Mono clonal antibodies that inhibit the rat liver receptor for asialo glyco proteins (original) (raw)

1983

Abstract

Publisher Summary A specific receptor exists in the plasma membrane of mammalian hepatocytes that mediates endocytosis of desialylated glycoproteins. The receptor has been isolated from Triton X-100 extracts of rat liver by affinity chromatography on ligand-Sepharose. The binding activity appears as a high MW entity by gel filtration, but sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) reveals a major band corresponding to 42,000 daltons and two less-prominent bands of slightly higher MW. This chapter describes an experiment where spleen cells from BALB/C mice immunized with a soluble receptor preparation were fused with P3X63–Ag8653 myeloma cells. Media from the resultant hybridomas were screened for their ability to bind a radioiodinated receptor. After subcloning, positive ascites was produced in BALB/C mice by i.p. injection of 106 cells. A clone was identified that produces antibody directed toward a determinant that is related to receptor function; it blocked the binding of ligand by plasma membranes. The SDS-PAGE pattern of the radiolabeled receptor preparation bound by this antibody was indistinguishable from the Coomassie-blue-staining pattern of the preparation used as antigen. However, because of the high apparent MW of the Triton X-100 extract, this result was viewed inconclusive. No reaction could be detected with the individual polypeptides recovered by a preparative SDS-PAGE using the gel system of Laemmli. Accordingly, a modified preparative SDS-PAGE system was devised that proved to be less destructive of antigenic reactivity. These results indicated that the recognized determinant is shared, at least in part, by the three polypeptides.

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