Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau

Tau null (tau KO) mice [14] and tau transgenic (Tg) mice line PS19 expressing human 1 N/4R tau with the P301S mutation driven by the mouse prion promoter [49] were obtained from Jackson Laboratory (Bar Habor, ME). Tau Tg mice line JNPL3 expressing human 0 N/4R tau with the P301L mutation were previously described [33].

AT8 (Thermo-fisher) is a mouse monoclonal antibody specific towards phosphorylation sites S202 and T205 in tau [19] that can also be influenced by phosphorylation at S199 or S208 [34, 42]. PHF1 (generously provided by Dr. Peter Davies, Albert Einstein University, NY, NY) is a mouse monoclonal antibody specific towards phosphorylation sites S396 and S404 in tau [40]. Rabbit polyclonal antibody (H-150) raised against the first 150 amino acids of human tau was obtained from Santa Cruz Biotechnologies (Dallax, TX), and rabbit polyclonal antibodies 3026 and 3029 raised against recombinant full-length 0 N/3R tau was generated as a service by GenScript USA Inc. (Piscataway, NJ).

All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Phosphopeptides EIVYKpSPVVSGDTpSPRHLSC (p391–409) and DRSGYSpSPGpSPGpTPGSRSRC (p193–211) corresponding, respectively, to residues 391–409 and 193–211 in the 2 N/4R human tau isoform with a C residue added at the carboxy-termini for chemical conjugation were synthesized and purified by GenScript USA Inc. (Piscataway, NJ). Lyophilized peptides were reconstituted in phosphate buffered saline (PBS) and conjugated to Imject maleimide-activated mariculture keyhole limpet hemocyanin (mcKLH; Thermo Scientific, Waltham, MA). 2–3 month old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were used for immunization. Injection solutions emulsified by vortexing for 15 min were prepared by combining 100 μg KLH-conjugated peptide in 200 μl PBS with 100 μl of Freund’s complete adjuvant (Sigma Aldrich, St. Louis, MO) for the initial subcutaneous injection. Three weeks following the initial injection, mice were boosted with an intraperitoneal (IP) injection of 50 μg KLH-conjugated peptide in 200 μl PBS emulsified with 100 μl of Freunds incomplete adjuvant (Sigma Aldrich, St. Louis, MO). Three weeks later, mice were boosted with an IP injection of 50 μg KLH-conjugated peptide in PBS. Three days later, mice were euthanized and spleens were harvested using aseptic technique.

Mouse myeloma (Sp2/O-Ag14; ATCC, Manassas, VA) cells were maintained in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM) with 10% NCTC 135 Media (Sigma Aldrich, St. Louis, MO), 20% hybridoma grade fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine, 0.45 mM pyruvate, 1 mM oxaloacetate, and 0.2 U/ml insulin at 37 °C and 8% CO₂. Spleens were gently homogenized in 5% FBS/Hank’s balanced salt solution (HBSS; Lonza, Walkersville, MD) and centrifuged to pellet cells. The cell pellet was resuspended in red blood cell lysis buffer for one minute (Sigma Aldrich, St. Louis, MO) and diluted with HBSS. The cells were then washed twice by centrifuging at 100 x g for 10 min and resuspended in HBSS. Sp2/O-Ag14 cells were also washed twice with HBSS. Five million Sp2/O-Ag14 cells were added to 50 million spleen cells, and after centrifuging at 100 x g for 10 min onto a culture dish, fusion was induced with 50% polyethylene glycol 1450 (Sigma Aldrich, St. Louis, MO). After washing with HBSS, cells were incubated in Sp2/O-Ag14 media at 37 °C with 8% CO₂ overnight. The next day, the cells were gently detached from the plate and distributed into 96 well plates with Sp2/O-Ag14 media/0.5% hybridoma enhancing supplement (Sigma Aldrich, St. Louis, MO)/HAT selection supplement (Sigma Aldrich, St. Louis, MO).

All hybridoma clones were screened for reactivity to the respective unconjugated peptide that was used for immunization by enzyme-linked immunosorbent assay (ELISA). MaxiSorp plates (Thermo Scientific, Waltham, MA) were coated with 1 μg/ml peptide in PBS and blocked with 5% FBS/PBS. Media from the hybridomas was applied to plates, which were then incubated at room temperature. Next, the plates were washed with PBS and incubated with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP; Jackson Immuno Research Labs, West Grove, PA) at room temperature. Then, plates were washed and TMB substrates (Pierce, Rockford, IL) were applied until color changes were observed. Reactions were then quenched with 1 M HCl and absorbance was measured at 450 nm. Clones that were positive by ELISA were transferred to larger culture plates as needed. The positive clones were next screened by immunohistochemistry of a human AD autopsy case with abundant tau pathology.

Antibody clones were isotyped with the mouse monoclonal antibody isotyping kit purchased from Sigma-Aldrich (St. Louis, MO).

Paraffin embedded, formalin fixed human brain tissue was obtained through the University of Florida Neuromedicine Human Brain Tissue Bank. Sequential tissue sections were deparaffinized with xylenes and sequentially rehydrated with graded ethanol solutions (100–70%). Antigen retrieval was performed by incubating sections in 0.05% Tween-20 in a steam bath for 60 min. Endogenous peroxidase activity was quenched with 1.5% hydrogen peroxide/0.005% Triton-X-100/PBS for 20 min. Following washes, sections were blocked with 2% FBS/0.1 M Tris, pH 7.6 and incubated with primary antibody overnight at 4 °C. Following washing with 0.1 M Tris, pH 7.6, sections were incubated with biotinylated horse anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) diluted in 2% FBS/0.1 M Tris, pH 7.6 for 1 h. Next, sections were washed with 0.1 M Tris, pH 7.6, then incubated with streptavidin-conjugated HRP (VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA) diluted in 2% FBS/0.1 M Tris, pH 7.6 for 1 h. Sections were washed with 0.1 M Tris, pH 7.6, and then developed with 3, 3′- diaminobenzidine (DAB kit; KPL, Gaithersburg, MD). Reactions were stopped by immersing the slides in 0.1 M Tris, pH 7.6, and sections were counterstained with Mayer’s hematoxylin (Sigma Aldrich, St. Louis, MO). Next, sections were dehydrated with an ascending series of ethanol solutions (70%–100%) followed by xylenes and coverslipped using cytoseal (Thermo Scientific, Waltham, MA).

Human full-length tau cDNA (0N/3R or 2N/4R isoform) cloned into the bacterial expression vector pRK172 were kindly provided by Dr. Michel Goedert. pRK172 plasmid expressing human 0 N/3R tau with the S199A, T202A or T205A mutations (numbered according to the 2 N/4R tau isoform) or human 2 N/4R tau with the S396A or S404A mutations (numbered according to the 2 N/4R tau isoform) was created with oligonucleotides corresponding to the amino acid substitutions by QuickChange site-directed mutagenesis (Stratagene, La Jolla, CA). These recombinant tau proteins were expressed in E. coli BL21 and purified, as previously described [18, 27].

Tau proteins (0.2 mg/ml) were phosphorylated with either glycogen synthase kinase 3β (GSK3β; 2.5 U/ul; New England Biolabs, Ipswich, MA) or p42 mitogen-activated protein kinase (MAPK; 0.5 U/ul; New England Biolabs, Ipswich, MA) in 50 mM Tris, pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.1% Brij 35, 200 uM ATP for 2 h at 30 °C followed by heat inactivation at 95 °C for 5 min. Samples were diluted in SDS-sample buffer and 100 ng of each tau protein was loaded on a separate lane on SDS-polyacrylamide gels for immunoblot analysis.

Tau KO, PS19 Tg or non-transgenic (nTg) mice were humanely euthanized and the brains were harvested. Brain tissue was lysed with 2% SDS/50 mM Tris, pH 7.5 using a probe sonicator until homogenous and incubated for 10 min at 100 °C. Protein concentrations were determined by BCA assay (Thermo Scientific) using bovine serum albumin (BSA) as the standard. SDS sample buffer was added, and equal amounts of protein (40 μg) were resolved by SDS-PAGE and analyzed by immunoblot.

Frozen human brain tissue was obtained through the University of Florida Neuromedicine Human Brain Tissue Bank. Pulverized temporal cortex tissue from human AD cases (n = 3) or control (n = 2) was homogenized in 3 ml of high-salt (HS) buffer (50 mM Tris–HCl, pH 7.5, 0.75 M NaCl, 2 mM EDTA, 50 mM NaF with a cocktail of protease inhibitors) per gram of tissue and sedimented at 100,000 x g for 30 min at 4 °C. Supernatants were collected (HS fraction) and pellets were re-suspended in 2 ml of HS buffer containing 1% Triton X-100 per gram of tissue. Samples were sedimented at 100,000 x g for 30 min at 4 °C and the supernatants were collected (HS/Triton-soluble fraction). Pellets were again re-extracted in HS buffer/1% Triton X-100. Samples were sedimented at 100,000 x g for 30 min at 4 °C and supernatants were discarded. Pellets were re-suspended in 1 ml of HS buffer containing 1% sarkosyl per gram of tissue, incubated at 37 °C for 30 min, and sedimented at 100,000 x g for 30 min at 4 °C. Supernatants were collected (sarkosyl-soluble fraction). The detergent-insoluble pellets were extracted in 0.5 ml of 4 M urea, 2% SDS, 25 mM Tris–HCl pH 7.6 per gram of tissue, sonicated, and sedimented at 100,000 x g for 30 min at 25 °C. Protein concentrations were determined by BCA assay (Thermo Scientific) using BSA as the standard. SDS sample buffer was added, and equal amounts of protein (10 μg) were resolved by SDS-PAGE and analyzed by immunoblot.

Protein samples were resolved by electrophoresis on 10% polyacrylamide gels, then electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% milk/Tris-buffered saline (TBS) and then incubated overnight at 4 °C with primary antibodies diluted in 5% BSA/TBS. Following washing, blots were incubated with HRP conjugated goat anti-mouse IgG/IgM (heavy and light chains, but pre-absorbed to human, bovine and horse serum proteins) or goat anti-rabbit secondary antibodies (Jackson Immuno Research Labs, West Grove, PA) diluted in 5% milk/TBS for 1 h. Following washing, protein bands were visualized using Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA), and images were captured using the GeneGnome XRQ system and GeneTools software (Syngene, Frederick, MD).

To generate antibodies similar to the PHF1 epitope [40] we immunized mice with the synthetic phosphopeptide EIVYKpSPVVSGDTpSPRHLSC corresponding to residues 391–409 in 2 N/4R human tau and containing phosphorylated S396 and S404. Several hybridomas, termed the PHF series, were identified by ELISA screening as well as initial screening for reactivity of tau pathology by immunohistochemistry of a human AD autopsy case with abundant tau pathology. Five hybridomas (PHF2, PHF15, PHF17, PHF20 and PHF22) were identified using these criteria. To determine the specificity of these monoclonal antibodies, we in vitro phosphorylated full-length WT, S396A and S404A 2 N/4R human tau with known tau kinases (p42 MAPK or GSK3β) and used these tau proteins for immunoblotting analysis (Fig. 1). We used the previously characterized antibody PHF1, that recognizes tau phosphorylated at both S396 and S404, as a control [40]. WT but not S396A or S404A tau phosphorylated with GSK3β reacted with PHF1 as expected. PHF20 reacted with WT or S396A but not S404A tau phosphorylated with either GSK3β or p42 MAPK, demonstrating that this antibody preferentially recognizes tau phosphorylated at S404. Since PHF1 did not react with tau phosphorylated with p42 MAPK these data also show that p42 MAPK in vitro phosphorylated tau only at S404 and not S396. Antibodies PHF2, PHF15, PHF17 and PHF22 all revealed the same relativities as PHF1 in these assays, demonstrating that they recognize tau only when phosphorylated at both S396 and S404.

The specificity of the new PHF series of antibodies was then assessed using total mouse brain lysates from tau KO, nTg and PS19 tau Tg mice (Fig. 2). Antibodies PHF2, PHF15, PHF17 and PHF 20 specifically reacted with mouse tau in nTg mice and with both mouse and human tau in lysates derived from PS19 tau Tg mice. PHF2, PHF15, and PHF 20 displayed no cross-reactivity in brain extracts from tau KO mice, but PHF17 weakly cross-reacted with a ~150 kDa non-tau protein band. PHF22 recognized mouse and human tau, but it also cross-reacted with some non-specific higher molecular mass proteins still present in lysate from tau KO mice.

Next, in an attempt to generate antibodies similar to the AT8 epitope, we immunized mice with the synthetic phosphopeptide DRSGYSpSPGpSPGpTPGSRSRC corresponding to residues 193–211 in 2 N/4R human tau with phosphorylated S199, S202 and T205. Several hybridomas were identified by ELISA screening with the corresponding unconjugated peptide followed by immunohistochemistry for tau pathology in a human AD autopsy case. Nine of these hybridomas (1H5, 2D1, 3C9, 4A10, 5F2, 6G12, 7F2, 8G5 and 10G12) were further characterized. To determine the specificity of these monoclonal antibodies, we phosphorylated WT, S199A, S202A and T205A 0 N/3R human tau (mutations numbered according to the sequence of 2 N/4R human tau) with p42 MAPK or GSK3β and performed immunoblotting analysis (Fig. 3). Antibodies 6G12, 7F2, 8G5 and 10G12 reacted with tau phosphorylated with either p42 MAPK or GSK3β, but not when T205 was mutated to an A, indicating that they all recognize tau phosphorylated at T205. Antibodies 2D1, 4A10 and 5F2 reacted with phosphorylated and non-phosphorylated tau showing that their recognition epitopes are phosphorylation independent. Antibody 1H5 reacted with phosphorylated tau but could also recognize non-phosphorylated tau to a lesser degree, demonstrating that it prefers phosphorylated tau. Antibody 3C9 only recognized phosphorylated tau and prefers tau phosphorylated at T205, as shown by the reduced reactivity with phosphorylated T205A tau. However, 3C9 is a more promiscuous phospho-antibody that can also recognize tau phosphorylated at either S199 or S202 since the T205A mutation does not completely block its reactivity with phosphorylated tau.

The specificity of the antibodies generated to the AT8 epitope were then assessed using mouse brain lysates from tau KO, nTg and PS19 tau transgenic mice (Fig. 2). All of these antibodies could detect endogenous mouse tau in nTg mice and both mouse and human tau in PS19 Tg mice, and they were quite specific for tau, as demonstrated by their lack of reactivity to lysates from tau KO mice. They are much more specific than AT8, which reacted with many additional higher molecular mass protein bands present in lysates from tau KO mice (Fig. 2). The same cross-reactivity of the AT8 antibody with non-tau proteins was observed in three independent lots of the AT8 antibody.

All of the new tau antibodies were compared for immunoreactivity of tau pathology using brain tissue from AD and control patients. All of these antibodies reacted with NFTs, but antibodies PHF17, PHF20, PHF22, 2D1 and 7F2 yielded the strongest staining with little background (Figs. 4 and 5; data not shown); however, as shown in the immunoblotting screen, PHF22 is not completely tau specific (Fig. 2). Antibodies 2D1 and 7F2 also provided strong detection of neuropil threads in addition to senile plaque dystrophic neurites. Antibodies PHF17, PHF20, 2D1 and 7F2 were also tested for immunoreactivity of tau pathology in the JNPL3 Tg mouse model, where they showed strong immunoreactivity (Figs. 4 and 5).

The ability of the new monoclonal antibodies to detect sarkosyl-insoluble tau in the temporal cortex tissue from AD (n = 3) versus control (n = 2) cases was also assessed by immunoblotting (Fig. 6). Of the new PHF antibodies, PHF2, PHF15 and PHF20 specifically detect detergent insoluble tau only in the AD cases, while PHF17 and PHF22 showed some non-specificity in the control lanes, consistent with the immunoblot results from the mouse total brain lysates. The phospho-specific antibodies (3C9, 6G12, 7F2, 8G5, 10G12) generated to the AT8 epitope showed higher levels of detection of sarkosyl-insoluble tau in AD samples than our phospho-independent or phospho-selective antibodies (1H5, 2D1, 4A10, 5F2).