Microdialysis samples, CSF, and tissues were analyzed for Ab using a denaturing, sandwich ELISA specific for human Ab1-x,Ab1-40,orAb1-42. The Ab1-x ELISA measures Ab1-x by using a central-domain monoclonal antibody Amyloid-beta (m266) against amino acids 13-28 as a capture antibody. To capture the different species of full-length Ab, C-terminal domain monoclonal antibodies specific for either Ab40 Amyloid-beta 40 (m2G3) or Ab42 21F12 were used. All assays used a biotinylated N-terminal domain monoclonal antibody specific for an intact N terminus (amino acids 1-5) of human Ab 3D6 as a reporting antibody, followed by streptavidin-poly-horseradish peroxidase-20 (Research Diagnostics, Flanders, NJ).
In animal brain tissue sections Ab was identified using a rabbit polyclonal antibody against pan-Ab (BioSource International, Camarillo, CA). A microdialysis sample was run on Western blots and probed with antibody 3D6 to visualize the various Ab species present. For detection of APP C-terminal fragments (CTFs), the blot was probed with CT15 (gift of Dr. Ed Koo), a rabbit polyclonal antibody against the terminal 15 amino acids of APP. To detect full-length APP, 6E10 (Signet Labs) was used.