ABSTRACT
Iodination with lactoperoxidase - 125I- - H2O2 was used to label surface components of rat epidermal cells. Lysis of the cells in non-idet P40 resulted in the solubilization of tissue-specific antigens and of concanavalin A receptors. These specificities were demonstrated using a radio-immunoprecipitation method. The tissue-specific antigens were recognized using absorbed rabbit anti-rat epidermal cell sera (Lloyd & Darnule 1974); they reacted with two low molecular weight components in the lysate (9,000 and 12,000 daltons). Concanavalin A reacted with three major components. Two had high molecular weights (75,000 and 95,000 daltons). The possibility that one of these components was radioiodinated lactooperoxidase, which would have reacted with concanavalin A, was disproved. Another component (which occasionally appeared as two peaks) was similar in size to the species detected by the tissue-specific antisera. Their non-identity was, however, demonstrated by the finding that the two specificities could be precipitated independently of each other.
Subject(s)
Antigens , Concanavalin A , Epitopes , Receptors, Drug , Skin/immunology , Animals , Binding Sites, Antibody , Cell Membrane/immunology , Chemical Precipitation , Concanavalin A/immunology , Concanavalin A/metabolism , Iodine Radioisotopes , Methods , Rats , Skin/cytologyABSTRACT
A monolayer of a clone of endothelial cells derived from rat lung cells (RLE) was overlaid with 1 M urea to extract the surface proteins. Hydrolysis and SDS-gel electrophoresis of the urea extracted cell surface proteins (UCSP) yielded four peptides of 350,000, 84,000, 66,000 and 18,500 molecular weight. Of these only the 66,000 and 18,500 molecular weight peptides reacted with antibodies raised in rabbit against rat lung endothelial cells (RLE). The 18,500 mol. wt. antigenic peptide was a serum protein associated with the cell surface, whereas the 66,000 mol. wt. peptide was the surface antigen synthesized and released into the medium by the rat lung endothelial cells. On rocket immunoelectrophoresis, the 66,000 mol. wt. rat kidney fibroblast surface peptide produced only a single rocket whereas peptides of RLE produced two rockets, suggesting the presence of an additional antigenic peptide which could serve as a marker for endothelial cells.
Subject(s)
Antigens, Surface/isolation & purification , Endothelium/immunology , Animals , Clone Cells/immunology , Epithelium/immunology , Fibroblasts/immunology , Kidney/immunology , Lung/immunology , Membrane Proteins/immunology , Membrane Proteins/isolation & purification , Molecular Weight , RatsABSTRACT
When guinea pigs instead of rabbits were used as the host animals, 8--16 times higher antibody titers against human lung elastin peptides were produced with only 1/20 the amount of antigen per unit body weight. This corresponds to a 200-fold enhancement of the immune response.
Subject(s)
Antibody Formation/drug effects , Peptide Fragments/immunology , Animals , Elastin/immunology , Guinea Pigs , Humans , Mice , Rabbits , Rats , Species SpecificityABSTRACT
Antibodies were developed in rabbits against an established line of endothelial cells from normal adult rat lung. Pre- and postimmunization sera were tested for antibody activity by the indirect immunofluorescence technique. Preimmunization serum failed to react with the endothelial cells, whereas the antibody titer of postimmunization serum from two rabbits was 1:512. Organ specificity and species specificity were assessed by absorbing the serum with packed dissociated cells from different organs of the rat and lung cells of other species. Only cells obtained from rat lung absorbed the antibodies completely. The antiserum showed some crossreactivity with the other cultured cells but the pattern of fluorescence was different. In the presence of complement the antiserum was found to be cytotoxic to cultured rat lung endothelial cells but not to the other crossreacting cells.