Human amyloid beta 1-42 assay principle

The human amyloid beta 1-42 assay measures human amyloid 1-42 peptide in a cell or tissue supernatant or lysate. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the human beta amyloid 1-42 peptide. In presence of human beta 1-42 peptide in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing any changes caused by experimental variability under a no-wash assay format.

Human amyloid beta 1-42 assay protocol

The Human Amyloid beta 1-42 kit features a two-plate assay protocol, where cells are plated, stimulated, and optionally lysed in the same culture plate. Cell supernatants and/or lysates are then transferred to an assay plate for the detection of Human amyloid beta 1-42. This protocol enables the cells' viability and confluence to be monitored. The antibodies labeled with HTRF fluorophores may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

Human Amyolid beta 1-42 assay specifications

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and the %CV was calculated for each sample.

Inter-assay (n=4)

Each of the samples was measured in 4 independent experiments by 4 different operators, and the % CV was calculated for each sample.

Human Amyloid beta 1-42 secretion from Swedish APP overexpressing SH-SY5Y cells

Human SH-SY5Y APPsw samples were generated by Dr Chami Mounia, Molecular and Pharmacology Institute CNRS-UMR 7275 (Nice, France). The neuroblastoma cells (CRL-2266; American Type Culture Collection) stably expressing amyloid precursor protein harboring the Swedish double mutation (K670N / M671L; APPswe) constructs were generated following standard protocols (Oules et al. 2012 [1]), and maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin in the presence of 400 μg geneticin. Cell supernatants were supplemented with a protease inhibitor cocktail and stored at -80 ° C until analysis. For each sample, several dilutions were assessed to determine the most appropriate conditions to minimize dilution biases while remaining in the linear range of the assays. 16 µL of supernatant was assessed in triplicates in both human amyloid beta 1-40 and 1-42 HTRF kits, following the procedure given in the HTRF kit package inserts. Results show the extreme amyloid concentrations, higher than 10,000 pg/mL, that are expected for this cell line stably expressing the Swedish double mutation, known to generate high cleavage events of APP into amyloid peptides.

[1] Oules , B., D. Del Prete , B. Greco, X. Zhang, I. Lauritzen, J. Sevalle , S. Moreno, P. Paterlini-Brechot , M. Trebak , F. Checler , F. Benfenati & M. Chami (2012) Ryanodine receptor blockade reduces amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer Disease. J Neurosci , 32, 11820-34.

Human Amyloid beta 1-42 secretion from Swedish APP overexpressing HEK293 cells

Human HEK293 APPsw cells were cultivated in a flask at a density of 660k cells/mL in 10% FCS supplemented DMEM medium, for 48 hours. Supernatants were supplemented with a protease inhibitor cocktail to prevent amyloid peptide degradation, and then stored at -80 ° C until analysis. For each sample, several dilutions were assessed to determine the most appropriate conditions to minimize dilution biases while remaining in the linear range of the assays. 16 µL of supernatant was assessed in triplicates in both human amyloid beta 1-40 and 1-42 HTRF kits, following the procedure given in the HTRF kit package inserts. Results show the high amyloid concentrations that are expected for this cell line stably expressing the Swedish mutation, known to generate high cleavage events of APP into amyloid peptides.