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.
Luk
Connie
C
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
Giovannoni
Gavin
G
Williams
David R
DR
Lees
Andrew J
AJ
de Silva
Rohan
R
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