The Preparation and Characterization of Alkaline Phosphatase—Thyroxine Conjugates and Their Use in the Enzyme Immunoassay of Thyroxine

Abstract

Alkaline phosphatase from calf intestine was conjugated to thyroxine using cyanuric chloride, glutaraldehyde and 1-cyclohexyl-3(−2 morphoinoethyl)-carbodiimide as coupling reagents. The conjugates produced were studied for incorporation of thyroxine, retention of enzyme activity and immunoreactivity in a thyroxine radioimmunoassay system against a range of thyroxine antisera. The use of these conjugates in thyroxine enzyme immunoassay was studied using second-antibody separation. Comparison of the results with radioimmunoassay data was satisfactory and indicates the potential of these conjugates for thyroxine immunoassay. The use of cyanuric chloride as a coupling reagent was unsatisfactory, but good results were obtained with glutaraldehyde. Although satisfactory immunoreactivity of carbodiimide conjugates was observed in a radioimmunoassay system, these results were not reflected in the enzyme immunoassay systems studied.