An enzymeimmunoassay for total thyroxine using avidin-biotin separation system and thyroxine-peroxidase conjugate

An avidin-biotin enzymeimmunoassay for total thyroxine in serum is described. Avidin was adsorbed to biotinylated bovine serum albumin coated tubes prepared with glutaraldehyde as coupling agent. In the enzymeimmunoassay, affinity purified biotinylated anti-thyroxine IgG, sample or standards, and thyroxine-horseradish peroxidase were simultaneously added to the avidin coated tubes. The bound enzymatic activity was then measured with o-phenylenediamine and H2O2. Results showed that the assay has good precision (within-assay CV% less than 10% and between assay 11.7% in hypo- and 6.9% in hyperthyroid range), good assay range (0-800 nmol/L), good sensitivity (4 nmol/L), and can be performed in 2.5 hours. The results obtained correlate well (r = 0.93) with those of an RIA.