P2-155: Development of sensitive and specific ELISAs for three-repeat and four-repeat tau isoforms

Abstract

Tau has been investigated in CSF as a potential diagnostic biomarker in neurodegenerative disorders associated with tau inclusions. Commercially available ELISA kits for quantification of total and phosphorylated tau have shown increased CSF t–tau and p–tau levels in Alzheimer's disease. In other tauopathies such as FTDP–17, four–repeat (4R) isoforms are abnormally elevated in brain. In such cases, the quantity and ratio of three–repeat (3R) and four–repeat (4R) tau isoforms could be reflected in CSF and could potentially serve as an early diagnostic biomarker. Previously, we developed two monoclonal antibodies; RD3 and RD4, specific for 3R and 4R tau isoforms, and these were effectively validated in several neuropathological studies. To develop highly specific and sensitive ELISAs for three–repeat (3R) and four–repeat (4R) tau isoforms. The current study describes the development of both competitive and sandwich ELISAs for 3R and 4R tau isoforms using RD3 and RD4. Peptide–protein conjugates are used as plate coating antigens in competitive ELISAs. Recombinant 3R and 4R tau isoforms are used as standards to compete against the solid phase antigen for antibody binding. In the sandwich format, several polyclonal and monoclonal antibodies have been tested to capture the tau isoforms, while RD3 and RD4 are respectively used as detector antibodies. In both assay formats, HRP has been used as label for quantification of bound antibodies onto the solid phase. The titres of RD3 and RD4 are found to be 1 in 150,000 and 1 in 6,000 respectively using peptide–protein conjugates as plate coating antigens. Both 3R and 4R tau isoforms are shown to effectively inhibit the binding of RD3 and RD4 towards the appropriate plate coating antigen in competitive ELISAs. We have achieved minimum detection limits for the tau isoforms in competitive ELISAs in the pg/mL range. Results of this study suggest that the current 3R tau and 4R tau ELISAs are feasible for rapid and sensitive detection of tau isoforms in CSF and corresponding biological matrices from patients with suspected tauopathies. These tests can potentially aid the diagnosis of tauopathies associated with differential expression of 3R and 4R tau isoforms.