This study was undertaken to localize fibronectin and type IV collagen in the cultured retinal pigment epithelial cell by means of immunofluorescent staining and immunocytochemical methods. Immunofluorescent staining and immunocytochemical methods revealed fibronectin and type IV collagen localized on the extracellular membrane of the cultured retinal pigment epithelial cell. Ultrastructural immunocytochemical technique also revealed fibronectin associated with extracellular tissue. This study demonstrated that fibronectin and type IV collagen are an integral component of the extracellular matrix of the human retinal pigment epithelial cell in vitro.
Cells, Cultured ; Collagen/*analysis ; Extracellular Matrix/*analysis/immunology ; Fibronectins/*analysis ; Fluorescent Antibody Technique ; Humans ; Immunohistochemistry ; Pigment Epithelium of Eye/*analysis

OBJECTIVETo prepare the polyclonal antibody to amelogenin.

METHODSThe fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test.

RESULTSThe Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000.

CONCLUSIONThe antibody prepared in this study can be used for the detection of amelogenin.

Amelogenin ; Animals ; Animals, Newborn ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Dental Enamel ; chemistry ; Dental Enamel Proteins ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; immunology ; Immunoglobulin G ; analysis ; biosynthesis ; Rabbits ; Swine ; Tooth Germ ; chemistry