Influence of Fusion Cell Ratio and Cell Plating Density on Production of Human-Human Hybridomas Secreting Anti-DNA Autoantibodies from Patients with Systemic Lupus Erythematosus

Massicotte, H; Rauch, Joyce; Shoenfeld, Yehuda; Tannenbaum, H

doi:10.1089/hyb.1984.3.215

Cited by 30 publications

(9 citation statements)

References 15 publications

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“…Table 1 gives results obtained from this fusion. 20 Established clones (9 months in vitro) 19 Wells containing clusters of more than 100 cells Nearly all viable cells of 8 IgM-producing hybridoma cell lines tested were clg-positive, as documented by indirect immunofluorescence staining carried out with mouse monoclonal antibodies against human Ig heavy and light chains ( Table 2). All cells of a non-producer clone (No.…”

Section: Resultsmentioning

confidence: 99%

Cell Biology of Human IgM-Producing Hybridomas Derived from a Fusion of Human Spleen Lymphocytes with Mouse Myeloma Cells

Jahn¹,

Grunow²,

Kießig³

et al. 1987

Hybridoma

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Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.

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Section: Resultsmentioning

confidence: 99%

Cell Biology of Human IgM-Producing Hybridomas Derived from a Fusion of Human Spleen Lymphocytes with Mouse Myeloma Cells

Jahn¹,

Grunow²,

Kießig³

et al. 1987

Hybridoma

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“…The PBMC were fused with the GM 4672 human lymphoblastoid cell line obtained from the Cell Repository Institute of Medical Research (Camden, NJ) at a cell ratio of 1 : 1 with 44.4% polyethylene glycol (PEG). as previously described (14). Hybridoma clones that produce IgM-RF were found to be monospecific (clones 5501, 5602, 5603, 8808. and 18141; all derived from patients with RA) or polyreactive (clones 1859 and 1872; derived from patients with RA and clones 103.4.…”

Section: Methodsmentioning

confidence: 99%

Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding

Newkirk

Lemmo

Rauch

1990

Arthritis & Rheumatism

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We investigated the influence of carbohydrate on the binding of human rheumatoid factors (RF) to the Fc fragment of IgC. The monoclonal RF studied were derived from the serum of patients with mixed cryoglobulinemia or from hybridomas generated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. Polyclonal RF were derived from patients with RA. The carbohydrate located on the Fc fragment, regardless of whether it contained different amounts of mannose or reduced amounts of galactose, or was removed, did not affect the binding of the RF. In contrast, the isotype of the Fc was found to be critical. Two groups of hybridoma RF could be delineated. One group bound preferentially to IgCl and/or IgG2, and a second group (primarily from patients with RA) bound preferentially to IgG3 and/or IgG4. Our results indicate that the isotype of the Fc fragment, and not the extent of galactosylation, influences the binding of the RF.Human rheumatoid factors (RF), a group of antibodies to gamma globulins, are highly associated with rheumatoid arthritis (RA) (1). These antibodies have been demonstrated to bind region(s) in the Fc fragment of gamma globulins, with good evidence to suggest the Cy2-Cy3 cleft as a likely site for binding

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“…A single cell suspension of lymphocytes derived from the pleural effusion of the patients was fused with GM 4672 lymphoblastoid cell line according to the method described previously by us [18,19,21]. These cells (106/ml) were incubated with 1:100 dilu tion of pokeweed mitogen (Gibco Laboratories, Grand Island Biological Co., Grand Island.…”

Section: Production O F Human Monoclonal Antibodiesmentioning

confidence: 99%

“…Established cell lines were grown in RPMI 1640 tissue culture medium supplemented with 10% fetal calf serum (FCS) [18][19][20], Culture fluids from each cell line were pooled, and the immunoglobulins were precipitated in 50% saturated ammonium sulfate, re-Serum dilution Fig. 1 a, b.…”

Section: Purification O F Hybridoma Antibodiesmentioning

confidence: 99%

“…De spite the facts pointing to the existence of the relationship between MMTV and human breast cancer, contradictory results have been described [17], Recently, we have reported the produc tion of human monoclonal antibodies estab lished by two different methods which react with MMTV. One of the monoclonal anti bodies (B11) was generated by the humanhuman hybridoma technique [18][19][20], fusing a human lymphoblastoid cell line GM-4672 with lymphocytes derived from axillary lymph nodes of a patient with breast cancer. In the primary tumor of this patient, MMTVgp-52-like antigen was detected [21].…”

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confidence: 99%

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Human Monoclonal Antibodies Derived from Pleural Effusion Lymphocytes of a Patient with Breast Carcinoma React with Human Breast Cancer-Associated Antigens and Mouse Mammary Tumor Virus Polypeptides

et al. 1987

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Four human hybridoma cell lines (PEB_1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 µg/ml/l0⁶ cells). These monoclonal antibodies (PEB_1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB_1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB_1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmal-ignant cells. The PEB_1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.

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Influence of Fusion Cell Ratio and Cell Plating Density on Production of Human-Human Hybridomas Secreting Anti-DNA Autoantibodies from Patients with Systemic Lupus Erythematosus

Cited by 30 publications

References 15 publications

Cell Biology of Human IgM-Producing Hybridomas Derived from a Fusion of Human Spleen Lymphocytes with Mouse Myeloma Cells

Cell Biology of Human IgM-Producing Hybridomas Derived from a Fusion of Human Spleen Lymphocytes with Mouse Myeloma Cells

Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding

Human Monoclonal Antibodies Derived from Pleural Effusion Lymphocytes of a Patient with Breast Carcinoma React with Human Breast Cancer-Associated Antigens and Mouse Mammary Tumor Virus Polypeptides

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