ABSTRACT
Radioiodine-labelled synthetic adrenocorticotrophic hormone (ACTH), human growth hormone (HGH), human chorionic somato-mammotrophin (HCS), and human (HTSH) and bovine (BTSH) thyroid stimulating hormones were studied by disc-electrophoresis and sucrose gradient centrifugation before and after incubation with corresponding antisera. All antisera contained 7 S antibodies. After incubation, soluble antigenantibody complexes besides a small amount of precipitate were observed in the incubation mixture, characteristic of each hormone. The complexes migrated like gamma globulins or more slowly on disc-electrophoresis. and on sucrose gradient centrifugation showed patterns dependent on the time of incubation. Light 7 or 9 S, or < 12 S complexes occurred mostly after incubation for several minutes (up to 30 min) before analysis. When incubation was prolonged to 24 h and more, these relatively light complexes disappeared or diminished in favour of heavier soluble or precipitating complexes. Reproducibly obtainable sedimentation patterns of the soluble complexes suggested some definite recombination of antigen molecules with 7 S antibodies. The complexes did not occur on incubation with other sera than an antiserum to a given hormone. They were not influenced by EDTA. Displacement of the radioactivity of the complexes into the free hormone peak was obtained by addition of a non-labelled hormone identical with the labelled one. Sucrose gradient centrifugation and disc-electrophoresis are recommended for the study of immunoreaction of diluted materials and for a separate analysis of different steps of the radioimmunoassay.
Radioimmunoassay was introduced for the measurement of protein hormones by Yalow & Berson (1960). The method, described originally for insulin, was later adapted to the detection of a number of protein and polypeptide hormones. On incubation of the hormone with its antiserum, a soluble antigenantibody complex is formed, which is separated from an excess of the free hormone by various methods, e. g. chromatoelectrophoresis, precipitation with a second antibody, adsorption on a solid phase etc. (Hunter 1967).
Sucrose gradient centrifugation and disc-electrophoresis were occasionally used to follow some isolated aspect of radioimmunoassay (Fitschen 1965; Monjardino et al. 1968).
We are demonstrating that these methods made it possible to analyze the radioimmunoassay step by step and thus may be useful for practical purposes as well as in a study of the immunoreaction of diluted materials.