A multifunctional ELISA to measure oxidised proteins: oxPin1 in Alzheimer's brain as an example

BACKGROUND: Oxidative stress occurs in many neurodegenerative diseases including Alzheimer's disease (AD) and evidence suggests that specific proteins are oxidised in individual diseases. Thus measures of oxidised proteins such as in human biological samples could represent potential disease-specific biomarkers. Protein carbonylation is considered to be an important marker of oxidative stress. In AD in particular, the peptidyl prolyl isomerase, Pin1, has been shown to be sensitive to metal-catalysed oxidation with the addition of carbonyl side-chains. METHODS: Based on this protein modification we developed a novel, enzyme-linked sandwich immunoassay for the quantification of oxidised Pin1 (oxPin1) in human brain tissue samples. RESULTS: We successfully developed an ELISA for the measurement of oxidised Pin1 in biological samples and measured oxPin1 in hippocampal tissue extracts from controls and AD, which showed an increased ratio of oxPin1 to total Pin1 in patients with early AD pathology compared with controls. CONCLUSIONS: We show that oxidised proteins, in this case oxPin1, can be measured using the developed ELISA. In addition, our results support the presence of increased oxidative stress in the early stages of AD pathology and show that the oxPin1/Pin1 ratio could indicate early stage pathology. This warrants further investigation in other biological fluids. GENERAL SIGNIFICANCE: Importantly, further development and adaption of the assay design will enable multi-functional use for the quantification of oxidised proteins in tissues and biological fluids that may be used in investigating the role of oxidised proteins in a range of neurodegenerative diseases, particularly in which disease-specific protein oxidation has been implicated.