FORMATION OF IMMUNOGLOBULINS BY HUMAN TISSUES IN VITRO .I. METHODS AND THEIR SPECIFICITY

Abstract

The formation of immuno-globulins by human tissues was studied by 3 techniques: Autoradiography of the immunoelectrophoretic pattern of culture fluids of tissue fragments or cell suspensions in a medium with radioactive amino acids; immunofluorescent staining of the tissue sections or cell suspensions, before incubation, with antisera specific for one immunoglobulin; and histological and cytological study of the samples. The paper gives a detailed description of the techniques applied and evaluates the specificity of the immunological methods used. Various control experiments were performed: Incubation of dead tissues or normal serum in the radioactive culture medium excluded the adsorption of [Cl4]amino acids on to immunoglobulins. Cultures of tissue fragments in the presence of puromycin showed an inhibition of the synthesis of [Cl4] immunoglobulins. Autoradiography of a "fingerprint" of radioactive IgG demonstrated radioactive amino acids present in different peptides of the synthesized molecule. From these control experiments it can be concluded that the labelling of the immunoglobulins is based on the incorporation of [Cl4]amino acids during incubation in vitro. The specificity of the immunofluorescent staining was evaluated by immuno-diffusion of the antisera and conjugates, by blocking procedures and by specific staining of bone-marrow samples from patients with various immunoglobulin abnormalities.