The research described in this thesis has centered on the development of a nonradioactive iodine label for use in immunoassays. A protein can be iodinated with nonradioactive iodine, used in an immunoassay, and the iodine label detected by a chemical method.
A microassay was developed to measure iodine by means of its catalytic effect on the
oxidation of antimony(III) by cerium(IV). The reaction was monitored both spectrophotometrically, by measuring the absorbance of cerium(IV), and fluorimetrically, by measuring the fluorescence of cerium(III) or by measuring the fluorescence produced by the oxidation of 8-hydroxyquinoline-5-sulphonic acid with cerium(IV). The detection of potassium iodide, iodine-containing organic compounds and iodinated proteins was possible using the microassay. Iodinated Bolton-Hunter reagent, an iodine-containing hapten which is used to iodinate proteins, could also be detected using the microassay. The catalytic activity of iodide is greater than that of iodine in iodocompounds, therefore, it was not possible to achieve the same sensitivity using IBHR as with potassium iodide.
A two-site immunoassay for the measurement of human IgG was set up and the effectiveness of non-radioactive iodine as a label in the immunoassay was illustrated using several assay formats. Initially, IBHR-labelled antibodies were used and directly compared to enzyme-labelled antibodies. The cerium (IV) - antimony(III) reaction was successfully used to detect the iodine label. The results obtained were comparable to those obtained using an enzyme label, with respect to accuracy and precision of the standards and with respect to the results obtained for serum samples. However, the use of enzyme-labelled antibodies enabled measurement of lower concentrations of human IgG.
In order to improve the detection limit when using the iodine label, the use of the avidin-biotin system and of bispecific F(ab’)2 antibodies was investigated. IBHRlabelled avidin and IBHR-labelled biotin were prepared, and used in a labelled avidinbiotin (LAB) immunoassay and bridged avidin-biotin (BRAB) immunoassay, respectively. Bispecific F(ab’)2 antibodies were prepared by a chemical method, and were used in an immunoassay as bridging agents between human IgG and iodinated BSA. An improvement in the detection limit was achieved using IBHR-labelled avidin in the LAB immunoassay.
An immunoassay for the measurement of thyroxine, which eliminates the necessity to prepare labelled derivatives of antigen or antibody, was developed. In this assay format, anti-thyroxine antibodies were used to capture thyroxine. Thyroxine contains four iodine atoms which served as the label, and these were monitored using the cerium (IV) - antimony (HI) reaction.