Receptor-mediated transport of IgG

The intestinal epithelium of the neonatal rat is a model system for the study of receptor-mediated endocytosis in which large amounts of IgG are transferred intact across polarized cells. This review summarizes the ultrastructural pathway followed by IgG during cellular transit and several important properties of the membrane receptor that recognizes the IgG

PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent Km of 0.2 M, an apparent vmax of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of ∼2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km of 0.4 M, an apparent vmax of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits

ULTRASTRUCTURAL LOCALIZATION OF CALCITONIN IN THE PARAFOLLICULAR CELLS OF PIG THYROID GLAND WITH CYTOCHROME c-LABELED ANTIBODY FRAGMENTS

Parafollicular cells in mammalian thyroid glands are thought to be responsible for the secretion of calcitonin. In this study, calcitonin was localized in pig thyroid gland by an indirect immunocytochemical technique using rabbit antiserum directed against synthetic porcine calcitonin for the first step, and sheep Fab fragments prepared against rabbit Fab and coupled to cytochrome c for the second step. The antigenic determinants of calcitonin were present only in the parafollicular cells, whose secretory granules were heavily labeled. Labeling of the cytoplasmic matrix is thought to indicate a possible leakage of the polypeptide from the granules. A striking observation was the complete absence of labeling in the cisternae of the rough-surfaced endoplasmic reticulum and of the Golgi apparatus. It is concluded that the secretory granules of parafollicular cells contain calcitonin; the mechanism of synthesis of this peptide is not clearly understood

Ultrastructural localization of intracellular antigen using enzyme-labeled antibody fragments

The efficiency of small enzyme-labeled tracers for the demonstration of intracellular antigen was investigated in tissues fixed with picric acid-formaldehyde. The influence of fixation on the immunological activity was tested in vitro by radial immunodiffusion. The experimental model consisted of newborn pig jejunum after absorption of ferritin from the intestinal lumen. Ferritin was located after 1 hr in vacuoles scattered in the cytoplasm of the absorptive cells and represented an easily recognizable intracellular antigen. After immunohistochemical treatments with antiferritin preparations, the distribution of labeling enzyme reaction product was examined by morphometry. The ratio of the labeled volume to the total volume of vacuoles containing ferritin indicated the degree of specific labeling of the antigen. In both direct and indirect methods, the degree of labeling was low when enzyme-labeled immunoglobulin G was the tracer. With antigen binding fragments (Fab), the labeling was significantly increased. In the indirect method, the degree of labeling was influenced by the first-step reagents. Onlywhen the serum titer was optimum was a high degree of labeling obtained. With antigen binding fragments or papain-digested serum the effect of the titer was negligible and maximum labeling was achieved. In both methods, with peroxidase as the labeling enzyme, a diffuse nonspecific deposition of reaction product was observed. This could be avoided by using cytochrome c instead