Isolation of Functional Human Coagulation Factor V by Using a Hybridoma Antibody
Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mi...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1981-01, Vol.78 (1), p.162-166 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Katzmann, Jerry A. Nesheim, Michael E. Hibbard, Lyndon S. Mann, Kenneth G. |
| description | Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M Nacl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mrcomparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule. |
| doi_str_mv | 10.1073/pnas.78.1.162 |
| format | Article |
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Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M Nacl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mrcomparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.78.1.162</identifier><identifier>PMID: 6941242</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>Animals ; Antibodies ; Antibodies - immunology ; Antibody Specificity ; Ascitic Fluid ; Biochemistry ; Blood plasma ; Clone Cells - immunology ; Factor V - immunology ; Factor V - isolation & purification ; Factor V - metabolism ; Gels ; Glycols ; Humans ; Hybrid Cells - immunology ; Hybridomas ; Mice ; Multiple Myeloma ; Neoplasms, Experimental ; Polyethylenes ; Radioimmunoassay ; Spleen ; Sulfates ; Thrombin - metabolism ; Ungulates</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1981-01, Vol.78 (1), p.162-166</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c451t-b28e43cda7b73166d4f3fb90759cd04190ccf77baec3bef6393bb67ba8abc2703</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/78/1.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/10003$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/10003$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/6941242$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Katzmann, Jerry A.</creatorcontrib><creatorcontrib>Nesheim, Michael E.</creatorcontrib><creatorcontrib>Hibbard, Lyndon S.</creatorcontrib><creatorcontrib>Mann, Kenneth G.</creatorcontrib><title>Isolation of Functional Human Coagulation Factor V by Using a Hybridoma Antibody</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M Nacl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mrcomparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.</description><subject>Animals</subject><subject>Antibodies</subject><subject>Antibodies - immunology</subject><subject>Antibody Specificity</subject><subject>Ascitic Fluid</subject><subject>Biochemistry</subject><subject>Blood plasma</subject><subject>Clone Cells - immunology</subject><subject>Factor V - immunology</subject><subject>Factor V - isolation & purification</subject><subject>Factor V - metabolism</subject><subject>Gels</subject><subject>Glycols</subject><subject>Humans</subject><subject>Hybrid Cells - immunology</subject><subject>Hybridomas</subject><subject>Mice</subject><subject>Multiple Myeloma</subject><subject>Neoplasms, Experimental</subject><subject>Polyethylenes</subject><subject>Radioimmunoassay</subject><subject>Spleen</subject><subject>Sulfates</subject><subject>Thrombin - metabolism</subject><subject>Ungulates</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1981</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kUFP2zAYhq1pqJSy4y6ISb7ALeVz7NrJgQOqKEVCggPsatmO06VK7BIn0_rvcdWsKhdOtvU8r_3JL0I_CUwJCHqzcSpMRTYlU8LTb2hMICcJZzl8R2OAVCQZS9kpOgthDQD5LIMRGvGckZSlY_TyGHytuso77Eu86J3Z7VWNl32jHJ57teoHvlCm8y3-jfUWv4XKrbDCy61uq8I3Ct-5rtK-2J6jk1LVwf4Y1gl6W9y_zpfJ0_PD4_zuKTFsRrpEp5ll1BRKaEEJ5wUraalzELPcFMBIDsaUQmhlDdW25DSnWvN4zpQ2qQA6Qbf7eze9bmxhrOtaVctNWzWq3UqvKvmZuOqPXPm_ksa7CYn56yHf-vfehk42VTC2rpWzvg9SzHjGacqjmOxF0_oQWlse3iAgdw3IXQNSZJLI2ED0fx0PdrCHL4_8cuC72H96FL_6Asuyr-vO_uuid7H31iG2cjQTAKUfAGmjwA</recordid><startdate>19810101</startdate><enddate>19810101</enddate><creator>Katzmann, Jerry A.</creator><creator>Nesheim, Michael E.</creator><creator>Hibbard, Lyndon S.</creator><creator>Mann, Kenneth G.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19810101</creationdate><title>Isolation of Functional Human Coagulation Factor V by Using a Hybridoma Antibody</title><author>Katzmann, Jerry A. ; Nesheim, Michael E. ; Hibbard, Lyndon S. ; Mann, Kenneth G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c451t-b28e43cda7b73166d4f3fb90759cd04190ccf77baec3bef6393bb67ba8abc2703</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1981</creationdate><topic>Animals</topic><topic>Antibodies</topic><topic>Antibodies - immunology</topic><topic>Antibody Specificity</topic><topic>Ascitic Fluid</topic><topic>Biochemistry</topic><topic>Blood plasma</topic><topic>Clone Cells - immunology</topic><topic>Factor V - immunology</topic><topic>Factor V - isolation & purification</topic><topic>Factor V - metabolism</topic><topic>Gels</topic><topic>Glycols</topic><topic>Humans</topic><topic>Hybrid Cells - immunology</topic><topic>Hybridomas</topic><topic>Mice</topic><topic>Multiple Myeloma</topic><topic>Neoplasms, Experimental</topic><topic>Polyethylenes</topic><topic>Radioimmunoassay</topic><topic>Spleen</topic><topic>Sulfates</topic><topic>Thrombin - metabolism</topic><topic>Ungulates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Katzmann, Jerry A.</creatorcontrib><creatorcontrib>Nesheim, Michael E.</creatorcontrib><creatorcontrib>Hibbard, Lyndon S.</creatorcontrib><creatorcontrib>Mann, Kenneth G.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Katzmann, Jerry A.</au><au>Nesheim, Michael E.</au><au>Hibbard, Lyndon S.</au><au>Mann, Kenneth G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation of Functional Human Coagulation Factor V by Using a Hybridoma Antibody</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1981-01-01</date><risdate>1981</risdate><volume>78</volume><issue>1</issue><spage>162</spage><epage>166</epage><pages>162-166</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M Nacl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mrcomparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>6941242</pmid><doi>10.1073/pnas.78.1.162</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0027-8424 |
| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1981-01, Vol.78 (1), p.162-166 |
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| language | eng |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Animals Antibodies Antibodies - immunology Antibody Specificity Ascitic Fluid Biochemistry Blood plasma Clone Cells - immunology Factor V - immunology Factor V - isolation & purification Factor V - metabolism Gels Glycols Humans Hybrid Cells - immunology Hybridomas Mice Multiple Myeloma Neoplasms, Experimental Polyethylenes Radioimmunoassay Spleen Sulfates Thrombin - metabolism Ungulates |
| title | Isolation of Functional Human Coagulation Factor V by Using a Hybridoma Antibody |
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