Quantification of sub-femtomole amounts of Alzheimer amyloid beta peptides

We evaluated methods for the quantitative Western blot analysis of A beta(1 -40) and A beta(1-42). Both chromogenic and chemi-luminescent detection met hods gave similar sensitivities (0.15 fmol of A beta(1-40) and 0.3 fmol of A beta(1-42)); however, the chromogenic method was move rapid simpler, less expensive and gave fewer background problems; consequently it yielded more reliable results. Adsorption to various types of laboratory plasticware ca n greatly interfere with the accurate measurement of A beta but this can be prevented by the addition of SDS or bovine serum albumin. Among several me thods for concentrating A beta from biological materials, immunoadorption t o Sepharose-bound antibodies was the most efficient. It yielded 50% recover y of 1 pM A beta(1-42) or A beta(1-40) and so was a suitable method to meas ure A beta levels in human plasma. Through combined immunoadsorption and We stern blotting we could determine the amounts of A beta isoforms secreted f rom 1 x 10(6) cells after a culture period as short as 1 h. This eliminates the need to use radiolabelling or over-expression to study A beta precurso r processing. Bovine serum contains subnanomolar A beta levels similar to t hose that reportedly stimulate cell proliferation. That cultured cells quic kly secrete these levels of A beta suggests that the peptide might exert an autocrine effect.