Intracellular localization of antigens recognized by anti-vimentin monoclonal antibodies (mAbs): cross-reactivities of anti-vimentin mAbs with other cellular components.

ABSTRACT

Monoclonal antibodies were raised against immunoaffinity-purified fusion regulatory protein (FRP)-1 complex from membrane fraction of HeLa cells. Immunoblotting and immunoprecipitation studies showed all ten antibodies reacted with a 55 kDa band of cell lysate and purified vimentin. Interestingly, one of the antibodies (mAb57) cross-reacted with purified tropomyosin and myosin. Further analyses using vimentin chemically cleaved by 2-nitro-5-thio-cyanobenzoic acid, and lambda gt 11 cDNA which encoded a partial sequence of vimentin indicated that six mAbs recognized epitopes between amino acids 1 and 313 and the other four mAbs recognized epitopes in the area between residues 314 and 326. Indirect immunofluorescence microscopy using 3% formalin-fixed, 0.1% Triton X-100 treated HeLa cells revealed that seven antibodies stained various intracellular components other than vimentin, while three antibodies stained vimentin filaments alone. Furthermore, flow cytometric analysis showed one of the antibodies (mAb25) clearly stained the surface of unfixed HeLa cells. All immunofluorescent findings were the same when HeLa, baby hamster kidney (BHK) and murine L229 cells were examined. These results indicate that we could obtain unique anti-vimentin mAbs which show cross-reactivities with previously undescribed cell surface and intracellular molecules including tropomyosin and myosin. Taken together, there are two possibilities that explain our findings: (1) The unknown molecules may have structural similarity to vimentin. (2) Our anti-vimentin mAbs can react specifically with structurally distinct epitopes present on both unknown molecules and vimentin. In either case, our cross-reactive mAbs, which recognized undescribed epitopes on vimentin, maybe provide useful tools for studying intermediate filaments and related cellular components.