In the T3 CLIA, as explained by Rongen et al. (1994), a certain
amount of anti-T3 antibody was coated on micro titer
wells. A measured amount of chicken serum and a constant
amount of T3 conjugated with horseradish peroxidase were
added to the micro titer wells. During incubation, T3 in the
samples and conjugated T3 competed for the limited binding
sites on the anti-T3 antibody of the wells. After 60 min of incubation
at room temperature, the wells were washed 5
times by wash solution to remove unbound T3 conjugate. A
solution of chemiluminescent substrate was then added and
relative light units in a Luminometer were read. The intensity
of the emitting light was proportional to the amount of enzyme
present and was inversely related to the amount of
unlabelled T3 in the sample. By reference to a series of T3
standards assayed in the same way, the concentration of T3
in the unknown sample was quantified (Rongen et al.,
1994). The precise method for T3 was repeated for T4.