Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis.
A protein with properties of laminin has been isolated from human placental extracts by using monoclonal antibodies. Placental tissue was extracted with 0.5 M NaCl and high molecular weight proteins were isolated from the extract by salt precipitation and gel filtration on Sepharose 6B. The resulting protein fraction which contained material cross-reactive with anti-sera to rat laminin was used as immunogen to prepare hybridomas. Thirteen hybrids produced antibodies which reacted with basement membrane-associated antigens in indirect immunofluorescence of tissues. One of these, 4E10, was characterized in detail. This monoclonal antibody reacted with human laminin as shown by several lines of evidence. Immunoprecipitation from metabolically labeled culture media of a human amniotic epithelial cell line with the 4E10 antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed polypeptides with Mr similar to those of rat laminin. Immunochromatography of placental extracts obtained by limited pepsin digestion yielded material with main polypeptides at 160 and 130 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. These peptic fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated with the fragments.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001681:Biological Assay; D005260:Female; D006801:Humans; D007106:Immune Sera; D005779:Immunodiffusion; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000032:analysis; D007797:Laminin / Q000276:immunology / Q000302:isolation & purification*; D008854:Microscopy, Electron; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007797:Laminin