Analysis of Heterogeneous βA4 Peptides in Human Cerebrospinal Fluid and Blood by a Newly Developed Sensitive Western Blot Assay

Abstract

The betaA4 peptide, a major component of senile plaques in Alzheimer's disease (AD) brain, has been found in cerebrospinal fluid (CSF) and blood of both AD patients and normal subjects. Although betaA4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed betaA4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of betaA4 1-40 and 1-42, and used them for the specific Western blot detection. After optimizing the assay conditions, these antibodies detected low picogram amount of betaA4, and both betaA4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. Blood levels of betaA4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF betaA4 1-42 level is lower in AD patients compared with non-demented controls, although there was a significant overlap between the groups. The level of betaA4 1-40 in CSF, and of betaA4 1-40 as well as betaA4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length betaA4 band, we could also detect several N-terminal variants of betaA4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated betaA4 species migrating at the position of 3.3 and 3.7 kDa were found in CSF, while 3.7- and 5-kDa forms were found in plasma. The relative abundance of these various species were considerably different in the CSF and plasma, suggesting that the cellular source and/or clearance of betaA4 is different in these two compartments.

Rapid cellular uptake of Alzheimer amyloid βA4 peptide by cultured human neuroblastoma cells

“…Samples were transferred to fresh eppendorf tubes, incubated on ice for 15 min and centrifuged at 13000 rpm at 4°C for 10 min. 13A4 in the supernatant (cell lysate) was first immunoprecipitated with anti-13A4 monoclonal antibody (clone W0-2 [13]) and protein G-agarose (Boehringer Mannheim, Germany), and analyzed by Western blotting.…”

“…Western blot detection and quantitation of [3A4 was performed as described elsewhere [13]. Briefly, heat-denatured samples were separated by 10-20% or 16% Tris-Tricine SDS-PAGE.…”

“…13A4 concentrations were quantitated by densitometric analysis of the bands using MacBas program (Fuji, Japan). The following monoclonal antibodies were used for the detection [13]; clone W0-2 (anti-13A4 1-16) for the detection of 13A4 1-40, 1-42 and 1-28; clone G2-10 (anti-13A4 C-terminus 40) for the detection of 13A4 6-40 and 17-40 and clone G2-11 (anti-13A4 C-terminus 42) for the detection of 17-42 and internalized ~A4 1-42.…”

Cerebrospinal fluid biomarkers of Alzheimer’s disease

“…Many studies demonstrated that the levels of CSF Aβ42 in AD are decreased compared with controls [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] . CSF Aβ42 can differentiate AD from controls with 89% sensitivity and 90% specificity [51] .…”

Catalytic antibodies to amyloid β peptide in defense against Alzheimer disease

“…2 In peripheral blood, the major species is Aβ40. 4 Soluble Aβ oligomers impair neuronal function by altering the expression of memory-related receptors, 5 inducing aberrant neuronal responses to electrical stimulation 6 and inducing neural death. 3,7 Aβ has emerged as a promising target for immunotherapeutic intervention in AD.…”