Production of monoclonal antibodies to fibronectin, type IV collagen and other antigens of the human glomerulus.

A series of monoclonal antibodies to human glomerular antigens was prepared by immunisation of a mouse with isolated whole glomeruli, followed by boosting with particulate glomerular basement membrane and fusion of murine spleen cells with the NSI-myeloma line. Hybridoma supernatants were screened jointly by a radioimmunoassay involving binding to isolated glomeruli, and by a 4-layer immunoperoxidase technique applied to polyester wax-embedded sections. Seven monoclonal antibodies with different specificities (PHM7-PHM13) were established and repeatedly cloned. Each antibody displayed a distinctive distribution within the glomerulus, including different patterns of staining of mesangial cells, mesangial matrix and glomerular basement membrane, in addition to extra-glomerular basement membranes and extracellular matrix. All antibodies also stained cellular outgrowths of isolated glomeruli cultured in vitro, and showed additive binding to cultured cells by radioimmunoassay. Physical characterization using absorptions with purified substrates, plus specific chemical and enzymatic digestions, indicated that PHM12 is directed against type IV collagen. PHM13 is directed against fibronectin as shown by absorption with purified fibronectin and immunoprecipitation of a 220 000 MW glycoprotein. The remaining 5 monoclonal antibodies, which react with carbohydrate (PHM7) or protein (PHM8-PHM11) determinants, were shown to be nonreactive with type IV collagen, fibronectin or other known glomerular components including sialic acid, laminin, amyloid P-component or various glycosaminoglycans. These monoclonal antibodies therefore appear to define a new series of human glomerular antigens, or possibly closely related antigenic determinants, which are synthesized by glomerular cells and incorporated into the mesangium and glomerular basement membrane. These antibodies, by providing markers for at least 2 antigens known to be important in glomerular cell-matrix interactions, should prove useful in research into the mechanisms involved in renal pathology.