Ultrasensitive thyrotropin immunoassay based on enzymatically amplified time-resolved fluorescence with a terbium chelate.
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.
Papanastasiou-Diamandi
A
A
CyberFluor Inc., Toronto, Ontario, Canada.
Christopoulos
T K
TK
Diamandis
E P
EP
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D013050:Spectrometry, Fluorescence; D019809:Streptavidin; D013725:Terbium; D013972:Thyrotropin / Q000097:blood*
D001426:Bacterial Proteins; D013725:Terbium; D013972:Thyrotropin; D019809:Streptavidin; D000469:Alkaline Phosphatase