Vasilevko V, Xu F, Previti ML, Van Nostrand WE, Cribbs DH. Experimental investigation of antibody-mediated clearance mechanisms of amyloid-beta in CNS of Tg-SwDI transgenic mice. J Neurosci. 2007 Dec 5;27(49):13376-83. PubMed Abstract

REAGENTS/MATERIAL:
Antibodies:
Fibrillar forms ofAβ42 (fAβ42) andAβ40-DI (fAβ40-DI) were prepared as described previously (Cribbs et al., 2003). The epitope vaccine containing two copies of Aβ1–11 in tandem with the PADRE T-cell epitope (Epi-Aβ) was synthesized as a multiple antigenic peptide. For active immunization, 100μg of fAβ40-DI, fAβ42 peptides, or 50μg of Epi-Aβ vaccine were formulated with 50μg (initial injection) or 20μg (subsequent injections) of Quil-A adjuvant (Brenntag Biosector, Frederikssund, Denmark) in a total volume of 100 μl in PBS per injection. Purified mouse polyclonal anti-Aβ1–11 or anti-Aβ1–42 antibodies (from epitope vaccine or fAβ42 immunized animals, respectively) were transferred to PBS buffer,pH7.2, and concentration was adjusted to 1 mg/ml.
In the sandwich ELISAs, Aβ40 and Aβ42 were captured using their respective C-terminal-specific antibodies m2G3 and m21F12, and biotinylated m3D6, specific for human Aβ, was used for detection (De-Mattos et al., 2002b). Various forms of Aβ40-DI peptide were detected with mouse monoclonal anti-Aβ1-17 (6E10) (Sigma Aldrich) or purified anti-Aβ1–11 antibodies isolated from immunized Tg-SwDI mice
Immunohistochemistry: Primary antibodies included a horseradish peroxidase-conjugated MAb66.1 recognizing amino acid residues 1–5 of human Aβ, monoclonal anti-Aβ MAb20.1 recognizing amino acid residues 1–8 of Aβ, monoclonal anti-Aβ 6E10, and rabbit anti-collagen type IV (RDI Fitzgerald).
Antibody response: To circumvent the problem of inadequate peripheral levels of anti-Aβ antibodies we decided to use an Epi-Aβ vaccine, which we have shown previously induces very high anti-Aβ antibody titers in mice (Agadjanyan et al., 2005).