RESUMO
Tyrosine hydroxylase (TH) catalyses the rate-limiting step in the biosynthesis of catecholamines. TH expression is regulated in a tissue-specific manner during neuronal development and differentiation. Because of its key regulatory role in central and peripheral catecholamine synthesis, TH is associated with the pathogenesis of several neurological and psychiatric diseases, including Parkinson's disease, dystonia, schizophrenia, affective disorders, and cardiovascular diseases. Therefore, developing a quantitative method to monitor the changes in TH expression in disease models could facilitate the identification and characterisation of neuromodulatory and neuroprotective therapeutic agents. The present report describes the generation and characterisation of a new set of monoclonal TH antibodies and the development of a novel sandwich ELISA for the quantitative detection of the TH protein in rodent brain tissue. This ELISA exhibits excellent reproducibility and good linearity in the analysis of complex brain tissue lysates. The cross-validation of the TH ELISA using semi-quantitative TH Western blot methods and HPLC measurement of dopamine levels suggests that the new TH ELISA is sufficiently sensitive to detect small-to-moderate region-specific differences, developmental changes, and Parkinson's disease-related changes in TH expression in rodent brains. This new TH ELISA also offers greater flexibility than conventional HPLC-based dopamine assays because the optimal tissue lysis buffer used for the detection of TH in brain tissue is also compatible with the analysis of other proteins associated with Parkinson's disease, such as α-synuclein, suggesting that this TH ELISA could be used in a multiplexed format.
