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. 1984 Sep;17(3):173–179. doi: 10.1007/BF00205482

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

II. Identification of the cellular target structures by immunoprecipitation and SDS-PAGE analysis

Staffan Paulie 1, Hannu Koho 1,✉, Hedi Ben-Aissa 1, Yngve Hansson 1, Marie-Louise Lundblad 1, Peter Perlmann 1
PMCID: PMC11039125  PMID: 6383601

Abstract

The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads. The precipitates were separated by sodium dodecyl sulfate- gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. The antibodies 4B5, 7E9, and 14B11 have previously been found to react in a similar way with TCC-targets and some non-TCC tumor cells, but not with normal urothelial cells or cells of hematopoietic origin. When tested with lysates of a TCC- cell line (TCCSuP) a strong 92K band and a weak 23K band were precipitated with any one of these antibodies. These polypeptides were expressed on the cell surface and were not linked by disulfide bonds. Depletion experiments confirmed that the three antibodies recognized the same antigens. Another antibody (4E8) probably directed to a differentiation antigen present on both urothelial and melanoma cells detected two high molecular polypeptides, 190K and 170K. Antibodies from the S2C6 hybridoma, which displayed a distinct dual specificity for TCC- targets and for malignant or transformed cells of B cell origin, precipitated a 50K component from extracts of either TCC- or B cell-derived cell lines. Antibodies produced by the S2A9 hybridoma were shown to bind to a framework epitope of the HLA-A, B, C heavy chain.

Keywords: Melanoma, Staphylococcus Aureus, Sodium Dodecyl Sulfate, Melanoma Cell, Transitional Cell

Footnotes

Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; PBS, phosphate-buffered saline, pH 7.2; SpA, protein A from Staphylococcus aureus (strain Cowan); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; K, kilodaltons; ELISA, enzyme-linked immunosorbent assay; ADCC, antibody-dependent cell-mediated cytotoxicity; TAA, tumor-associated antigen; ConA, concanavalin A

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