Demonstration of Antibodies to Thyroxine and Monoiodothyronine by Mixed Haemadsorption

Abstract

A TECHNICAL procedure for the application of the mixed haemadsorption technique¹ to soluble antigens has recently been described². This technique, which is essentially a mixed antiglobulin reaction, is being investigated because it is potentially a means of titrating antibodies to thyroglobulin in the sera of patients suffering from thyroiditis and other thyroid diseases. In the course of this work, it became of interest to find out whether the reaction with thyroglobulin may depend to some extent on the thyroxine determinants present on the thyroglobulin molecules. Pure thyroxine coated over a glass slide should have its antigenic determinants exposed much in the same way as when thyroglobulin is similarly exposed. Glass slides were therefore coated with a layer of thyroxine applied either as a suspension of microcrystals in phosphate buffered saline (PBS), pH 7.4, containing 1 per cent of normal rabbit serum (NRS) or as a solution in ethanol. Monoiodothyronine was applied as a solution in PBS, pH 7.4, containing 1 per cent of NRS. Human thyroglobulin was applied as a solution in PBS, pH 7.4. The various antigen films were dried at room temperature and all slides except those coated with thyroxine in ethanol were fixed for 5 min in acetone. The antigen films were covered with layers of agar and ‘Perspex’ disks provided with holes, which served as serum reservoirs. Two-fold serial dilutions of the sera containing antibody were added to the holes and the antibodies were allowed to diffuse through the agar layer. The antibody zones produced when the diffusing antibody attached itself to the antigen film on the slide were marked out by red cells provided with an exterior coating of antiglobulin so that they were able to adsorb specifically on to antibodies from the animal species concerned^2,3.