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

The βA4 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 βA4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed βA4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of βA4 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 βA4, and both βA4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. Blood levels of βA4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF βA4 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 βA4 1-40 in CSF, and of βA4 1-40 as well as βA4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length βA4 band, we could also detect several N-terminal variants of βA4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated βA4 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 βA4 is different in these two compartments. The βA4 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 βA4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed βA4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of βA4 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 βA4, and both βA4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. Blood levels of βA4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF βA4 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 βA4 1-40 in CSF, and of βA4 1-40 as well as βA4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length βA4 band, we could also detect several N-terminal variants of βA4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated βA4 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 βA4 is different in these two compartments.