T4 radioimmunoassay with ion-exchange resin separation

An explanation is provided for the reports of falsely low values of serum-thyroxine (T4) measured by radioimmunoassay (RIA), particularly in sera containing raised concentrations of thyroxine-binding globulin (TBG) (Burr, W.A., Evans, S.E., Hogan, T.C., 1977, Lancet, April 2, 757). A re-examination of the assay technique used in commercial RIA kits ('RIA-gnost T3' and 'RIA-gnost T4', Behring Diagnostics) showed that blocking of binding proteins by 8-anilino-1-naphthalene sulphonic acid (ANS) was incomplete in sera with raised TBG levels. Spectroscopic determination of the blocker concentration during the time the solution was in contact with the ion exchange resin showed that 98% of the ANS was removed from solution by 10 min contact with the resin, yet 60 min was required to absorb the free T4. There was therefore ample time for unblocked TBG to recombine with free T4 which was then misclassified as antibody-bound T4. The assay technique used in the kits was therefore modified. The less polar TBG blocking agent, ethylmercurithiosalicylate (merthiolate), replaced ANS and it was demonstrated that accurate T3 and T4 measurements in high TBG sera could be made by resin-separation RIA without resorting to prior denaturation or alcohol extraction. (U.K.)