TAU AGGREGATION ASSAY D. JAGA

Transcription

1 TAU AGGREGATION ASSAY D. JAGA

2 Contents General introduction Assay format Assay protocol Product specifications Assay performance Ordering information 2

3 TAU aggregation : General Introduction TAU (Tubulin Associated Unit) are abundant in neurons of the in central nervous system. TAU and other MAPs (microtubule associated protein) stabilize microtubules by binding to the MT surface and promote their selfassembly from tubulin subunits. TAU are phosphoproteins (80 potential sites) regulated by kinases/phosphatases. A hyperphosphorylation decreases their affinity for microtubules resulting in disruption of microtubule organization. This could play an important role in neuron apoptosis Hyperphosphorylation of the TAU protein can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of Alzheimer's disease and other taupathies.. Microtubule TAU protein 3

4 TAU aggregation : General Introduction TAU can be phosphorylated by several kinases (PKN,PKA,GSK3 are often reported) The aggregation of the protein tau into fibrils is known to be involved in the causation of the neurodegenerative taupathies and the progression of cognitive decline in Alzheimer s disease. 4

5 TAU aggregation : Assay format Anti-Tau d2 conjugate Anti-Tau Tb conjugate Non Aggregated Tau Aggregated Tau This assay is designed for the quantitative determination of Tau aggregation. Tau aggregation is measured in a sandwich assay format using the same monoclonal antibody, labelled with Tb Cryptate (donor) or with d2 (acceptor). The intensity of the signal obtained is proportional to the aggregated Tau complexes formed. 5

6 Assay protocol The reagents are dispensed in a white plate (384 wells small volume) as described below in the following order Incubate 2h to ON at RT Read on an HTRF compatible reader 10 µl Sample 5 µl anti TAU-d2 conjugate 5 µl anti TAU-Tb conjugate 6

7 TAU aggregation assay: product specification Detection of TAU aggregation on chemically aggregated TAU or crude brain extract from transgenic mice. Robustness of the assay and good sensitivity in brain extract tested (might even need sample dilution sometimes) Incubation: 2h to overnight (to get a better sensitivity) Reactivity tested : human only Research areas: CNS, Alzheimer disease, Taupathies 7

8 DeltaF % TAU aggregation: assay performance The experiment was read on a PHERAstar FS The curve was done on recombinant TAU protein (full length) chemically aggregated diluted in diluent (linear range : 1.9 to 500 ng/ml) Comparison of non aggregated TAU & aggregated TAU Non aggregated TAU Aggregated TAU [Human TAU] ng/ml 8

9 DeltaF % TAU aggregation: assay performance & validation TAU recombinant (full length) was chemically aggregated with our customer s protocol (24h) We asked them to produce TAU aggregated and stop at different time, to evaluate the kinetics of aggregation on their protocol. TAU aggregation detection & Kinetics of aggregation h Non-Aggregated TAU (sample) Aggregated TAU (sample) Non-Aggregated TAU Aggregated TAU after 1h Aggregated TAU after 2h Aggregated TAU after 4h Aggregated TAU after 6h Aggregated TAU after 24h h 4h 6h h Non-Aggreg [Aggregated TAU] ng/ml 9

10 DeltaF % TAU aggregation: assay performance & validation TAU aggregation was tested on brain extracts from transgenic mice (wild type, control, and mice sick from «Alzheimer disease» like ). The samples were thawed, diluted 1/50 and tested at +4 C or RT. Samples extracted from brain transgenic mice diluted 1/ c incubation RT incubation WT/WT ctrl AD mice WT/WT ctrl AD mice 10

11 DeltaF % TAU aggregation: assay performance & validation TAU aggregation was tested on brain extracts from triple transgenic mice (TAU/PSEN2/APP) These mice will develop «Alzheimer disease» like overtime. The samples were tested at different stage to follow the evolution of the disease TAU aggregation on mouse brain extract (straight) Pherastar FS Laser T=21h 10 months old 14 months old 20 months old mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 16 mouse 17 mouse 18 mouse 19 mouse 20 mouse 1605 mouse 1606 mouse 1607 mouse

12 DeltaF % TAU aggregation: assay performance & validation Zoom on the previous slide : it was very interesting to be able to already detect a difference between the 10 months old mice and the 14 months old. To our customer, this showed the sensitivity of our kit. They usually have to wait 20 months. TAU aggregation on mouse brain extract (straight) Pherastar FS Laser T=21h months old 14 months old mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 16 mouse 17 mouse 18 mouse 19 mouse 20 12

13 TAU aggregation : Ordering Information The kit includes : - 1 tube of d2-conjugate - 1 tube of Tb-conjugate - 1 tube of control - 1 bottle of Diluent Part number Description name Number of tests per kit List price 6FTAUPEG TAU aggregation tests 500 $580/580 Euros 6FTAUPEH TAU aggregation 10, 000 tests 10,000 $4 900/5 145 Euros 13

14 Companion products Amyloid beta 1-40 (62B40PEB) MAP-TAU (63ADK008PEB) BACE 1 assay (with SA-XL and biotin APP-K 64APBKAA) Phospho protein detection 14

15 Bibliography of interest: Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K (June 2001). "Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments" Proc. Natl. Acad. Sci. U.S.A. 98 (12): Taniguchi T, Kawamata T, Mukai H, Hasegawa H, Isagawa T, Yasuda M, Hashimoto T, Terashima A, Nakai M, Mori H, Ono Y, Tanaka C (March 2001). "Phosphorylation of tau is regulated by PKN". J. Biol. Chem. 276 (13): Fiona Grueninger, Bernd Bohrmann, Christian Czech, Theresa Maria Ballard, Johann R. Frey, (Sept 2009) Phosphorylation of Tau at S422 is enhanced by Aβ in TauPS2APP triple transgenic mice» Neurobiology of Disease

16 Progesterone: Ordering Information The kit includes : - 1 tube of d2-conjugate - 1 tube of K-conjugate - 1 tube of Standard - 1 bottle of Diluent - 1 bottle of Conjugate buffer Part number Description name Number of tests per kit List price 6FPROPEB Progesterone tests 1000 $870/780 Euros The progesterone standard is available upon request (ref : 6FCUS000) 16

17 Companion products All other metabolic assays : Cortisol (62CO2PEB) Estradiol (62ESTPEB) Aldosterone (64ALDPEB) Testosterone (64TESTPEB) 17

18 Thank you