Quantification of sub-femtomole amounts of Alzheimer amyloid β peptides

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Abstract
We evaluated methods for the quantitative Western blot analysis ofAβ1-40 andAβ1-42. Both chromogenic andchemi-luminescent detection methods gave similar sensitivities (0.15 fmol ofAf31-40 and 0.3 fmol ofAβ1-42); however, the chromogenic method was more rapid, simpler, less expensive and gave fewer background problems; consequently, it yielded more reliable results. Adsorption to various types of laboratory plasticware can greatly interfere with the accurate measurement of Aβ, but this can be prevented by the addition of SDS or bovine serum albumin. Among several methods for concentrating Aβ from biological materials, immunoadsorption to Sepharose-bound antibodies was the most efficient. It yielded 50% recovery of I pM Aβ1-42 or Aβl-40andso was a suitable method to measure Aβ levels in human plasma. Through combined immunoadsorption and Western blotting we could determine the amounts of Aβ isoforms secreted from I x106 cells after a culture period as short as 1 h. This eliminates the need to use radiolabelling or over-expression to study Aβ precursor processing. Bovine serum contains subnanomolar A β levels, similar to those that reportedly stimulate cell proliferation. That cultured cells quickly secrete these levels of Aβ suggests that the peptide might exert an autocrine effect.