Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

Subramanian, Vijay; Blanckaert, Vincent; Schelling, Margaret E.

doi:10.1007/bf01409107

Cited by 3 publications

(4 citation statements)

References 14 publications

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“…Endothelial cell membrane fragments were produced according to our published protocol (13). lodinated FGF was crosslinked to FGF receptors on the endothelial cell membrane fragments utilizing the reuersible crosslinker EGS.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to exhibiting typical endothelial cobblestone morphology, the CUECs were confirmed to be endothelial by staining positiue for factor Ulli antigen and UER lectin (9). CUEC membrane fragments containing high affinity FGF receptors (10)(11)(12) were prepared according to our published protocol (13).…”

Section: Mrterirls Rnd Methodsmentioning

confidence: 99%

“…prior to membrane preparation as preuiously described (13). The membranes were solubilized with TBS-2% Triton X100 and the insoluble material was centrifuged at 25,000g, 1 hr., 4°C.…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…The second antibodies were incubated with the preparation ouernight at 4°C and were respectiuely: c) Goat anti-mouse IgM biotinylated for UBSI (1:1000) and d) Goat anti-mouse IgG biotinylated, for antiphosphotyrosine (1:1000). The streptauidin-peroxidase complex (1:750) was then added, incubated for 2 hr., and the deueloping was done as preuiously described (13).…”

Section: Immunoprecipitationmentioning

confidence: 99%

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Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization

Subramanian

Blanckaert²,

Schelling³

1992

Hybridoma

Self Cite

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Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the development of blood vessels). Monoclonal antibodies against the FGF receptor were made by in vitro immunization. Receptor was PAGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolved with acetonitrile, and the receptor used as antigen in an in vitro immunization system. Screening for FGF receptor positive clones was done using membrane preparations from Coronary Venular Endothelial Cells (CVEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. Culture supernatants of several clones tested positive for IgM or IgG monoclonal antibodies against the FGF receptor. Antibodies were affinity purified. Ascites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuvant (IFA). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a variety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway.

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Section: Resultsmentioning

confidence: 99%

Section: Mrterirls Rnd Methodsmentioning

confidence: 99%

“…prior to membrane preparation as preuiously described (13). The membranes were solubilized with TBS-2% Triton X100 and the insoluble material was centrifuged at 25,000g, 1 hr., 4°C.…”

Section: Immunoprecipitationmentioning

confidence: 99%

Section: Immunoprecipitationmentioning

confidence: 99%

See 2 more Smart Citations

Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization

Subramanian

Blanckaert²,

Schelling³

1992

Hybridoma

Self Cite

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Partial Characterization of Endothelial FGF Receptor Functional Domain by Monoclonal Antibody VBS-1

Blanckaert

Subramanian

Han

et al. 2002

Hybridoma and Hybridomics

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Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.

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Efficient Purification of Mouse Anti-FGF Receptor IgM Monoclonal Antibody by Magnetic Beads

Quitadamo¹,

Schelling²

1998

Hybridoma

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Affinity chromatography has been widely used for the purification of monoclonal antibodies (MAb). Traditionally, activated agarose beads conjugated with specific antisera have been used as a solid support in chromatographic protein purification. Magnetic beads conjugated with various antibodies have recently become an alternative method for the isolation of diverse proteins, nucleic acids, and cell types. In this study, murine anti-fibroblast growth factor receptor 1 (FGFR1) immunoglobulin M (IgM) was isolated from protein solutions to compare immunoaffinity column chromatography and magnetic bead IgM purification methods. Using immobilized rat anti-mouse IgM MAb, an UltraLink 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC)/diaminodipropylamine (DADPA) immunoaffinity column and polystyrene-coated magnetic beads were used for the purification of mouse IgM from bovine serum albumin/phosphate-buffered saline (BSA/PBS) as well as from crude ascites. Protein quantitation and percent IgM yield were determined by reducing SDS-PAGE electrophoresis followed by silver staining, then IgM and protein contaminants were quantitated using densitometry analysis. IgM anti-FGFR1 binding specificity and immunologic activity were determined by enzyme-linked immunosorbant assay (ELISA). This study demonstrates that magnetic bead isolation of IgM from ascites is more effective than traditional affinity chromatography purification as determined by greater IgM yield, purity, and immunologic activity.

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Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

Cited by 3 publications

References 14 publications

Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization

Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization

Partial Characterization of Endothelial FGF Receptor Functional Domain by Monoclonal Antibody VBS-1

Efficient Purification of Mouse Anti-FGF Receptor IgM Monoclonal Antibody by Magnetic Beads

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