Wick Chromatography for Rapid and Reliable Immunoassay of Insulin, Glucagon and Growth Hormone

Abstract

THE essential principle of competition for a given amount of anti-hormone between standard or unknown quantities of hormone and a fixed amount of labelled hormone in Yalow and Berson's¹ radio-immunoassay has been maintained in all modifications. The modifications have simplified the apparatus and have achieved a more rapid and complete separation of free and antibody-bound radioactive hormone after the immunological reaction has taken place. This has been achieved best by double-antibody techniques^2,3. The introduction of a second antibody has made these immunoassays sensitive to factors in serum and plasma^4,5 which may give rise to spuriously high, or spuriously low, or even “negative” plasma values⁶. Another drawback is the difficulty in evaluating and correcting for the increased denaturation in serum or plasma of labelled hormone. This is inherent in modifications where free radioactive hormone cannot easily be isolated. Yalow and Berson¹ called this phenomenon “incubation damage” and estimated the percentage breakdown in individual sera by incubation without anti-hormone. Their chromato-electrophoresis, as does all paper chromatography, allows for a correction, because free polypeptide hormone is isolated at the site of application. The evaluation of breakdown is important in assay of peptide hormones, particularly glucagon, because pancreatic glucagon is very rapidly destroyed in serum or plasma unless special precautions are taken.