Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-β3-42 (pE3-Aβ).

N-terminally truncated pyroglutamate amyloid-beta (A beta) peptide starting at position 3 represents a significant fraction of A beta peptides (pE(3)-A beta) in amyloid plaques of postmortem brains from patients with Alzheimer's disease (AD) and older persons with Down syndrome (DS). Studies in transgenic mouse models ofADalso showed that pE(3)-A beta is a major component of plaques, and mouse monoclonal antibody to pE(3)-A beta appears to be a desirable therapeutic agent for AD. Since small peptides do not typically elicit a good immune response in mice, but do so favorably in rabbits, our aims were to generate and partially characterize a rabbit monoclonal antibody (RabmAb) to pE(3)-A beta. The generated RabmAbwas found to be specific for pE(3)-A beta, since it showed no reactivity with A beta(16), A beta(40), A beta(42), A beta(3-11), and pE(11-17) A beta peptides in an enzyme linked immunosorbent assay (ELISA). The isotype of the antibody was found to be IgG class. The antibody possesses high affinity to pE(3)-A beta with dissociation constant (K-D) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE(3)-FRHD. In dot blotting, the optimal detection of pE(3)-A beta was at an antibody concentration of 0.5 mu g/ml. The threshold of pE(3)-A beta detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE(3)-A beta in sandwich ELISA. pE(3)-A beta was detected in AD and DS brain extracts in ELISA and immunoblotting. Immunohistological studies showed immunolabeling of plaques and blood vessels in brains from patients with AD, and DS showing AD pathology. Thus, the antibody can be widely applied in AD and DS research, and therapeutic applications.