Enzyme immunoassay of thyroxine in serum and dried blood samples on filter paper

A double antibody heterologous enzyme immunoassay was developed for measuring thyroxine (T4) in serum and in dried blood samples on filter paper for use in screening for neonatal hypothyroidism. In this method, ethanol extracts of 100 microliter of serum or dried blood spots of 9 mm in diameter were incubated with T4-alkaline phosphatase conjugate prepared with glutaraldehyde, and antiserum to T4-bovine serum albumin conjugate prepared with carbodiimide. The enzyme activity in the precipitates formed with second antibody was then determined photometrically at 500 nm. The measurable range of T4 was 1-32 micrograms/dl. A linear relation was obtained between the sample volume and the T4 value, and a high correlation was found between values for T4 determined by this method and those for T4 in serum determined by radioimmunoassay (coefficient of correlation: r=0.97 for serum and r=0.88 for dried blood samples).