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P3‐089: Measurement of ß‐amyloid isoforms (1‐38, 1‐40, 1‐42) in plasma by new sensitive and accurate elisas
Author(s) -
Stoops Erik,
Mauroo Kimberley,
Herbst Victor,
Demeyer Leentje,
Vanderstichele Hugo
Publication year - 2015
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2015.06.956
Subject(s) - analyte , biotinylation , immunoassay , chemistry , chromatography , multiplex , antibody , bioinformatics , biochemistry , immunology , medicine , biology
Background:The search for blood-based biomarkers in Alzheimer’s disease (AD) increased substantially due to the success rates of proteomic approaches. The quantification of proteins in blood with classical immunoassay technologies (ELISA) is difficult due to the low concentrations of the relevant analytes (1⁄4 the need for sensitivity) and the presence of interfering substances (e.g. heterophilic antibodies). New generation platforms that can measure the analytes in the femtogram range were able to identify several of these potentially interesting proteins and provided proof-ofconcept for their clinical utility. However, the complexity of the technologies limited the translation into the clinical workup. The present study describes the development of conventional colorimetric ELISAs for quantification of Amyloid b (Ab) 1-38, Ab1-40, and Ab1-42 in plasma. Pre-analytical aspects, including plasma type selection and stability, as well as assay-robustness, will be presented.Methods:The analytical sensitivity of the existing CSF ELISA test kits for quantification of Ab isoforms was significantly improved to allow detection of the analytes in EDTAplasma. Much attention was given to a reduction of the matrix interference and to harmonize the test procedures to allow a full automation of the test protocols. The performance characteristics of the assays were challenged for their aid as diagnostic tool for AD by testing samples from healthy controls and AD subjects. Results: The optimization of assay development parameters (e.g., degree of antibody biotinylation, reagent formulation, quality and concentration of critical raw materials, sample incubation procedures) allowed us to measure 10-fold lower concentrations of analytes in plasma as compared to the CSF protocol (lower pg range). The analytical accuracy of each assay was optimized by comparison of possibilities and final inclusion of reagents to block heterophylic antibody interference. Other Amyloid b isoforms could be detected using N-terminus specific detector antibodies (amyloid isoform profiling). Conclusions:The ELISAs were qualified to measure Ab1-38, Ab1-40, and Ab1-42 in EDTA-plasma samples. The test procedures were harmonized and automated. These assays are new tools for the quantitative determination of amyloid isoforms in blood samples.