Quantification of Alzheimer Amyloid β Peptides Ending at Residues 40 and 42 by Novel ELISA Systems

Abstract

Background: The amyloid β (Aβ) peptide is a key molecule in the pathogenesis of Alzheimer’s disease. Reliable methods to detect and quantify soluble forms of this peptide in human biological fluids and in model systems, such as cell cultures and transgenic animals, are of great importance for further understanding the disease mechanisms. In this study, the application of new and highly specific ELISA systems for quantification of Aβ40 and Aβ42 (Aβ peptides ending at residues 40 or 42, respectively) in human cerebrospinal fluid (CSF) are presented. Materials and Methods: Monoclonal antibodies W0-2, G2-10 and G2-11 were thoroughly characterized by (SPOT) epitope mapping and immunoprecipitation/mass spectrometry. We determined whether aggregation affected the binding capacities of the antibodies to synthetic peptides and whether components of the CSF affected the ability of the antibodies to bind synthetic Aβ1–40 and Aβ1–42 peptides. The stability of Aβ40 and Aβ42 in CSF during different temperature conditions was also studied to optimize sample handling from lumbar puncture to Aβ assay. Results: The detection range for the ELISAs were 20–250 pM. The intra-assay variations were 2% and 3%, and the inter-assay variations were 2% and 10% for Aβ40 and Aβ42, respectively. The antibodies specifically detected the expected peptides with equal affinity for soluble and fibrillar forms of the peptide. The presence of CSF obstructed the recognition of synthetic peptides by the antibodies and the immunoreactivity of endogenous CSF Aβ decreased with increasing storage time and temperature. Conclusions: This study describes highly sensitive ELISAs with thoroughly characterized antibodies for quantification of Aβ40 and Aβ42, an important tool for the understanding of the pathogenesis of Alzheimer’s disease. Our results pinpoint some of the difficulties associated with Aβ quantification and emphasize the importance of using a well-documented assay.