Measurement of Tyrosine Hydroxylase Apoenzyme Protein by Enzyme-Linked Immunosorbent Assay (ELISA): Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) on Striatal Tyrosine Hydroxylase Activity and Content.

摘要

An enzyme-linked immunosorbent assay (ELISA) has been developed for tyrosine hydroxylase (TH). The method uses a polyclonal antibody to trap TH, a monoclonal antibody to bind the immobilized TH, a biotinylated, anti-mouse immunoglobulin to bind the monoclonal antibody, and streptavidin covalently coupled to horseradish peroxidase (SA-HRP). The incubations are performed at 37 C and the antigen-antibody complex detected colorometrically following incubation with an HRP substrate. The method detects less than 1 ng (16 fmol) of TH and can be performed in 3 hours or less on multiples of 96. The high specificity of the assay is attributed to the use of both polyclonal and monoclonal antibodies, each of which are specific for TH. Data acquisition and reduction is rapid (10 seconds/plate) and linked directly to a common desktop computer. Levels of TH protein average 1 ng/micrograms protein in striatum and, following treatment with the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), are decreased to a similar extent as is catalytic activity. In contrast, MPTP did not alter TH homospecific activity. The monoamine oxidase B inhibitor deprenyl blocked both the decrease in activity and the decrease in immunoreactive protein caused by MPTP.