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Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon

mAbs against human IFN-gamma (huIFN-gamma) receptors were obtained by immunizing a BALB/c mouse with eluates from immobilized recombinant huIFN-gamma (rhuIFN-gamma) on which lysates of enriched Raji cell membranes had been adsorbed. mAbs were selected for competitive inhibition of receptor binding o...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1987
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2188584/
https://www.ncbi.nlm.nih.gov/pubmed/2435837
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description mAbs against human IFN-gamma (huIFN-gamma) receptors were obtained by immunizing a BALB/c mouse with eluates from immobilized recombinant huIFN-gamma (rhuIFN-gamma) on which lysates of enriched Raji cell membranes had been adsorbed. mAbs were selected for competitive inhibition of receptor binding of 125I-labeled rhuIFN-gamma. The following additional properties suggest that these antibodies are specific for huIFN-gamma receptors: they bind to the surface of human cells expressing IFN-gamma receptors but not to heterologous cells; this binding is inhibited competitively by addition of rhuIFN-gamma; the number of binding sites revealed by direct binding of 125I-labeled rhuIFN-gamma correlates with the amount of antigen recognized by the mAbs on different cell lines. A Triton X-100 extract of a membrane- enriched fraction of human Raji cells was affinity purified with these mAbs and the eluates from such columns were further purified on immobilized rhuIFN-gamma. As revealed by SDS-PAGE, the final eluate contained two major protein bands with approximate Mr of 90,000 (p90) and 50,000 (p50), respectively. Both proteins were able to specifically bind 125I-labeled rhuIFN-gamma upon electroblotting to nitrocellulose. This binding could be inhibited by the huIFN-gamma receptor mAbs, suggesting that the same epitopes are recognized on p90, p50, and on the cell surface. Therefore, these proteins most likely represent at least a part of huIFN-gamma receptors.
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spelling pubmed-21885842008-04-17 Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon J Exp Med Articles mAbs against human IFN-gamma (huIFN-gamma) receptors were obtained by immunizing a BALB/c mouse with eluates from immobilized recombinant huIFN-gamma (rhuIFN-gamma) on which lysates of enriched Raji cell membranes had been adsorbed. mAbs were selected for competitive inhibition of receptor binding of 125I-labeled rhuIFN-gamma. The following additional properties suggest that these antibodies are specific for huIFN-gamma receptors: they bind to the surface of human cells expressing IFN-gamma receptors but not to heterologous cells; this binding is inhibited competitively by addition of rhuIFN-gamma; the number of binding sites revealed by direct binding of 125I-labeled rhuIFN-gamma correlates with the amount of antigen recognized by the mAbs on different cell lines. A Triton X-100 extract of a membrane- enriched fraction of human Raji cells was affinity purified with these mAbs and the eluates from such columns were further purified on immobilized rhuIFN-gamma. As revealed by SDS-PAGE, the final eluate contained two major protein bands with approximate Mr of 90,000 (p90) and 50,000 (p50), respectively. Both proteins were able to specifically bind 125I-labeled rhuIFN-gamma upon electroblotting to nitrocellulose. This binding could be inhibited by the huIFN-gamma receptor mAbs, suggesting that the same epitopes are recognized on p90, p50, and on the cell surface. Therefore, these proteins most likely represent at least a part of huIFN-gamma receptors. The Rockefeller University Press 1987-04-01 /pmc/articles/PMC2188584/ /pubmed/2435837 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title_full Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title_fullStr Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title_full_unstemmed Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title_short Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
title_sort purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2188584/
https://www.ncbi.nlm.nih.gov/pubmed/2435837