The assay is a direct competitive EIA for the quantitative measurement
of Total Triiodothyronine (Total T3) in human serum or plasma. A specific
agent displaces T3 from binding proteins, making it available for antibody
binding. T3 then equilibrates with fluorescein labelled T3 (T3 Derivative)
in binding to a limited amount of alkaline phosphatase labelled anti-T3
antibody. An anti-fluorescein antibody bound to magnetic particles then
separates the T3/fluorescein T3-antibody complex from unbound components.
A further incubation with substrate produces colour in inverse proportion
to the amount of T3 present. The assay has four main stages:
Displacing agent, alkaline phosphatase labelled anti-T3
antibody, and fluorescein-T3 derivative are incubated with sample
(100 µL) for 15 minutes at 37°C.
Anti-fluorescein antibody coupled to magnetic particles is
added and incubated for 10 minutes at 37°C. Anti-T3 antibody is
then isolated by means of a magnetic wash step.
A coloured enzyme substrate is incubated with the particles for
15 minutes at 37°C. The presence of alkaline phosphatase causes
a colour change from yellow to pink.
The reaction is terminated by addition of Stop Solution.
The tubes are then placed in the spectrophotometer or colorimeter.
The optical density (at 550nm or 492nm) of each tube can be measured
and the results calculated using a data processing program.