The fluorescent antibody technique was adapted to the localization of native protein antigens in cells and tissues. This method was applied specifically to the localization of adrenocorticotropic hormone in the pituitary gland.
An antiserum to hog ACTH was produced in an adrenalectomized rabbit. The γ2-globulin fraction of the serum was conjugated with fluorescein. After purification, the fluorescent antibody solution stained selectively the cytoplasm of basophil cells of the hog pituitary. No cells of sheep or beef pituitary or of hog kidney were stained. A fluorescent globulin solution prepared from normal rabbit serum gave no selective staining in any of these tissues.
Immunochemical tests showed that the fluorescent antibody gave a precipitin reaction with a highly active ACTH preparation of low molecular weight. The supernatant solution from this reaction showed a loss of hormone activity.