Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques
Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) wer...
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| Veröffentlicht in: | European journal of immunology 1974-08, Vol.4 (8), p.553-560 |
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| creator | Freedman, M. H. Pincus, J. H. Yeger, H. McKenney, Judith A. Mage, Rose G. |
| description | Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum. |
| doi_str_mv | 10.1002/eji.1830040807 |
| format | Article |
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I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques</title><source>MEDLINE</source><source>Wiley Online Library All Journals</source><creator>Freedman, M. H. ; Pincus, J. H. ; Yeger, H. ; McKenney, Judith A. ; Mage, Rose G.</creator><creatorcontrib>Freedman, M. H. ; Pincus, J. H. ; Yeger, H. ; McKenney, Judith A. ; Mage, Rose G.</creatorcontrib><description>Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.</description><identifier>ISSN: 0014-2980</identifier><identifier>EISSN: 1521-4141</identifier><identifier>DOI: 10.1002/eji.1830040807</identifier><identifier>PMID: 4153482</identifier><language>eng</language><publisher>Weinheim: WILEY‐VCH Verlag GmbH</publisher><subject>Animals ; Antibodies, Bacterial - analysis ; Antibodies, Bacterial - isolation & purification ; Antigens, Bacterial ; Chromatography, Affinity ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Genotype ; Hydrogen-Ion Concentration ; Immunization ; Immunization, Secondary ; Immunoglobulin Fragments - isolation & purification ; Immunoglobulin Heavy Chains - isolation & purification ; Immunoglobulins - analysis ; Immunoglobulins - isolation & purification ; Isoantigens ; Isoelectric Focusing ; Polysaccharides, Bacterial - immunology ; Precipitin Tests ; Rabbits ; Streptococcus pneumoniae - immunology</subject><ispartof>European journal of immunology, 1974-08, Vol.4 (8), p.553-560</ispartof><rights>Copyright © 1974 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2557-df092f392fc8fd0680d75d37bc6a11ba85da3665d420ae79dd32c5cae1db7d3c3</citedby><cites>FETCH-LOGICAL-c2557-df092f392fc8fd0680d75d37bc6a11ba85da3665d420ae79dd32c5cae1db7d3c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Feji.1830040807$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Feji.1830040807$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27923,27924,45573,45574</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/4153482$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Freedman, M. H.</creatorcontrib><creatorcontrib>Pincus, J. H.</creatorcontrib><creatorcontrib>Yeger, H.</creatorcontrib><creatorcontrib>McKenney, Judith A.</creatorcontrib><creatorcontrib>Mage, Rose G.</creatorcontrib><title>Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques</title><title>European journal of immunology</title><addtitle>Eur J Immunol</addtitle><description>Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.</description><subject>Animals</subject><subject>Antibodies, Bacterial - analysis</subject><subject>Antibodies, Bacterial - isolation & purification</subject><subject>Antigens, Bacterial</subject><subject>Chromatography, Affinity</subject><subject>Chromatography, Ion Exchange</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Genotype</subject><subject>Hydrogen-Ion Concentration</subject><subject>Immunization</subject><subject>Immunization, Secondary</subject><subject>Immunoglobulin Fragments - isolation & purification</subject><subject>Immunoglobulin Heavy Chains - isolation & purification</subject><subject>Immunoglobulins - analysis</subject><subject>Immunoglobulins - isolation & purification</subject><subject>Isoantigens</subject><subject>Isoelectric Focusing</subject><subject>Polysaccharides, Bacterial - immunology</subject><subject>Precipitin Tests</subject><subject>Rabbits</subject><subject>Streptococcus pneumoniae - immunology</subject><issn>0014-2980</issn><issn>1521-4141</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1974</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks1u1TAUhA0ClUthy66SV-wSbCfOzxJVBS6qBBKwjhz7uHHlxMF2QOmqj8Az8iQ4vZefrlhYtjTjb0Y6B6EXlOSUEPYKrk1Om4KQkjSkfoh2lDOalbSkj9COEFpmrG3IE_Q0hGtCSFvx9gSdlJQXZcN2D84-xUUZCNhpPKwzeDOOywTYQ4jeyAgKi0nhWfhohLXrfSGan7c_5gmW0UknpbB4dnYNQspBeKPgztK7uwDt3YgTwsUUk74p0GZKlBmUufKQXl70vYkhx_scf_SQMkU03wBb83UxCpvgwILc0rF2cglmutpqx-FezlZ3ixepozc3CeGm37aEsGKrrjc5CQH3Kz5Q3Tw4DzHBI8hhSpkQnqHHWtgAz4_3Kfry5uLz-bvs8sPb_fnry0wyzutMadIyXaQjG61I1RBVc1XUvawEpb1ouBJFVXFVMiKgbpUqmORSAFV9rQpZnKKXB-7s3ZYbu9EECdaKCdwSuoZxlgbcJmN-MErvQvCguzlNTPi1o6Tb9qFL-9D93Yf04exIXvoR1B_7cQGS3h7078bC-h9ad_F-_w_7F0W1zns</recordid><startdate>197408</startdate><enddate>197408</enddate><creator>Freedman, M. H.</creator><creator>Pincus, J. H.</creator><creator>Yeger, H.</creator><creator>McKenney, Judith A.</creator><creator>Mage, Rose G.</creator><general>WILEY‐VCH Verlag GmbH</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></search><sort><creationdate>197408</creationdate><title>Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques</title><author>Freedman, M. H. ; Pincus, J. H. ; Yeger, H. ; McKenney, Judith A. ; Mage, Rose G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2557-df092f392fc8fd0680d75d37bc6a11ba85da3665d420ae79dd32c5cae1db7d3c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1974</creationdate><topic>Animals</topic><topic>Antibodies, Bacterial - analysis</topic><topic>Antibodies, Bacterial - isolation & purification</topic><topic>Antigens, Bacterial</topic><topic>Chromatography, Affinity</topic><topic>Chromatography, Ion Exchange</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Genotype</topic><topic>Hydrogen-Ion Concentration</topic><topic>Immunization</topic><topic>Immunization, Secondary</topic><topic>Immunoglobulin Fragments - isolation & purification</topic><topic>Immunoglobulin Heavy Chains - isolation & purification</topic><topic>Immunoglobulins - analysis</topic><topic>Immunoglobulins - isolation & purification</topic><topic>Isoantigens</topic><topic>Isoelectric Focusing</topic><topic>Polysaccharides, Bacterial - immunology</topic><topic>Precipitin Tests</topic><topic>Rabbits</topic><topic>Streptococcus pneumoniae - immunology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Freedman, M. H.</creatorcontrib><creatorcontrib>Pincus, J. H.</creatorcontrib><creatorcontrib>Yeger, H.</creatorcontrib><creatorcontrib>McKenney, Judith A.</creatorcontrib><creatorcontrib>Mage, Rose 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><jtitle>European journal of immunology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Freedman, M. H.</au><au>Pincus, J. H.</au><au>Yeger, H.</au><au>McKenney, Judith A.</au><au>Mage, Rose G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques</atitle><jtitle>European journal of immunology</jtitle><addtitle>Eur J Immunol</addtitle><date>1974-08</date><risdate>1974</risdate><volume>4</volume><issue>8</issue><spage>553</spage><epage>560</epage><pages>553-560</pages><issn>0014-2980</issn><eissn>1521-4141</eissn><abstract>Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.</abstract><cop>Weinheim</cop><pub>WILEY‐VCH Verlag GmbH</pub><pmid>4153482</pmid><doi>10.1002/eji.1830040807</doi><tpages>8</tpages></addata></record> |
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| subjects | Animals Antibodies, Bacterial - analysis Antibodies, Bacterial - isolation & purification Antigens, Bacterial Chromatography, Affinity Chromatography, Ion Exchange Electrophoresis, Polyacrylamide Gel Genotype Hydrogen-Ion Concentration Immunization Immunization, Secondary Immunoglobulin Fragments - isolation & purification Immunoglobulin Heavy Chains - isolation & purification Immunoglobulins - analysis Immunoglobulins - isolation & purification Isoantigens Isoelectric Focusing Polysaccharides, Bacterial - immunology Precipitin Tests Rabbits Streptococcus pneumoniae - immunology |
| title | Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques |
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