Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates.
We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.
Christopoulos
T K
TK
Department of Clinical Biochemistry, Toronto Western Hospital, Ontario, Canada.
Diamandis
E P
EP
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D015200:Fluoroimmunoassay; D012459:Salicylates / Q000737:chemistry; D013725:Terbium / Q000737:chemistry*; D000509:alpha-Fetoproteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D012459:Salicylates; D000509:alpha-Fetoproteins; D013725:Terbium; C075234:5-fluorosalicylic acid; D004492:Edetic Acid; D000469:Alkaline Phosphatase