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 chemiluminescent detection methods 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 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 A beta, but this can be prevented by the addition of SDS or bovine serum albumin. Among several methods for concentrating A beta from biological materials, immunoadsorption to Sepharose-bound antibodies was the most efficient. It yielded 50% recovery of 1 pM A beta 1-42 or A beta 1-40 and so was a suitable method to measure A beta levels in human plasma. Through combined immunoadsorption and Western blotting we could determine the amounts of A beta isoforms secreted from 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 precursor processing Bovine serum contains subnanomolar A beta levels, similar to those that reportedly stimulate cell proliferation. That cultured cells quickly secrete these levels of A beta suggests that the peptide might exert an autocrine effect.
Potempska
A
A
NYS Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
Mack
K
K
Mehta
P
P
Kim
K S
KS
Miller
D L
DL
D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods*; D019556:COS Cells; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011233:Precipitin Tests; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D016229:Amyloid beta-Peptides; D003470:Culture Media; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)