AD2 is a monoclonal antibody (Ig G1) directed against Paired Helical Filaments (PHF) isolated from an Alzheimer brain. It allows a biochemical typing of tauopathies (from type 0 to type V)

1. Epitope location
2. Specificity
3. Sensitivity
4. Immunohistochemistry
5. Experimental protocol

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Epitope location

AD2 binds on the C-terminal part of Tau proteins, more precisely on the phosphorylated Ser 396 and 404 present on PHF-Tau and normal Tau (rapidly processed) (Buée-Scherrer V, Goedert et al Molecular Brain Research, 1996, 39:79-88).
AD2 is the fruit of a collaboration between Sanofi/Diagnotics Pasteur (Pr B. Pau and Dr Gilles Mourton-Gilles) and INSERM, represented by Dr A. Delacourte.

 Representation of AD2 on tau

To obtain AD2, please contact BioRad France: ref catalog 56484. Anti-tau protein.  AD2 monoclonal antibody.  100 µg/50 µl

Sensitivity

Using the western blot approach, AD2 is able to detect directly the pathological Tau proteins among the resolved proteins from a brain homogenate treated with SDS Laemmli buffer.
Brain tissues are homogenized in Laemmli sample buffer 1:10 (wt/vol) containing 5% sodium dodecyl sulfate (SDS) and heat-treated. For immunoblot, total brain homogenates were loaded and 150 µg total proteins were resolved on 10-20% polyacrylamide gel gradients in presence of SDS and transferred onto nitrocellulose membranes. Protein A-purified AD2 is used at a dilution of 20-50 nanogram/ml. Detection of pathological Tau can be performed on as little as 15 µg of homogenates (1 to 5 µl of a brain homogenate (1 g/ 10 ml).
- An ELISA sandwich assay with AD2 has been set up for the rapid quantification of pathological Tau .

Exemple: Western blot of brain homogenates

CONDAMINES O., BUÉE-SCHERRER V., BOISSIER L., WATTEZ A., DELACOURTE A., PAU B., MOURTON-GILLES C. New immunoassay for the mapping of neurofibrillary degeneration in Alzheimer's disease using two monoclonal antibodies against human PHF-tau proteins. Neurosciences Letters. 1995;192:1-4.

Specificity

- Using western blots and the human brain tissue, AD2 binds to the triplet of pathological Tau proteins from Alzheimer brains (Tau 60, 64, 69). It does not detect normal Tau proteins from control brains, but immunodetects Tau proteins from rapidly-processed biopsies from control brains (Sergeant et al, NeuroReport, 1995). Explanation: normal Tau proteins are phosphorylated in a manner similar to Tau-PHF, but are rapidly dephosphorylated after death by phosphatases, unlike tau-PHF.

SERGEANT N, BUSSIERE T, VERMERSCH P, LEJEUNE J.P, DELACOURTE A. Isoelectric point differentiates phf-tau from biopsy-derived human brain Tau proteins. Neuroreport. 1995, 6: 2217-2220.

- AD2 immunolabels pathological Tau proteins found in different neurodegenerative disorders (see DELACOURTE A. Pathological Tau of Alzheimer's diseas as a marker of neurofibrillary degeneration. Biomedicine and Pharmacotherapy. 48:287-295, and more recent reviews).

- AD2 immunostains a doublet Tau 60 and Tau 64 in brain homogenates from Pick's disease

(DELACOURTE A, ROBITAILLE Y, SERGEANT. N, BUEE L, HOF P, WATTEZ A, LAROCHE-CHOLETTE A, MATHIEU J, CHAGNON P, GAUVREAU D. Specific pathological tau protein variants characterize Pick's disease. J. Neuropathol. Exp. Neurol. 1996; 55:151-159)

- AD2 immunostains a doublet Tau 64 and Tau 69 in brain homogenates from Progressive Supranuclear Palsy and Corticobasal degeneration

BUEE-SCHERRER V., HOF PR, BUEE L, LEVEUGLE B, VERMERSCH P, PERL DP, OLANOW CW, DELACOURTE A. Hyperphosphorylated Tau protein doublets in corticobasal degeneration and Pick's disease. Acta Neuropathol.1996. 91: 351-359.

- AD2 immunodetects a triplet of pathological Tau proteins (Tau 60, 64, 69) in brain homogenates from the ALS/ Parkinson/ dementia complex of Guam

BUEE-SCHERRER V., BUEE L., HOF P.R, LEVEUGLE B., GILLES C., LOERZEL A.J, PERL D.P, DELACOURTE A. Neurofibrillary degeneration in Amyotrophic Lateral Sclerosis / Parkinsonism- Dementia complex of Guam: immunochemical characterization of tau proteins. Am. J. Pathology. 1995, 68: 924-932.

- A triplet is also observed in the hippocampal area of aged non-demented controls over 75

VERMERSCH P., DAVID J-P FRIGARD B., FALLET-BIANCO C., WATTEZ A., PETIT H., DELACOURTE A. Cortical mapping of Alzheimer pathology in brains of aged non-demented subjects. Prog. Neuropsychopharmaco. & Biol Psychiatry. 1995, 19/6, 1035-1047.

- Other neurodegenerative disorders can also be characterized, using AD2:

VERMERSCH P., BORDER R., LEDOZE F., RUCHOUX M-M, CHAPON F., THOMAS P., DESTEE A., LECHEVALLIER B., DELACOURTE, A. Demonstration of a specific profile of pathological Tau proteins in FrontoTemporal dementia cases. C. R. Acad. Sci Paris. 1995, 318:439-445.

- AD2 detects a specific profile of pathological Tau proteins in myotonic dystrophy (main band Tau 60). This genetic disease alters transcription of tau isoforms with exon 2

VERMERSCH P., SERGEANT N., RUCHOUX M.M., HOFMANN-RADVANYI H., PETIT H., DEWAILLY P., DELACOURTE A. Specific Tau variants in the brain from patients with myotonic dystrophy. Neurology. 1996, 47:711-717.

Sergeant N, Sablonniere B, Schraen-Maschke S, Ghestem A, Maurage CA, Wattez A, Vermersch P, Delacourte A.

Related Articles

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1.
Hum Mol Genet. 2001 Sep 15;10(19):2143-55.
PMID: 11590131

 

 

1:

Seznec H, Agbulut O, Sergeant N, Savouret C, Ghestem A, Tabti N, Willer JC, Ourth L, Duros C, Brisson E, Fouquet C, Butler-Browne G, Delacourte A, Junien C, Gourdon G.

Related Articles

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.
Hum Mol Genet. 2001 Nov 1;10(23):2717-26.
PMID: 11726559

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AD2 and the cellular models of Alzheimer's disease neurodegeneration.

CAILLET-BOUDIN ML, DELACOURTE A. COURATIER P., LESORT M., MOURTON- GILLES C. DELACOURTE A., HUGON J. Neuronal protein kinase C activation enhances phosphorylated Tau protein immunoreactivity. Neurosciences Letters. 1996, 203:155-158.

SOULIÉ C, LEPAGNOL J, DELACOURTE A, CAILLET-BOUDIN M.L. Déphosphorylation studies of SKNSH-SY 5Y cell Tau proteins by endogeneous phosphatase activity. Neuroscience Letters. 1996, 206:189-192.

DUPONT-WALLOIS L., SAUTIERE P.E., COCQUERELLE C., BAILLEUL B., DELACOURTE A., CAILLET-BOUDIN ML. Shift from foetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated by Okadaic acid. FEBS Letters, 1995 357:197-201.

SAUTIÈRE P. E, CAILLET-BOUDIN M. L, WATTEZ A, DELACOURTE A Detection of Alzheimer-type Tau proteins in okadaic acid-treated SKNSH-SY5Y Neuroblastoma cells. Neurodegeneration. 1994, 3: 53-60.

IMMUNOHISTOCHEMISTRY.

- Tangles, dystrophic neurites and neuritic plaques from Alzheimer Disease are specifically immunostained.
Tangles and dystrophic neurites from PSP, CBD, PEP are immunostained, as well as Pick Bodies from Pick Disease.

(DELACOURTE A, ROBITAILLE Y, SERGEANT. N, BUEE L, HOF P, WATTEZ A, LAROCHE-CHOLETTE A, MATHIEU J, CHAGNON P, GAUVREAU D. Specific pathological tau protein variants characterize Pick's disease. J. Neuropathol. Exp. Neurol. 1996; 55:151-159)

See also Hof et al, 1994, 1995.

- PHF are immulabelled at the electron microscope level, on tissue sections.
REIG S., BUEE-SCHERRER V., DEFOSSEZ A., DELACOURTE, A., BEAUVILLAIN J-C., MAZZUCA, M. Immunogold labelling of Paired Helical Filaments and amyloid filaments by specific monoclonal and polyclonal antibodies. Acta Neuropathol. 1995. 90: 441-447.
 

- Excellent antibody for confocal studies

·Galván M,  David J.P, Delacourte A, Luna J, Mena R. Sequence of neurofibrillary changes in aging and Alzheimer's disease: a confocal study with phospho-tau antibody, AD2. Journal of Alzheimer's disease. Volume 3, Number 4, August 2001, 417-425

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Experimental protocol

Product : anti-human phosphotylated Tau protein Ab
Isotype/specy : Murine IG1
identification : AD2
Tube Concentration : 0,2mg/ml (glycerol 1 vo l/ PBS 1 vol) corresponding to dilution : 1/10e
Final working dilution : 1/10000e to 1/30.000



Protocol for AD2

Cerebral tissue was homogenized with a mechanical 2cm3 potter into 10 volumes Laemli with 5% SDS.
Equal amounts of proteins were subjected to gel electrophoresis at 150V constant.
Finally, the proteins were transferred onto nitrocellulose membrane (Hybond Amersham 0.22µm) using a liquid electroblotter (Biorad) at 100V constant for 30 minutes.
The membrane was blocked for 30 minutes at room temperature in TNT* buffer + 5% skimmed dry milk.
TNT buffer : 15 mM Tris buffer pH 8, 140mM NaCl, 0.05% Tween 20
TNT* : TNT buffer with 0.05% Tween 20 supplement
Then, it was incubated with AD2 mouse antibody diluted in TNT* + skimmed milk (1/10000) for 1 hour at room temperature. AD2 must be diluted in TNT* + milk not only in TNT* in order to get a good final result.
After thorough washes in TNT*, the membrane was treated with the corresponding horseradish peroxydase-linked secondary antibodies (anti-mouse PI-2000 Vector Labs antibody, 1/50000) diluted in TNT* for 1 hour at room temperature.
After several washes in TNT*, the membrane was processed for chemiluminescent detection using the ECL Amersham chemiluminescent substrate, according to the manufacturer instructions.
The blots were finally exposed to Amersham Hyperfilms for 5 minutes to 2 hours.

Checking the sensitivity and specificity of AD2

Compare the immunodetection of brain homogenates (frontal cortex if possible) from controls (young if possible) versus a full blown Alzheimer patient. Load the brain homogenate at different dilutions (10 microliter non diluted, diluted 1/5, diluted 1/25) as shown here: