P.M. Lansdorp

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells

An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the unlabeled antibody-enzyme method of Sternberger (1974). The cell-bound peroxidase could either be quantified per well or visualized on individual cells by the use of appropriate substrates for peroxidase. Experimental procedures are described in detail and results obtained with several monoclonal antibodies with specificity for different target cells are shown. Limitations and applications of the technique are discussed.

Stepwise amplified immunoperoxidase (PAP) staining. I. Cellular morphology in relation to membrane markers.

A novel procedure for the assay of monoclonal antibodies is described. The technique is based on a combination of three principles. Unlabeled (sheep) antiserum to mouse immunoglobulin (Ig) and complexes of peroxidase with mouse monoclonal antiperoxidase (monoclonal PAP complexes) are used as reagents in a variant of the unlabeled antibody enzyme (PAP) method, described by Sternberger. The amount of peroxidase eventually bound to a monoclonal antibody can be varied over a wide range by repetition of incubation cycles with anti-mouse Ig and monoclonal PAP complexes. During the assay, incubations and wash steps are performed by immersion of whole slides. The influence of repetitive incubation cycles with anti-mouse Ig and monoclonal PAP complexes on background staining and detection of monoclonal antibodies at low concentrations was quantitated in a model system. At a given primary antibody concentration, a linear relationship was found between peroxidase activity and the number of incubation cycles. Application of the technique to the detection of monoclonal antibodies bound to cell-surface antigens is described. Peripheral blood cells were labeled in suspension with monoclonal antibodies. Cytocentrifuge preparations of labeled cells were prepared, and such preparations were fixed before stepwise-amplified PAP staining. Cells showed intense specific staining. Morphological detail of stained and unstained cells is preserved, allowing morphological analysis of labeled cells and rapid analysis of monoclonal antibody specificity. Because the reagents used in the assay can be produced in large quantities with uniform quality, the technique can be readily automated. This, together with the possibility to increase the sensitivity of antibody detection in a controlled, stepwise fashion to levels that cannot be reached with single-step techniques, may further expand the applications of monoclonal antibodies.

Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas.

The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes. A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient. The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved. In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided. The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.

Serological and Biochemical Characterization of Human Myeloid-Associated Antigens and Their Expression in Human-Mouse Cell Hybrids

Recently many monoclonal antibodies (MoAb) which define new granulocyte- and/or monocyte-specific antigens have been described. These antibodies have been applied to the study of myeloid and monocyte differentiation [8, 9, 11], as well as to the typing of the cells of patients with leukemia [5, 10]. In this report we describe three new monoclonal antibodies, two reacting with granulocytes and one reacting with granulocytes, monocytes, and a population of non-T lymphocytes. The molecular identity of the antigens recognized by the antibodies and the expression of the antigens in hybrids derived from fusion of human and mouse myeloid cells was studied. They were also compared with monoclonal antibodies against granulocytes from other laboratories.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

Colony-forming cells in chronic granulocytic leukemia - I. Proliferative responses to growth factors☆

Peripheral blood cells from 2 patients with chronic granulocytic leukemia were separated by density centrifugation. Mononuclear cells of low density (d less than 1.062g cm-3) with blast-cell morphology were cryopreserved before culture in vitro. Upon culture in conventional colony assays, up to 20% of the cells formed hemopoietic colonies. Although the spectrum of colony types resembled that of normal bone-marrow cells, there were large differences between the patients with respect to the number, type and size of colonies that were observed. Colony formation required the addition of hemopoietic growth factors, such as colony-stimulating activity and erythropoietin to the culture medium. The cells were used to assay hemopoietic regulatory molecules. Both erythropoietin and colony-stimulating activity induced a strong proliferative response as measured by thymidine incorporation. Maximal stimulation was observed when erythropoietin and the supernatant of mixed lymphocyte cultures were added simultaneously. The difference between the cells from the 2 patients in clonal assays was reflected by the different response to individual hemopoietic regulators. The time course of maximal stimulation followed distinct patterns dependent on the source of stimulator. The stimulation was linearly dependent on the input cell number. Taken together, cryopreserved blast cells from patients with chronic granulocytic leukemia appear to be very useful for the characterization of factors regulating hemopoiesis, as well as for studies of hemopoiesis in general.

Mixed megakaryocytic-granulocytic differentiation during diffusion chamber culture of peripheral blast cells from the blast crisis of chronic myelocytic leukemia

Morphologically and cytochemically undifferentiated peripheral blast cells from three patients in blast crisis of Ph-positive chronic myelocytic leukemia were analysed morphologically, immunologically and cytogenetically prior to and during in-vivo diffusion chamber culture (DC) to investigate their differentiation capacity. In two patients immunological markers characteristic of the megakaryocytic lineage were found on the original cells, but lineage-specific differentiation markers were absent on the blast cells of the third patient. During DC culture multilineage differentiation capacity could be demonstrated immunologically and morphologically in all three patients with expression of megakaryocytic and granulocytic markers as well as terminal differentiation along these lineages. Cytogenetic analysis prior to and during DC culture provided evidence that the cells differentiating in culture were derived from the blast crisis clones and that the event leading to blast crisis might have occurred in a pluripotent precursor cell which retained its differentiation capacity along several lineages.

Human-Mouse Hybridoma Cells Producing Antibodies

Abstract 1 Human Endothelial Culture Supernatant (HECS) has a growth-promoting activity toward hybridoma cells and increases the stability of human-mouse hybridoma cells. 2 The recovery of human-mouse hybridoma cells after fusion is strongly increased when the human lymphocytes are stimulated in vitro prior to the fusion. 3 The highest yields of hybridoma cells are obtained when the human lymphocytes are cultured with the antigen, in the presence of endothelial cells, prior to the fusion with mouse myeloma cells. 4 The recovery of stable human-mouse hybridoma cells is increased by selecting for clones producing antibodies, which can be achieved by cloning at low cell density.

Analysis of Monoclonal Antibody Binding Using Stepwise-Amplified Immunoperoxidase Staining

Immunofluorescence techniques in combination with flow cytofluorometry are established tools for the analysis of monoclonal antibody binding. Drawbacks of the immunofluorescence techniques are that the cellular preparations are not permanent and are lacking in morphological detail. This is a particular problem when the cells under study have a low frequency and/or when morphological information is important (e. g., recognition of atypical cells) Immunoperoxidase techniques have been proposed as an alternative to immunofluorescence for these reasons [8, 9]. We have described immunoperoxidase procedures to detect monoclonal antibodies against cell-surface antigens [4], using a variant of the unlabeled antibody-enzyme method [12]. In this technique, binding of monoclonal mouse antibody to cells is detected using unlabeled antiserum to mouse immunoglobulin and peroxidase-antiperoxidase (PAP) complexes prepared with a monoclonal mouse antiperoxidase antibody. Adaptation of this technique for high-power microscopy of cytocentrifuged cells has now been introduced. Cells are labeled with monoclonal antibodies in suspension prior to cytocentrifugation and fixation. Sensitivity of immunoperoxidase staining is increased by repeating incubations with anti-mouse Ig and monoclonal PAP. Results obtained using such stepwise amplified immunoperoxidase staining with monoclonal antibodies which have not been previously described but were included in the workshop are given here.

Reactivity of Monoclonal Anti-HLA Antibodies with Blood Platelets

Abstract Binding pattern of mouse monoclonal antibodies specific for determinants on HLA-A, -B and -C glycoproteins were studied. The reactivity of the antibodies was quantitated by an “unlabeled antibody” immunoperoxidase technique. Blood platelets from 4 donors were used as target cells. The following results were obtained: 1) Observed binding patterns of monoclonal anti-HLA-A2 and anti-HLA-B7 was in agreement with results of conventional HLA-typing of lymphocytes; 2) Binding curves, obtained at different dilutions of a monoclonal antibody against a non-polymorphic “backbone” HLA-determinant, showed little variation between platelets of the different donors; 3) A a saturating amount of antibody, binding of monoclonal anti-HLA-A2 to platelets showed a gene-dose effect: platelets of an HLA-A2 homozygous individual bound approximately twice the amount of anti-HLA-A2 antibody compared to platelets from a HLA-A2 heterozygous donor.