann b a Paul Flechsig Institute for Brain Research, b Department of Bioanalytics, Center for Biotechnology and Biomedicine (BBZ), c Interdisciplinary Center of Clinical Research (IZKF) at the Faculty of Medicine, University of Leipzig and d Priontype GmbH & Co. KG, Research & Development, BioCity, Leipzig, Germany Correspondence and requests for reprints to Dr Wolfgang HÌrtig, Paul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany Tel: + 49 341 9725772; fax: + 49 341 9725749; e-mail: hartig@medizin.uni-leipzig.de * These authors contributed equally to the present study. Sponsorship: Parts of this work were supported by the BMBF (Grant 03DU03LE) the Interdisciplinary Center of Clinical Research (IZKF) at the Faculty of Medicine of the University of Leipzig (project C1) and the SAB (Grant 8879). Received 31 January 2006; accepted 22 February 2006 Neuro¢brillary tangles as a neuropathological hallmark of Alzheimer’s disease are mainly composed of abnormally phosphorylated microtubule-associated protein tau. The present work was primarily focused on the immunohistochemical characterization of recently developed monoclonal antibodies directed against disease-associated epitopes. Anti-phospho- threonine 212/phospho-serine 214 (HPT-1), anti-phospho-threonine 231/phospho-serine 235 (HPT-101) and their biotinylated derivatives were shown to be sensitive markers for the immunohistochemical detection of neuropathological alterations during Alzheimer’s disease. Triple carbocyanine immuno£uores- cence labelling was based on digoxigenylated, £uoresceinated and biotinylated primary antibodies. AT8 -immunolabelling of phospho-serine 202 and phospho-threonine 205 combined with HPT-1 and HPT-101-staining revealed similar distribution patterns of the three double-phosphorylated tau epitopes in the neocortex of patients with Alzheimer’s disease. NeuroReport 17:869^ 874  c 2006 Lippincott Williams & Wilkins. Keywords: Alzheimer’s disease, anti-digoxin, anti-£uorescein, carbocyanine, digoxigenin^anti-digoxigenin, hapten^anti-hapten technique, neocortex, tau pathology, triple immuno£uorescence labelling Introduction The microtubule-associated protein tau is abundant in the central nervous system and predominantly expressed in the axons of neurons but is barely detectable in astrocytes and oligodendrocytes [1]. Human tau occurs in six isoforms and comprises up to 441 amino acids [2]. About 30 hydroxyl groups from serine (Ser) and threonine (Thr) residues of tau can be phosphorylated by numerous kinases [1,2]. Vice versa, the phosphorylation status of tau is also determined by phosphatases catalysing its dephosphoryla- tion. Abnormally phosphorylated protein tau is a main component of neurofibrillary tangles that occur during Alzheimer’s disease primarily in the entorhinal cortex and hippocampal formation and then spread throughout the brain [3]. Several highly specific antibodies recognize defined epitopes only if their Thr and Ser residues are phosphory- lated, but not their dephosphorylated counterparts; for example, the monoclonal antibody (mAb) AT8 was shown to bind tau phosphorylated at Ser 202 (202P) and Thr 205 (205P) [4]. The generation of the AT8 epitope was considered an early event during the pathogenesis of Alzheimer’s disease [5,6]. In contrast, Thr 231 was found to be already partially phosphorylated in fetal and adult brains [7], whereas Hoffmann et al. [8] suggested that the double phosphorylation of Thr 231 and Ser 235 may be unique to paired helical filaments in Alzheimer’s disease. Moreover, it was shown that the mAb AT100 recognizes 212P/214P only in tau molecules in disease-specific con- formation [8,9]. The time course of the occurrence of different phospho-epitopes was previously only detected by single staining of different markers for phospho-tau on consecutive sections (see e.g. [6,10]). Thus, a main goal of the present study was the simultaneous detection of the three double-phosphorylated epitopes so as to also reveal slight differences between their distribution patterns in tangles, neuritic plaques and neuropil threads in the neocortex of patients with Alzhei- mer’s disease. As a result of the lack of specific primary antibodies from different animal host species, differently haptenylated mAbs to be detected by anti-hapten immunoreagents instead of cross-reacting secondary AGEING NEUROREPORT 0959-4965  c Lippincott Williams & Wilkins Vol17 No 9 26 June 2006 869 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.