Competitive ELISA for the measurement of tau protein in Alzheimer's disease.
Tau protein is a major component of paired helical filaments (PHFs) which constitute the characteristic neurofibrillary tangle lesions observed in Alzheimer's disease. Two tau mAbs have been produced which show distinct patterns of immunoreactivity with intact human tau and with tau incorporated in PHFs. The mAb 423 recognises PHFs but not human tau on immunoblots whereas mAb 7/51 reacts with human tau but its epitope is buried within the PHF and is only exposed after formic acid treatment. A competitive ELISA has been developed for both of these mAbs and these have been used to quantify the two distinct tau epitopes in PHFs. Samples containing antigen are incubated with horseradish peroxidase-conjugated mAb at 4 degrees C for 16 h and non-adsorbed antibody then measured by binding, at 37 degrees C for 1 h, to a fragment of tau coated on microtitre plates. Bound enzyme-labelled antibody is measured kinetically using a spectrophotometer capable of automatically mixing the samples throughout a 2-min incubation with substrate and chromogen. The interfacing of the plate reader with a computer permits competitive curves to be plotted automatically using Softmax. Curves are fitted using a 4-parameter logistic algorithm which allows one to determine the relative immunoreactivity for different samples. The application of these assays to monitoring biochemical fractions and quantifying distinct immunochemical presentations of tau protein with these two mAbs is described.
University of Cambridge, Department of Psychiatry, U.K.
C R
CR
Harrington
P C
PC
Edwards
C M
CM
Wischik
D000544:Alzheimer Disease / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001923:Brain Chemistry; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D006735:Horseradish Peroxidase; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D016875:tau Proteins
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D016875:tau Proteins; D006735:Horseradish Peroxidase