The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites.
We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 microliters) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.
Barnard
G
G
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Kohen
F
F
Mikola
H
H
Lovgren
T
T
D004970:Estrone / Q000031:analogs & derivatives* / Q000652:urine; D005260:Female; D005453:Fluorescence; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D012015:Reference Standards; D013050:Spectrometry, Fluorescence
C015856:estrone-3-glucuronide; D004970:Estrone