Anal. Chem. 1994,66, 1837-1840
Bioluminescence Binding Assay for Biotin with Attomole Detection Based on Recombinant Aequorin Allan Wltkowskl, Srldhar Ramanathan, and Sylvla Daunert' Department of Chemistry, Universiw of Kentucky, Lexington, Kentucky 40506-0066
Biotinylated recombinant aequorinwas used in the development of a homogeneous bioluminescence assay for biotin. It was demonstrated that the Caz+-triggeredluminescent reaction of biotinylated aequorin can be inhibited by the presence of avidin, and this inhibition in bioluminescence intensity can be used to determine the biotin concentration in a sample. The optimized assay thus developed had a detection limit for biotin of 4 amol (equivalent to 1 X 10-14M in the assay system), which to our knowledge is the lowest detection limit reported to date for a homogeneous competitive binding assay. Fluorophore-linked binding assays have found limited use because they are often seriously impaired by interferences from biological samples (e.g., background fluorescence of serum). Some of the limitations of fluorescent labels may be circumvented by employing chemiluminescence or bioluminescence methods. In that respect, several heterogeneous chemiluminescence-linked binding assays have been developed which allow for the determination of biomolecules at low levels.'-3 Indeed, a comparative study of enzymatic assays reported much better detection limits when chemi- or bioluminescence methods of detection were used instead of fluorescence- or absorbance-based methods.2 One of the drawbacks of chemiluminescent labels is that they often require high pH for the optimum emission of the luminescence signal. In some cases, this can seriously hinder the interaction between the analyte (ligand) and its binder (antibody or binding protein). Although conditions can be found where the chemiluminescence can be triggered without affecting the association between the ligand and the binder, i.e., at pH
