Human melanoma associated antigens: A solid-phase assay for detection of specific antibody (original) (raw)
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International Journal of Cancer, 1976
Antibody-dependent cell-mediated cytotoxic assays have been used to examine antigens on human melanoma cells obtained either directly from patients or from long-term melanoma cell lines. A panel of melanoma antisera was selected from human subjects which could be shown not to have significant reactivity to histocompatibility antigens. With these antisera extensive cross-reactions between melanoma cells were found. However, the cross-reactivity was incomplete and the pattern of reactivity was dijerent for each antiserum tested. These results were not consistent with a common melanoma antigen on human melanoma cells but rather indicated heterogeneity of melanoma antigens and multiple antibody specificities in the sera tested. This appeared to be confirmed by extensive cross-absorption studies which indicated limited crossreactivity of antigens on melanoma cells from either long-term or short-term cultures. Several changes in the antigenic profile of melanoma cells in vitro from both long-term and short-term cultures were documented which resulted from contamination of the melanoma cell lines with non-melanoma cells and fibroblasts. Melanoma antisera tnay therefore be useful to monitor changes in long-term cultures which would otherwise give spurious results in in vitro tests. These results appear to have considerable significance for understanding tumourjhost relationships and for the establishment of rational imrnunotherapeutic procedures and diagnostic tests in melanoma.
Identification of Melanoma-associated Antigens Using Fixed Tissue Screening of Antibodies
Cancer Research, 1984
Early culture supernatants from hybridomas that were ob tained through fusions of mouse myeloma cells with lymphocytes of melanoma-immunized mice were screened for their reactivity with a paraffin-embedded cell block of a melanoma cell line, using a biotin:avidin immunoperoxidase procedure. Eleven monoclonal antibodies were derived that define several new melanomaassociated antigens. The antigens include a neutral glycolipid, gangliosides, membrane-associated proteins, cytosolic proteins, and strongly secreted proteins. These antibodies, which detect antigens that withstand tissue fixation and embedding proce dures, were tested for reactivity in fixed cell lines, as well as in melanoma biopsies. These antibodies may provide powerful tools in diagnostic studies of human malignant melanoma biopsy material.
Common human melanoma-associated antigen(s) detected by monoclonal antibodies
Cancer research, 1980
Hybridoma cells have been derived from a fusion between mouse myeloma cells (P3-NSI/1Ag4) and spleen cells from a mouse immunized with membrane-enriched fractions from the human melanoma cell line Me-43. Of the 26 hybrids obtained, seven secreted antibodies which reacted with the melanoma cell line used for immunoassay. The specificity of the antibodies produced by the seven positive hybrids was further investigated on 16 melanoma cell lines, 15 other tumors, and 14 lymphoblastoid cell lines. The antibodies from four positive hybrids showed a broad reactivity, whereas those from three hybrids reacted exclusively with melanoma cells. The antibodies from two of these three hybrids, alpha-Mel/5 and alpha-Mel/14, seem to be directed against common melanoma antigen(s) since they reacted with all (with one exception) of the 16 melanoma cell lines tested only with five of the 16 melanoma lines. Reciprocal binding inhibition tests using [3H]leeucine-labeled antibodies showed that alpha-Mel/...
Cell surface antigens of human melanoma identified by monoclonal antibody
Proceedings of the National Academy of Sciences, 1979
Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10
Cancer research, 1979
To characterize the antigen present on the surface of cultured human malignant melanoma, three monkey xeno geneic antisera were raised. After appropriate absorption with pooled human erythrocytes, peripheral blood leuko cytes, and liven homogenate, no blood group or HLA neac tivity was detectable. Analysis of melanoma antigenic spec ificity was performed by the mixed hemadsonption microas say on live monolayer cells in conjunction with quantitative microabsorption analysis. To eliminate reactivity against nonmelanoma lines, 2 of the 3 antisera required further absorption with cells of the KB oral carcinoma line. The absorbed antisera reacted with 9 of 10 long-term estab lished lines, 3 of 3 short-term cultures of human malignant melanoma, and i of 10 nonmelanoma epithelial and fibro blastic cell lines. Absorption experiments using a variety of cultured cells and fresh tissue homogenates of adult human melanoma and nonmelanoma sources further substantiated the results obtained with the direct tests. While each mela noma line showed a differing degree and pattern of neactiv ity, the antisera were most reactive against their respective immunizing lines as revealed by both the direct tests and absorption analysis. By quantitative absorption analysis, no evidence for individually specific melanoma antigens was obtained. The only positive absorption with nonmelanoma adult tissues was obtained with a retinoblastoma cell line, indicating the presence of antigens shared by tumors of common neunoectodermal origin. Extensive absorption with two xenogeneic malignant melanoma (munine and porcine) homogenates, normal human adult skin, and spleen tissues, or Bacillus Calmette-Guéninfailed to reduce the reactivity against melanoma-associated antigens. Fur then absorption with human fetal tissues of 8 to 20 weeks of gestation removed part but not all of the reactivity. These studies with xenogeneic monkey antisera provide evidence for the existence of common melanoma-associated anti gens as well as distinct but shared human fetal antigens on human melanoma cells. , This is Paper 8 of the series, â€oeCharacterization of Human Malignant MelanomaCell Lines.― The work was supportedby the Medical Research Council of Canada and the Ontario Cancer Treatment and Research Foun dation.
Molecular Immunology, 1983
Normal human sera were analyzed for the presence and molecular form of two human melanoma-associated antigens (MAAs), the 250 "melanoma-specific" glycoprotein and the 100 K "common tumor antigen". The 250K MAA was not synthesized by any cultures other than human melanoma and was not detectable in normal human serum. In contrast, the 100 K MAA, which is present in spent medium of cultured human melanoma, carcinoma and fetal melanocytes but not of adult normal cells, was found in normal human serum in nanogram quantities. This serum form of the 100 K MAA was also found in pooled sera of higher apes but not of lower species. The cell-derived form of the 100 K MAA, present in spent culture medium, had a similar phylogenetic distribution. The molecule was produced by cultured brain glia from gorilla, but not by melanoma cells from miniature swine or dog. The 100 K MAA from gorilla glia had a mol. wt identical to the molecule produced by human melanoma cells. Molecular characterization of this MAA in normal human serum showed that it was heterogeneous in size and was present in fractions > 100 kd after analytical HPLC gel sieving under non-denaturing conditions. In contrast, MAA from spent culture medium of melanoma cells was 100 kd or less in chemically defined medium (CDM) with no protein supplement, but had a higher mol. wt in CDM with BSA or fetal calf serum supplement, simila; to the se&m form of the mole&le. An association of the 100 K MAA with albumin was demonstrated bv analvtical HPLC gel sieving and SDS-PAGE analvsis of monoclonal antibody immunoprecipitates. Tie 100-K MAA wasldissociated from albumin in nor&al human serum by treatment with SDS and fractionation by gel sieving. Under these conditions 100 K MAA from serum co-migrated with similarly treated 100 K from melanoma cells. These results indicate that the 100 K MAA is a normal serum constituent which forms a strong, non-covalent association with albumin and is evolutionarily restricted to higher apes or humans.
Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue
Cancer Immunology Immunotherapy, 1987
Immunohistology provides a necessary but insufficient criterion for selecting monoclonal antibodies (MAbs) capable of tumour targeting; in vivo. Additional selection procedures have been evaluated using a panel of anti-melanoma MAbs, including immunoreactivity of (labelled) MAbs, antibody affinity, kinetics of binding and release, apparent antigen density and accumulation in nude mouse transplants. According to these criteria, MAbs M.2.7.6 and M.2.9.4 showed the most favourable properties, i.e. high immunoreactivity and pronounced internalization into melanoma ceils. With MAbs M.2.10.15 and KG 6-56, moderate immunoreactivity and a binding pattern characterized by temperature dependence in the absence of internalization was observed. According to the paired label assay, all four MAbs showed specific accumulation into solid melanoma tissue. However, application in the patient still requires evaluation of the side effects of antigen cross-expression on normal human tissues.