Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.
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.
Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Franck R
FR
Petry
Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Jérôme
J
Pelletier
Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Alexis
A
Bretteville
Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Françoise
F
Morin
Université Laval, Faculté de Pharmacie, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Frédéric
F
Calon
Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Sébastien S
SS
Hébert
Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, New York, New York, United States of America.
Robert A
RA
Whittington
Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.
Emmanuel
E
Planel
D000544:Alzheimer Disease; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D016477:Artifacts; D004195:Disease Models, Animal; D055786:Gene Knockout Techniques; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000276:immunology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D016875:tau Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D016875:tau Proteins