Development of a sensitive ELISA for quantification of three- and four-repeat tau isoforms in tauopathies.
Tau protein plays an important role in stabilising and assembling neuronal microtubules. Pathological changes in expression and aggregation of tau isoforms containing three (3R-tau) and four (4R-tau) microtubule-binding repeat domains are associated with several tauopathies. This paper describes novel sandwich ELISAs for quantification of 3R- and 4R-tau in brain. The assays are constructed using well-characterised isoform-specific antibodies (RD3 and RD4) as capture antibodies and an affinity-purified HRP-anti-tau peptide antibody and biotin-tyramide amplification for detection. For 3R-tau, we achieved a minimal detection limit in buffer of 460 pg mL(-1) and a recovery of 81.0% using 500 pg mL(-1) recombinant 3R-tau spiked in diluted brain homogenate. Mean intra- and inter-assay variation of the 3R-tau ELISA was 8.8 and 10.5%, respectively. For 4R-tau, the detection limit was 780 pg mL(-1) and the recovery of 5 ng mL(-1) spiked recombinant 4R-tau was 86.0% and the mean intra- and inter-assay variation was 10.4 and 15.6%, respectively. With these assays, we showed that in progressive supranuclear palsy (PSP) brains, 4R-tau is significantly increased in frontal cortex and caudate, the two regions that are usually associated with 4R-tau-dominant pathology. This increase was not observed in occipital lobe, a region that is spared of tau inclusions. No differences in 3R-tau levels were found between PSP and control brains in all regions tested. With this, we have for the first time developed ELISAs for quantification of 3R- and 4R-tau isoforms in pathological samples. These could prove useful in the pathological investigation and differential diagnosis of tauopathies.
Reta Lila Weston Institute of Neurological Studies and Department of Molecular Neuroscience, UCL Institute of Neurology, 1 Wakefield Street, London WC1N1PJ, UK. c.luk@ion.ucl.ac.uk
Connie
C
Luk
Gavin
G
Giovannoni
David R
DR
Williams
Andrew J
AJ
Lees
Rohan
R
de Silva
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity / Q000276:immunology; D001921:Brain / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D001923:Brain Chemistry; D002421:Caudate Nucleus / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005625:Frontal Lobe / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism / Q000473:pathology; D011237:Predictive Value of Tests; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D034561:Sheep, Domestic; D013494:Supranuclear Palsy, Progressive / Q000175:diagnosis / Q000378:metabolism / Q000503:physiopathology; D024801:Tauopathies / Q000175:diagnosis* / Q000378:metabolism / Q000503:physiopathology; D016875:tau Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D020033:Protein Isoforms; D016875:tau Proteins