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Clinical Chemistry 38: 545-548, 1992;
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Clinical Chemistry, Vol 38, 545-548, Copyright © 1992 by American Association for Clinical Chemistry

Ultrasensitive thyrotropin immunoassay based on enzymatically amplified time-resolved fluorescence with a terbium chelate

A Papanastasiou-Diamandi, TK Christopoulos and EP Diamandis
CyberFluor Inc., Toronto, Ontario, Canada.

We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid- phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high- throughput automated analyzers.




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Copyright © 1992 by the American Association for Clinical Chemistry.