Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-ß3-42 (pE3-Aß).

N-terminally truncated pyroglutamate amyloid-ß (Aß) peptide starting at position 3 represents a significant fraction of Aß peptides (pE3-Aß) 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 of AD also showed that pE3-Aß is a major component of plaques, and mouse monoclonal antibody to pE3-Aß 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 pE3-Aß. The generated RabmAb was found to be specific for pE3-Aß, since it showed no reactivity with Aß16, Aß40, Aß42, Aß3-11, and pE11-17 Aß 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 pE3-Aß with dissociation constant (KD) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE3-FRHD. In dot blotting, the optimal detection of pE3-Aß was at an antibody concentration of 0.5 µg/ml. The threshold of pE3-Aß detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE3-Aß in sandwich ELISA. pE3-Aß 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.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-ß Peptide 1-37 (Aß37).

Secreted soluble amyloid-ß 1-37 (Aß37) peptide is one of the prominent Aß forms next to Aß40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aß37 levels in combination with Aß38, Aß40, and Aß42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aß37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aß37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aß37, and 2) to determine whether the antibody detects changes in Aß37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aß37 was found to be specific to Aß37, since it did not react with Aß36, Aß38, Aß39, Aß40, and Aß42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aß37. The antibody was sensitive enough to measure CSF and plasma Aß37 levels in ELISA. Immunohistological studies showed the presence of Aß37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aß37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

Increased serum neopterin levels in adults with Down syndrome.

We quantitated serum neopterin levels in Down syndrome (DS), normal controls, Alzheimer's disease, multiple sclerosis and other neurological diseases. We then analyzed the relationships with age, sex, apolipoprotein E (Apo E) phenotype, and amyloid beta protein 1-40 (Abeta40) and 1-42 (Abeta42) levels. Neopterin levels were higher in DS than all other groups. Levels in young DS (< 40 years of age) and old DS (> 41 years) were similar. There was no significant correlation between neopterin levels and age, sex, Apo E phenotype, and Abeta40 or Abeta42 levels in DS. This lack of correlation between neopterin and Abeta levels suggests that the higher neopterin concentrations in DS group reflect inflammatory cell activation rather than AD neuropathology.

Generation of Rabbit Monoclonal Antibody to Amyloid-ß38 (Aß38): Increased Plasma Aß38 Levels in Down Syndrome.

Secreted soluble amyloid-ß (Aß)38 is the second most prominent Aß form next to Aß40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aß38 levels in combination with those of Aß40 and Aß42 to support the diagnosis of Alzheimer's disease (AD), and other neurodegenerative diseases, and to facilitate drug discovery studies. However, the availability of reliable and specific Aß38 monoclonal antibody is limited. Our first aim was to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aß38. The antibody was specific to Aß38, since it did not react with Aß37, Aß39, Aß40, or Aß42 in ELISA or immunoblotting. The antibody was sensitive enough to measure Aß38 levels in plasma. Our second aim was to quantitate Aß38 levels in plasma from older Down syndrome (DS) persons and age-matched controls. Persons with DS (35 years and older) have neuropathological changes characteristic of AD. Studies have shown that plasma Aß40 and Aß42 levels are higher in older persons with DS than in controls. However, none examined Aß38 levels in DS. Our quantitation data showed that, like Aß40 and Aß42 plasma levels, Aß38 plasma levels were higher in DS than in controls. Longitudinal studies will determine whether plasma Aß38 levels in combination with levels of Aß40 and Aß42 are useful to predict early signs of AD in DS.

Plasma amyloid beta protein 1-42 levels are increased in old Down Syndrome but not in young Down Syndrome.

Plasma amyloid beta protein 1-40 (Abeta40) and Abeta42 levels were quantitated from 28 young Down syndrome (DS) (20-40 years old), 28 age-matched controls, 32 old DS (41-65 years old) and 32 age-matched controls in a sandwich enzyme-linked immunosorbent assay. Abeta40 levels were higher in young DS and old DS than controls. Abeta42 levels in young DS and controls were similar, however Abeta42 levels were higher in old DS than controls or young DS. The higher Abeta42 levels in old DS suggests that Abeta42 is selectively increased in plasma concurrently with the development of Alzheimer disease neuropathology.

Amyloid beta 38, 40, and 42 species in cerebrospinal fluid: more of the same?

Various C-terminally truncated amyloid beta peptides (Abeta) are linked to Alzheimer's disease (AD) pathogenesis. Cerebrospinal fluid (CSF) concentrations of Abeta38, Abeta40, and Abeta42 were measured by enzyme-linked immunosorbent assay in 30 patients with AD and 26 control subjects. CSF Abeta42 levels was decreased in patients with AD, whereas CSF Abeta38 and Abeta40 levels were similar in patients with AD and control subjects. All three Abeta peptides were interrelated, particularly CSF Abeta38 and Abeta40. Diagnostic accuracy of CSF Abeta42 concentrations was not improved by applying the ratios of CSF Abeta42 to Abeta38 or Abeta40.