Rodent beta Amyloid 1-40 ELISA

Additional Information

Assay Background

The first case of Alzheimer’s disease was defined and reported in 1907 by the German scientist, Dr. A. Alzheimer. His studies have shown that this is the main cause of dementia in the elderly. The plaques which appear in the brains of Alzheimer’s disease patients are mostly constituted by the Amyloid beta protein (A-beta). A-beta is a peptide which consists of 40 or 42 (43) amino acids, and reports show that this is cleaved by beta- and gamma- secretase from the amyloid precursor protein. In addition, the presence of numerous variant A-beta molecules has been demonstrated in the culture fluid of mouse neuroblastoma cells transfected with cDNA coding human amyloid precursor protein (APP). Furthermore, in 1995, a dominant and differential deposition of distinct beta amyloid peptide species, A-beta (N3pE), in senile plaques was found by Saido et al. This modified molecule, starting at the 3rd amino terminal residue, glutamate, was discovered to convert to pyroglutamate through intramolecular dehydration.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 100 μl of standards (0-100 pg/ml) and samples into wells (Assay Diluent serves as zero standard). Add 100 μl Assay Diluent into additional two wells as blank. Cover with enclosed foil, incubate for overnight at 4°C.
3. Aspirate each well and wash, repeating the process 6 times for a total 7 washes. Wash by filling each well with 1× Wash Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher and keep the wash buffer in the well for 15-30 seconds. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
4. Add 100 μl of 1X HRP-labeled antibody solution to each well (except blank). Cover wells and incubate for 1h at 4°C.
5. Aspirate each well and wash the microtiter plate 9 times as step 3.
6. Add 100 μl of TMB substrate to each well. Incubate for 30 minutes at room temperature in dark. (Take the required quantity of TMB substrate into a disposable test tube before use, and do not return the rest of the test tube to original TMB substrate bottle to avoid contamination.) The liquid will become blue by addition of TMB Substrate Solution.
7. Add 100 μl of STOP Solution to each well. The color of the solution should change from blue to yellow.
8. Read the OD with a microplate reader at 450 nm immediately. The measurement should be done within 30 minutes after addition of STOP Solution.

Typical Standard Curve