Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label.
Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.
T L
TL
Kjellström
L G
LG
Bachas
D000243:Adenosine Deaminase / Q000378:metabolism*; D001360:Avidin; D001667:Binding, Competitive; D001710:Biotin; D004563:Electrochemistry; D004798:Enzymes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D009700:Nucleoside Deaminases / Q000378:metabolism*
D004798:Enzymes; D007202:Indicators and Reagents; D001360:Avidin; D001710:Biotin; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase