NONCOMPETITIVE IMMUNOASSAY OF THYROXINE USING A LIQUID-PHASE BINDING ASSAY

A thyroxine immunoassay was developed based on the principle of a liqu id-phase binding assay. Thyroxine and peroxidase-(POD-) labeled anti-t hyroxine monoclonal antibody (Fab'-POD) solutions ape mixed, incubated , and analyzed directly by high-performance liquid chromatography usin g a cation-exchange column and a postcolumn enzyme reaction apparatus. This system Separates antigen-antibody immune complex from free label ed antibody. The amount of labeled antibody is determined fluorophotom etrically by assay of POD enzymatic activity. The amount of thyroxine- was determined from the chromatographic peak height of the immune comp lex. All thyroxine molecules formed immune complexes upon addition of excess Fab'-POD. A linear thyroxine dose-response curve was obtained b ecause thyroxine molecules bound peroxidase-antibody conjugates in a 1 :1 ratio. This simple, rapid, and convenient immunoassay method does n ot require an analyte derivative and can be used to measure other low molecular weight antigens.