The development and use of a non-radioactive iodine-labelling system for microassays

The research described in this thesis has centred on the development, detection and evaluation of a non-radioactive iodine-label for assay purposes. Traditionally, radioactively-labelled iodine has been used as a reporter group for the detection of analytes in test samples. The catalytic effect of iodide on the oxidation of arsenic III by cerium IV was used to measure iodine-containing compounds. The reaction can be followed spectrophotometrically by measuring the intensity of the cerium yellow colour after a defined time period. The assay procedure was optimised with, respect to temperature, incubation time, detection wavelength, concentration of reagents and acidity of reacting medium. The iodine labelling system was then successfully applied to the detection of immunoglobulin G levels in biological samples from human and mouse sources using a sandwich type immunoassay. The performance of the iodine label was then evaluated against an enzyme label Attempts were made to increase the sensitivity of the iodide immunoassay through the conjugation of poly-L-lysme to second antibody. This procedure should increase the number of iodide binding sites available. The successful application of the iodide assay to the detection of proteolytic activity is also described. The assay system described uses small quantities of reagents, is suitable for use with a photometric microplate reader, can test many samples simultaneously, eliminates problems associated with the use of radioisotopes and has good sensitivity and reproducibility