Biotinidase radioassay using an 125I-biotin derivative, avidin, and polyethylene glycol reagents.
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.
Evangelatos
S A
SA
National Centre for Scientific Research Demokritos, Athens, Greece.
Livaniou
E
E
Kakabakos
S E
SE
Evangelatos
G P
GP
Ithakissios
D S
DS
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D001360:Avidin; D001710:Biotin / Q000031:analogs & derivatives*; D043523:Biotinidase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008239:Lysine / Q000031:analogs & derivatives; D011092:Polyethylene Glycols; D013388:Succinimides
D006902:Hydroxymercuribenzoates; D007457:Iodine Radioisotopes; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; C053607:N-(4-hydroxy-3-iodophenylethyl)biotin amide; D001360:Avidin; D011092:Polyethylene Glycols; D001710:Biotin; D000581:Amidohydrolases; D043523:Biotinidase; C013411:biocytin; D008239:Lysine