Modified Aβ42 peptides as immunoassay calibrators improve the measurement of native Aβ42 peptides in clinical samples

Amyloid peptides that are 42 amino acids long (Aß 1-42) are putative neurotoxic species in Alzheimer's disease either alone or as aggregates. Assays used to measure Aß 1-42 peptides in clinical studies are typically made quantitative by the use of Aß1-42 full length synthetic peptides as reference material to generate standard curves. However, Aß 1-42 peptides have strong amyloidogenic properties leading to the formation of aggregates in aqueous solutions. The mechanism of oligomer formation has been shown to originate from a small, five amino acid region located at residues 16-20. Thus, we developed synthetic Aß peptides comprising an N-terminal immunoreactive region, a C-terminal immunoreactive region, and several different hydrophilic linker regions. Full length and modified versions of Aß 1-42 peptides were subjected to several biochemical analyses including dynamic light scattering and circular dichroism for the assessment of higher order structure and aggregation. These peptides were subjected to stability studies at different temperatures for up to 60 days. Peptides were run in an electrochemiluminescent ELISA format Meso Scale Discovery (MSD). Briefly, the immunoassays incorporated a biotinylated anti-C terminal Aß 1-42 antibody that was captured on streptavidin plates. The native and modified peptides were added as calibrators followed by a ruthenium tagged antibody specific for the N-terminus of Aβ peptides. The antibody/modified Aß 1-42 complexes formed in each well were measured using an MSD instrument. The biochemical analysis suggests that, unlike native peptide the modified Aß 1-42 peptides do not aggregate or form any higher order structures. The calibration curves generated from modified Aß 1-42 peptides were comparable to those generated by native Aß 1-42 peptides, suggesting that the modified Aß 1-42 peptides can be used to quantitate the levels of Aβ peptides present in clinical samples. Only a 3.7% decrease in response was observed when the modified Aß 1-42 was tested from 0 to 60 days at 37°C. By contrast, the response of native Aß 1-42 was decreased up to 80%. Modified Aβ peptides offer better stability and comparable performance to native Aß 1-42 peptides as immunoassay standards. Thus, these modified Aß 1-42 peptides will provide better consistency in assays measuring Aß 1-42 peptides in normal and diseased patients.