Improved fusion technique. II. Stability and purity of hybrid clones

Westerwoudt, Regine J.; Naipal, A.; Harrisson, C.M.H.

doi:10.1016/0022-1759(84)90139-x

Cited by 29 publications

(10 citation statements)

References 6 publications

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“…3 ) . We had decided to go back to the parent culture to select variants from a population with the greatest potential heterogeneity, as had been shown by others (2,5,24). We clone-sorted (one plate each) hybridoma cells from three sorting regions: "dim," "medium," and bright" subpopulations of these AMIGG-FITC reactors.…”

Section: Surface Immunoglobulin Expressionmentioning

confidence: 99%

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

et al. 1990

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Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescenceactivated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouseIgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell depositioin provides positive attributes of severall traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.

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Section: Surface Immunoglobulin Expressionmentioning

confidence: 99%

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

et al. 1990

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“…When a N P appears in culture, it does not always take over the whole culture (Gardner et al, 1985;Westerwoudt et al, 1984). Populations of producer (P) and N P may be balanced under certain culture conditions, depending on the relative growth rate of producing and nonproducing cells and the rate of loss of MAb productivity.…”

Section: Introductionmentioning

confidence: 99%

Application of Population Balance Model to the Loss of Hybridoma Antibody Productivity

Lee

Varma

Palsson

1991

Biotechnology Progress

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A simple dynamic model has been applied to explain the population dynamics of monoclonal antibody (MAb) producing (producer) and nonproducing hybridoma cells (nonproducer) coexisting in culture. The events of mutation or loss of genes associated with antibody synthesis have been incorporated into the model to account for the conversion of a producer to a nonproducer. The model shows that the cell population is not necessarily dominated by the nonproducer, and a steady balance of producer and nonproducer populations can be achieved. A nonproducer population is undesirable, and cultivation strategies to maximize MAb production are suggested, taking into account the dynamics of a nonproducer population.

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“…However, by recloning for antibody-producing cells every 6 to 10 weeks, we have successfully maintained several hybridomas for over a year. The generation of antibody-negative cells with culture has also been noted for hybridomas of mouse lymphocytes and mouse myeloma cells (12) and human lymphocytes and heteromyelomas (13). Recloning presumably removes the nonsecreting cells that eventually outgrow the producing cells.…”

Section: Discussionmentioning

confidence: 95%

Altered Antigenicity of Human Monoclonal Antibodies Derived from Human-Mouse Heterohybridomas

Kan‐Mitchell¹,

Andrews²,

Gallardo³

et al. 1987

Hybridoma

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We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.

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Improved fusion technique. II. Stability and purity of hybrid clones

Cited by 29 publications

References 6 publications

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Application of Population Balance Model to the Loss of Hybridoma Antibody Productivity

Altered Antigenicity of Human Monoclonal Antibodies Derived from Human-Mouse Heterohybridomas

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