IgA hybridomas: A method for generation in high numbers

Colwell, Dawn E.; Gollahon, Katherine A.; McGhee, Jerry R.; Michalek, Suzanne M.

doi:10.1016/0022-1759(82)90067-9

Cited by 20 publications

(5 citation statements)

References 13 publications

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“…The cells were washed by centrifugation (1,000 rpm) in RPMI 1640. After washing, cells were resuspended in medium consisting of complete medium (RPMI 1640 supplemented with 2 mM t.-glutamine, 100 U/ml penicillin, 100 ~g/ml streptomycin, 5 × 10 -5 M 2-mercaptoethanol and 10% FCS)with 0.01 M hypoxanthine, 4 × 10 -7 M aminopterin, and 1.6 × 10 -6 M thymidine (28). After 3-4 wk of growth in selective medium, it was gradually removed and replaced by complete medium.…”

Section: Formation Of T-t Hybridomasmentioning

confidence: 99%

“…Formation of T-T Hybridomas. For the generation of hybridomas, PP Th A clones 1 or 9 were fused with R 1.1 T lymphoma cells using polyethylene glycol, tool wt 4,000 (Sigma Chemical Co., St. Louis, MO), as previously described (8,27,28). Equal numbers of PP Th A cells and RI.1 cells were pelleted together and fused by adding 1 ml of 50% polyethylene glycol in RPMI 1640 (Gibco Laboratories, Grand Island, NY).…”

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confidence: 99%

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Isotype-specific immunoregulation. IgA-binding factors produced by Fc alpha receptor-positive T cell hybridomas regulate IgA responses.

Kiyono¹,

Mosteller-Barnum²,

Pitts³

et al. 1985

The Journal of Experimental Medicine

103

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T-T hybridomas, produced by fusions between R1.1 T lymphoma and cloned T helper cells that promote IgA responses (Th A cells) were characterized in this study. A total of 85 cloned cell lines were produced, and their supernatants were assessed for support of antigen-dependent IgA (and IgM and IgG) responses. 16 of 85 culture fractions supported IgA anti-sheep red blood cell, -horse red blood cell, or -trinitrophenyl responses in either lipopolysaccharide-triggered splenic B cell, or normal Peyer's patch B cell cultures, and the responses were specific for the antigen used for in vitro immunization. None of the supernatants from the cell lines induced significant polyclonal responses in these B cell cultures. Interestingly, the 16 hybridomas that produced supernatants with IgA-promoting properties had Fc receptors for IgA (Fc alpha R), but did not express Fc mu R or Fc gamma R. When supernatants from Fc alpha R+ T cell lines were subjected to IgA affinity chromatography, the IgA-promoting activity bound to IgA (IBF alpha) and was recovered in the eluate. No binding of active fractions occurred when supernates were passed through IgM or IgG immunoadsorbent columns. High concentrations of purified IBF alpha suppressed T-dependent IgA responses, while an optimal level was required for enhancement of this isotype response. These results suggest that Fc alpha R+ hybridomas derived from Th A cells release IBF alpha into the culture medium, and that these molecules regulate IgA responses to various T-dependent antigens.

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Section: Formation Of T-t Hybridomasmentioning

confidence: 99%

mentioning

confidence: 99%

Isotype-specific immunoregulation. IgA-binding factors produced by Fc alpha receptor-positive T cell hybridomas regulate IgA responses.

Kiyono¹,

Mosteller-Barnum²,

Pitts³

et al. 1985

The Journal of Experimental Medicine

103

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“…Human pIgA was isolated on jacalin affinity columns (Pierce, Rockford, Ill.) from sera of patients with acute infectious mononucleosis as previously described (47). To prepare a murine MAb of the IgA isotype specific to the major membrane glycoprotein of EBV, gp350, naturally glycosylated gp350 isolated by fast protein liquid chromatography (FPLC) from membranes of productively infected B95-8 cells (12) was used in a standard immunization protocol (11,27,32). Cells from a mesenteric lymph node fused to the murine myeloma cell line X63-Ag8.653 (25) produced a stable, gp350-specific, IgA-secreting hybridoma, designated 1556F2, after multiple subclonings.…”

Section: Mice and Virusmentioning

confidence: 99%

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus

Gan

Chodosh

Morgan

et al. 1997

J Virol

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Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocatedIgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.

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“…The probability of getting IgM clones may also be increased by immunizing once only with a large dose of immunogen 3/4 d before fusion. IgA and IgE hybridomas are rare from spleen cell fusions following normal immunization, but IgAsecreting clones may be obtained either by using Peyer's patches for fusion following normal immunization, or administering antigen by gastric intubation and using the spleen for fusion (Colwell et al 1982). A number of different myeloma lines is in use, but there is no general agreement about which is the best.…”

Section: Technology Of Monoclonal Antibody Productionmentioning

confidence: 99%

Monoclonal antibodies and their use in microbiology

Tiffin

1987

Journal of Applied Bacteriology

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IgA hybridomas: A method for generation in high numbers

Cited by 20 publications

References 13 publications

Isotype-specific immunoregulation. IgA-binding factors produced by Fc alpha receptor-positive T cell hybridomas regulate IgA responses.

Isotype-specific immunoregulation. IgA-binding factors produced by Fc alpha receptor-positive T cell hybridomas regulate IgA responses.

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus

Monoclonal antibodies and their use in microbiology

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