Development of a sensitive ELISA for quantification of three- and four-repeat tau isoforms in tauopathies.

Abstract

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.

• Authors: Connie Luk, Gavin Giovannoni, David R Williams, Andrew J Lees, Rohan de Silva
• Journal: Journal of neuroscience methods (J Neurosci Methods) Vol. 180 Issue 1 Pg. 34-42 (May 30 2009) ISSN: 1872-678X [Electronic] Netherlands
• PMID: 19427527 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies
• Protein Isoforms
• tau Proteins

Topics

• Animals
• Antibodies
• Antibody Specificity (immunology)
• Brain (metabolism, pathology, physiopathology)
• Brain Chemistry
• Caudate Nucleus (metabolism, pathology, physiopathology)
• Enzyme-Linked Immunosorbent Assay (methods)
• Frontal Lobe (metabolism, pathology, physiopathology)
• Humans
• Inclusion Bodies (metabolism, pathology)
• Predictive Value of Tests
• Protein Isoforms (analysis, immunology, metabolism)
• Sensitivity and Specificity
• Sheep, Domestic
• Supranuclear Palsy, Progressive (diagnosis, metabolism, physiopathology)
• Tauopathies (diagnosis, metabolism, physiopathology)
• tau Proteins (analysis, chemistry, immunology)