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    Anal Biochem. 1991 Aug 1;196(2):385-9.

    Biotinidase radioassay using an 125I-biotin derivative, avidin, and polyethylene glycol reagents.

    Evangelatos SA, Livaniou E, Kakabakos SE, Evangelatos GP, Ithakissios DS.

    Source

    National Centre for Scientific Research Demokritos, Athens, Greece.

    Abstract

    A radioassay for determining biotinidase activity in human serum was developed, using N-[beta-(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 gamma-emitting 125I-biotinamide (= tracer) was synthesized by coupling (pH 8.5, 20-22 degrees 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 less than 3 h.

    PMID:
    1776689
    [PubMed - indexed for MEDLINE]

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