BIOTINIDASE RADIOASSAY USING AN I-125 BIOTIN DERIVATIVE, AVIDIN, AND POLYETHYLENE-GLYCOL REAGENTS

A radioassay for determining biotinidase activity in human serum was developed, using N-[β-(4-OH-3-125I-phenyl)ethyl]-biotinamide in combination with biocytin as the substrate, avidin as a binding protein, and polyethylene glycol as a separation reagent. The γ-emitting 125I-biotinamide (=tracer) was synthesized by coupling (pH 8.5, 20-22°C, 90 min) N-hydroxysuccinimidobiotin to 125I-tyramine. Using polyethylene glycol as a separation reagent, it was possible to eliminate several problems that were encountered when other separation reagents were used. Biotinidase activity was evaluated following the cleavage of the 125I-biotinamide and expressed in fmol of tracer cleaved · min-1 · ml-1 in the presence of 9 nmol of biocytin. Under the conditions used, the time response of the assay was linear up to 3 h. The method is simple to perform, more sensitive than the previously described methods, and reproducible (intra- and interassay CVs of 4.9 and 10.2%, respectively) and allows the simultaneous handling of more than 100 samples in <3 h. © 1991.