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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm2 flasks, incubated at 37 degrees C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning. No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.
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PMID:Rapid isolation of monoclonal hybridoma cultures by a 'fusion-cloning' method: the requirement for aminopterin. 195

Hybridoma lines frequently lose their ability to produce ascites upon extended cultivation in vitro. We have reintroduced genes for tumor formation into three independent hybridoma lines by backcrossing them to the parental myeloma. The parental myeloma cells were eliminated by a brief in vitro selective period in HAT medium, after which the cells were inoculated into pristane-primed mice. In two out of three cases the resultant lines were able to produce ascites without further subcloning or other manipulations. The antibody retained its specificity as judged by immunoblotting. This method is a rapid and efficient approach for the reestablishment of ascites production in hybridoma lines.
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PMID:Ascites produced in recalcitrant hybridomas by backcrossing to a parental myeloma. 196 Apr 21

The combination of Epstein-Barr-Virus (EBV)-permitted immortalization and somatic hybridization (fusion with a myeloma partner) may be the method of choice to produce human monoclonal antibodies. We show here that the fusion of EBV-infected human B-lymphocytes to the HAT-sensitive, ouabain-resistent heteromyeloma (human x mouse) fusion line CB-F7, resulted in stable growing hybridomas producing much more immunoglobulin than the parental lymphoblastoid lines. A more efficient clonability was shown for hybridoma cultures too. The loss of B cell markers (HLA-class II antigen, CD-22, CD-37) was detected. Limiting dilution experiments showed a better fusionability of IgM-producing EBV-transformed B cells in comparison to IgG-secreting counterparts.
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PMID:The hybridization of EBV-immortalized human B-lymphocytes with a human-mouse heteromyeloma cell line. 196 78

To obtain suitable cell lines for the immortalisation of human lymphocytes, we constructed a heteromyeloma between the murine myeloma Ag8 and human lymphocytes from a highly malignant polymorphic, centroblastic B-cell lymphoma. The thioguanine-resistant and HAT-sensitive heteromyeloma HAB-1 neither secretes nor contains cytoplasmatic immunoglobulins, the cells being EBV negative but positively stained for HLA-BC and the human proliferation marker Ki-67. The karyotype consists of about 50 murine and 20 human chromosomes. The HAB-1 cells grow in suspension and have a doubling rate of about 25-30 h. In fusion experiments with spleen cells from stomach carcinoma patients HAB-1 cells show a 5-7 times higher fusion efficiency than murine Ag8 cells or another heteromyeloma SPM4-0 and give stable antibody producing products. The cell line will be made available to interested scientists.
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PMID:HAB-1, a new heteromyeloma for continuous production of human monoclonal antibodies. 222 77

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.
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PMID:Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry. 241 47

Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, heparin-affinity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell-lines producing monoclonal antibodies were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali-burned corneas contained collagenase, whereas stromal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.
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PMID:Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas. 243 Jul 58

Human monoclonal antibodies to human endocrine cells have been obtained following the generation of immunoglobulin-secreting interspecies lymphocyte hybridomas. Peripheral blood lymphocytes from an adult patient presenting with acute onset, Type I, diabetes mellitus were fused in vitro with mouse myeloma cells of the NS1 cell line. Initial selection of resulting hybridomas was made by their ability to proliferate in HAT medium. Those hybridomas secreting human immunoglobulins were identified by radioimmunoassay and, thereafter, cloned at frequent intervals to ensure continued antibody production. Human monoclonal antibodies selected in this manner are being employed to identify those epitopes which are common antigenic targets during initial stages of autoimmune-mediated diabetes mellitus and associated multiple endocrinopathies. Of these antibodies, one (HML 3.22) recognizes an epitope present on the human TSH receptor and a second (HML 3.21) identifies a component of thyroglobulin. The potential value of human monoclonal antibodies as probes for analyzing autoimmune-mediated endocrine diseases is discussed.
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PMID:Human monoclonal antibodies to thyroid antigens derived by hybridization of lymphocytes from a diabetic patient. 243 25

An IgG monoclonal antibody to recombinant human granulocyte colony-stimulating factor (G-CSF), designated HG1, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. This HG1 was capable of neutralizing the colony-stimulating activity of G-CSF in vitro, and it did not cross-react with human granulocyte/macrophage colony-stimulating factor (GM-CSF). An enzyme-linked immunosorbent assay (ELISA) for measurement of G-CSF was developed using HG1 and a polyclonal antibody against G-CSF raised in a rabbit. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml of recombinant G-CSF. This assay system therefore warrants further attention.
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PMID:Measurement of human G-CSF by enzyme-linked immunosorbent assay using monoclonal antibody. 247 2

A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.
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PMID:A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres. 248 Sep 79

An IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGM1, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGM1 and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.
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PMID:Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay. 248 32


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