Enzyme immunoassay of thyroxin with a centrifugal analyzer

We have applied a homogeneous enzyme immunoassay for determination of thyroxin in serum to the "Cobas Bio" centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing "Lipex," an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37 degrees C. To 20 microL of sample mixture is added 125 microL of reagent (thyroxin antibodies and NAD+) and this mixture is incubated for 10 s. Then 25 microL of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 micrograms of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.