Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions

Abstract

Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.

Dendritic Spine Loss and Chronic White Matter Inflammation in a Mouse Model of Highly Repetitive Head Trauma

“…For this purpose, tau phosphorylation was analyzed by Western blot analysis using a panel of specific antitau antibodies that we have previously characterized. 22 We found no changes in tau phosphorylation state in multiple brain regions, including the cortex (Figure 8), hippocampus, and midbrain ( Figure 9). …”

“…22 Proteins (5 to 10 mg) were separated on SDS-PAGE gels, blotted onto nitrocellulose membranes, and blocked 1 hour at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween 20. Membranes were probed with primary antibodies (listed below) diluted in SuperBlock blocking buffer (Thermo Fisher Scientific, Rockford, IL), washed, and incubated for 1 hour at room temperature with the corresponding horseradish peroxidaseelinked secondary antibody.…”

“…For tau analysis, a secondary antibody directed against light chain of immunoglobulins was used to detect monoclonal primary antibodies to avoid non-specific signals coming from the heavy chains of Igs. 22 Membranes were revealed by enhanced chemiluminescence (ECL Plus; GE Healthcare Biosciences, Piscataway, NJ) in a Fujifilm LAS4000 imaging system (Fujifilm USA, Valhalla, NY). Densitometric analyses were performed with Image Gauge analysis software version 3.0 (Fujifilm USA).…”

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

“…In fact, many commercially available PTM-specific antibodies have not been validated for their specificity. Recent evidences using various approaches, including immunoassays using synthetic peptides and peptide arrays, have shown that indeed a significant fraction of existing PTMtargeting antibodies lack specificity (30)(31)(32)(33)(34). These studies have identified major species that nonspecifically bind to the antibodies, including nonmodified epitopes with the target amino acid sequence and those with modifications at non-target sites (31,34).…”

Extracellular Tau Oligomers Induce Disruption of Endogenous Tau Distribution, Invasion of Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction

“…Differential seeding of normal or mutant tau, resulting from the conformational diversity and location of the first pathogenic seeds to form in the brain, could produce a great deal of heterogeneity in both the clinical presentation and morphology of tau pathology [214,222,223,275]. AD tau has long been known to be phosphorylated at multiple sites that are rarely phosphorylated in normal tau [140,[276][277][278], and to adopt immunologically sensitive conformational changes [248][249][250]279]. Interestingly, we found that extracellular tau oligomers did not alter the immunoreactivity of intracellular tau with two monoclonal antibodies, Alz50 [248,249] and MC1 [250], that detect AD tau conformations, nor with the PHF1 monoclonal, which recognizes tau phosphorylated at S396/S404 [171,251] (Fig.…”