Detection of cell surface Ig allotypes and isotypes by rosetting with antibody-coated erythrocytes. Most rabbit Ig-bearing cells have surface IgM.
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Biomedical subjects
Publications and source records attributed to G A Molinaro.
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Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC* assays. MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.
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Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.
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In vitro treatment of goat red blood cells (GRBCs) with the sulphydryl compound 2-aminoethylisothiouronium bromide (AET) increases their specific reactivity with human T lymphocytes without affecting the specificity of the reaction. AET-GRBCs bind to only part of T lymphocytes rosetting with AET-sheep red blood cells (SRBCs): the receptors for both types of RBCs are very simular if not identical, but display higher affinity for AET-SRBCs than for AET-GRBCs. Rosetting of T lymphocytes with AET-GRBCs may be useful to enumerate T lymphocyte subsets in patients with abnormality of the immune system and to fractionate T lymphocyte subpopulations.
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We developed a hemolytic radial immunodiffusion assay for identifying immunoglobulin (Ig) isotypes, allotypes and idiotypes by using gels containing erythrocytes coated with anti-Ig antibody or erythrocytes coated with Staphylococcal protein A. These indicator cells lysed specifically when treated sequentially with Ig antigen, the appropriate anti-Ig antiserum (developer) and complement. To identify these Ig subpopulations, we used monospecific indicator cells, e.g. erythrocytes coated with antibody specific for an Ig isotype, and developers with broader specificities ('multispecific'), e.g. antiserum to Fab. Alternatively, we used 'multispecific' indicator cells, e.g. erythrocytes coated with antibody to Fab and monospecific developers, e.g. antiserum to Ig idiotype. To identify Ig subpopulations specifically, either the indicator cells or the developer need to be monospecific. When both the indicator cells and the developer were monospecific, e.g. to allotype and to isotype, the specificity was determined by both reagents and ultimately restricted by the reagent with the narrower specificity, that is, reacting with the smallest Ig subpopulation. This sensitive hemolytic assay may be used to quantitate subpopulations of Ig molecules and may be modified into a reverse plaque forming cell assay to count lymphocytes secreting a given Ig class, type, allotype and idiotype.
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We used a new immunocytoadhesion method to test 15 different tumors of the human nervous system to see whether medulloblastomas share tumor-associated surface antigens (TSA) with other tumors. Antisera against medulloblastoma cells were raised in rabbits. One antiserum was adsorbed extensively with tonsil and adenoid cells and with cerebral tissues. Freshly isolated cells from 15 different tumors were first sensitized with the adsorbed antiserum and then rosetted with erythrocytes coated with purified antibody to rabbit immunoglobulin. We found that the antimedulloblastoma antiserum detected TSA on the immunizing medulloblastoma cells and on cells from 2 other medulloblastomas, but apparently not on cells from other tumors (astrocytomas, glioblastomas, neurinoma, meningiomas and craniopharyngioma). In addition, the antiserum probably detected the medulloblastoma TSA on some other tumors (oligodendroglioma, ependymoma and malignant glioma of mixed type).
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Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.
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