MONOCLONAL-ANTIBODY OF A 43000 MR SURFACE PROTEIN OF A HUMAN-LEUKEMIA CELL-LINE (THP-1) CROSSREACTS WITH THE FIBROBLAST INTERMEDIATE FILAMENT PROTEIN VIMENTIN

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 73  (FEB) , 87-103
Abstract
Monoclonal antibodies were produced against surface antigens of live cells from a human acute monocytic leukemia cell line (THP-1). One clone, VIC-C2, when assayed by immunofluorescence microscopy, brightly stained the surface of THP-1 cells and the cytoplasm of Langerhans cells, fibroblasts and melanocytes in sections of human skin. The immunoreactive cytoplasmic structures were filamentous and resembled intermediate filaments. By double immunofluorescence microscopy using VIC-C2 and polyclonal antibodies to vimentin, the VIC-C2 antigen was shown to be located on intermediate filaments of cultured fibroblasts and to follow these filaments during various drug-induced rearrangements. As demonstrated by immunoprecipitation, antibody gel overlay and immunoblotting of 2-dimensional polyacrylamide gels, VIC-C2 recognized 2 different antigens in extracts of THP-1 cells: one of MW = 43,000 and pI [isoelectric point] = 7, the other MW = 57,000. In extracts from various cultured fibroblast cells only the 57,000 MW antigen was detected. This 57,000 MW protein was identified as vimentin by immunoblotting of rat glioma C6 cytoskeletons on 2-dimensional gels. When vimentin was digested with chymotrypsin, only fragments containing parts of both helical rod pieces and the connecting non-helical spacer-region were strongly antigenic, whereas the helical rods alone were only weakly cross-reactive. Immunoprecipitation revealed that VIC-C2 preferentially reacted with native compared to denaturated vimentin.

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