25] Radioimmunoassay and immunotitration of human serum dopamine β-hydroxylase

Abstract

Publisher Summary This chapter describes the radioimmunoassay and immunotitration of human serum dopamine β-hydroxylase. Dopamine β-hydroxylase (DBH) is the enzyme that catalyzes the conversion of 3,4-dihydroxy-phenylethylamine (dopamine) to the neurotransmitter norepinephrine is released with catecholamines from vesicles in the adrenal medulla and sympathetic nerves, and is found circulating in blood. The immunotitration of DBH, like other immunotitration procedures, is based on the assumption that increasing quantities of antibody will be required to bind a given fraction of antigen as the quantity of antigen is increased. In practice, several different quantities of anti-DBH antibody are incubated with aliquots of the sample being analyzed. Antibody-bound and free DBH may be separated by precipitation of the anti-DBH antibody with a second antibody. Immunotitration of human serum DBH was performed with a two-stage procedure. Incubation tubes for the first stage contained 50 μl of human serum diluted 1:6 and 50 μl of rabbit serum diluted 1:250. The observation suggests the existence of a rare variant form of DBH with decreased enzymatic activity per molecule of enzyme protein. This observation was facilitated by the ability to assay many samples simultaneously and could not have been made without the ability to measure DBH protein.