Development of homogeneous 384-well high-throughput screening assays for Abeta1-40 and Abeta1-42 using AlphaScreen technology.
Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.
Szekeres
Philip G
PG
Eli Lilly and Co. Ltd, Lilly Research Centre, Windlesham, Surrey, UK. szekeresp@lilly.com
Leong
Kaitlin
K
Day
Theresa A
TA
Kingston
Ann E
AE
Karran
Eric H
EH
D000544:Alzheimer Disease / Q000175:diagnosis*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001923:Brain Chemistry; D002138:Calibration; D002478:Cells, Cultured; D003362:Cost-Benefit Analysis; D003470:Culture Media / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D008403:Mass Screening / Q000191:economics / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014020:Tissue Extracts / Q000032:analysis / Q000737:chemistry
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D003470:Culture Media; D010446:Peptide Fragments; D014020:Tissue Extracts; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)