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The analysis of human myelogenous leukemia cells in the fluorescence-activated cell sorter

University of British Columbia

A cell surface protein from human acute myelogenous leukemia (AML) cells has been purified. (Al-Rammahy et al., Cancer Immunol. Immunother. 9:181, 1980; Malcolm et al., J. Immunol. 128:2599, 1982). This material was used to immunize rabbits. The resulting antiserum (anti-AML) showed myelogenous leukemia specificity in that it reacted with myelogenous leukemia cell extracts and did not react with cell extracts of normal individuals or patients with non-myelogenous leukemia or other malignant disorders in the enzyme-linked immunosorbent assay (ELISA). Bone marrow and peripheral blood leucocytes (PBL) from either patients with myelogenous leukemia, other disorders or normal individuals were analysed in the fluorescence-activated cell sorter (FACS IV) after labelling with anti-AML, normal rabbit serum (NRS), or antiserum raised to normal human membrane antigens. Of 40 cell samples from patients with AML, 39 reacted strongly with the anti-AML. Similarly, all of 15 specimens from patients with chronic myelogenous leukemia (CML) reacted with the anti-AML. When 42 bone marrow or PBL samples from patients with a variety of lymphoproliferative disorders were examined, only 2 specimens reacted with the antiserum, both from individuals with diagnoses of acute lymphocytic leukemia (ALL). None of the 14 normal bone marrow or PBL donor specimens tested reacted with the anti-AML. It was also found that essentially all samples from patients in clinical remission from AML had high numbers of cells reactive with the anti-AML. When cells from such individuals were labelled and sorted on the FACS IV, it was found that the cell population fluorescing strongly with the anti-AML contained cells of both myeloid and lymphoid origin. The AML antigen was used to produce AML specific monoclonal antibody. Spleens from AML-antigen immunized Balb/c mice were fused to NS-1 myeloma parental cells and a myelogenous leukemia specific monoclonal antibody was selected from the hybrid colonies produced. This monoclonal antibody (MAL-1) as well as the rabbit anti-AML has been used to identify myelogenous leukemia patient samples in the FACS IV. In addition, this monoclonal also demonstrates positive fluorescence binding to HL-60 (a promyelocytic leukemia cell line), while there is no binding to lymphocytic leukemia cell lines, CCRF-SB-ALL-B and CCRF-CEM-ALL-T. The MAL-1 monoclonal has been shown to be specific for myelogenous leukemia cell extracts in the ELISA and has been successfully used as an immunoadsorbent for the isolation of the AML antigen from cell extracts. No equivalent antigen was found when cell extracts from normal cells, lymphocytic leukemia cells and lymphoma cells were similarly absorbed. These findings indicate that both the rabbit anti-AML serum and MAL-1 monoclonal show specificity for an antigen associated with myelogenous leukemia cells.

Text

2010-05-02

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http://hdl.handle.net/2429/24325

Microbiology and Immunology

University of British Columbia

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