In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen
Identifieur interne : 001D92 ( Istex/Corpus ); précédent : 001D91; suivant : 001D93In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen
Auteurs : Laszlo Otvos Jr. ; Anne Marie Pease ; Krisztina Bokonyi ; Wynetta Giles-Davis ; Mark E. Rogers ; Paul A. Hintz ; Ralf Hoffmann ; Hildegund C. J ErtlSource :
- Journal of Immunological Methods [ 0022-1759 ] ; 2000.
English descriptors
- Teeft :
- Accessory cells, Amino, Amino acid concentration, Amino acid derivatives, Amino acid solutions, Amino acids, Antigenic potency, Assay, Assay medium, Assay plate, Binding peptide mixtures, Carrier cellulose membrane, Cellulose membrane, Cellulose membranes, Contact sites, Degradation rate, Dependent epitopes, Deprotection, Deprotection steps, Different batches, Eagles medium, Elsevier science, Entire length, Epitope, Error bars, Ertl, Febs lett, Final volume, Helper cell activity, Helper cell epitopes, Helper cell hybridoma, Helper cells, High performance, Human serum, Hxthymidine incorporation, Hybridoma, Immunodominant epitope, Immunological, Immunological methods, Immunosorbent assay, Lowest amounts, Lymphokine release, Major histocompatibility, Mass spectra, Mass spectrometry, Membrane, Negative control, Negative control peptide, Next step, Nitrocellulose immunoblots, Nmole amounts, Nucleoprotein, Open triangles, Otvos, Paper dots, Peptide, Peptide amount, Peptide array, Peptide dots, Peptide fragments, Peptide libraries, Peptide spot, Peptide spots, Peptide synthesis, Rabies, Rabies virus, Rabies virus nucleoprotein, Same medium, Simultaneous synthesis, Solid carrier, Solid line, Solid phase, Solution controls, Solution techniques, Splenocyte culture, Splenocytes, Spot size, Standard assay, Standard deviation, Stimulation assay, Stimulation assays, Stimulatory activity, Synthetic peptides, Target peptides, Test samples, Trifluoroacetic acid, Triplicate assay data, True reaction, Tryptic fragments.
Abstract
Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.
Url:
DOI: 10.1016/S0022-1759(99)00194-5
Links to Exploration step
ISTEX:C7833F77814D3441F6B65AF955DC4908B86719AFLe document en format XML
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Rogers</name><affiliation><mods:affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</mods:affiliation></affiliation></author><author><name sortKey="Hintz, Paul A" sort="Hintz, Paul A" uniqKey="Hintz P" first="Paul A" last="Hintz">Paul A. Hintz</name><affiliation><mods:affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</mods:affiliation></affiliation></author><author><name sortKey="Hoffmann, Ralf" sort="Hoffmann, Ralf" uniqKey="Hoffmann R" first="Ralf" last="Hoffmann">Ralf Hoffmann</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Ertl, Hildegund C J" sort="Ertl, Hildegund C J" uniqKey="Ertl H" first="Hildegund C. J" last="Ertl">Hildegund C. J Ertl</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:C7833F77814D3441F6B65AF955DC4908B86719AF</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0022-1759(99)00194-5</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001D92</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001D92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title><author><name sortKey="Otvos Jr, Laszlo" sort="Otvos Jr, Laszlo" uniqKey="Otvos Jr L" first="Laszlo" last="Otvos Jr.">Laszlo Otvos Jr.</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Pease, Anne Marie" sort="Pease, Anne Marie" uniqKey="Pease A" first="Anne Marie" last="Pease">Anne Marie Pease</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Bokonyi, Krisztina" sort="Bokonyi, Krisztina" uniqKey="Bokonyi K" first="Krisztina" last="Bokonyi">Krisztina Bokonyi</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Giles Davis, Wynetta" sort="Giles Davis, Wynetta" uniqKey="Giles Davis W" first="Wynetta" last="Giles-Davis">Wynetta Giles-Davis</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Rogers, Mark E" sort="Rogers, Mark E" uniqKey="Rogers M" first="Mark E" last="Rogers">Mark E. Rogers</name><affiliation><mods:affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</mods:affiliation></affiliation></author><author><name sortKey="Hintz, Paul A" sort="Hintz, Paul A" uniqKey="Hintz P" first="Paul A" last="Hintz">Paul A. Hintz</name><affiliation><mods:affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</mods:affiliation></affiliation></author><author><name sortKey="Hoffmann, Ralf" sort="Hoffmann, Ralf" uniqKey="Hoffmann R" first="Ralf" last="Hoffmann">Ralf Hoffmann</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author><author><name sortKey="Ertl, Hildegund C J" sort="Ertl, Hildegund C J" uniqKey="Ertl H" first="Hildegund C. J" last="Ertl">Hildegund C. J Ertl</name><affiliation><mods:affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Immunological Methods</title><title level="j" type="abbrev">JIM</title><idno type="ISSN">0022-1759</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">233</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="95">95</biblScope><biblScope unit="page" to="105">105</biblScope></imprint><idno type="ISSN">0022-1759</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-1759</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Accessory cells</term><term>Amino</term><term>Amino acid concentration</term><term>Amino acid derivatives</term><term>Amino acid solutions</term><term>Amino acids</term><term>Antigenic potency</term><term>Assay</term><term>Assay medium</term><term>Assay plate</term><term>Binding peptide mixtures</term><term>Carrier cellulose membrane</term><term>Cellulose membrane</term><term>Cellulose membranes</term><term>Contact sites</term><term>Degradation rate</term><term>Dependent epitopes</term><term>Deprotection</term><term>Deprotection steps</term><term>Different batches</term><term>Eagles medium</term><term>Elsevier science</term><term>Entire length</term><term>Epitope</term><term>Error bars</term><term>Ertl</term><term>Febs lett</term><term>Final volume</term><term>Helper cell activity</term><term>Helper cell epitopes</term><term>Helper cell hybridoma</term><term>Helper cells</term><term>High performance</term><term>Human serum</term><term>Hxthymidine incorporation</term><term>Hybridoma</term><term>Immunodominant epitope</term><term>Immunological</term><term>Immunological methods</term><term>Immunosorbent assay</term><term>Lowest amounts</term><term>Lymphokine release</term><term>Major histocompatibility</term><term>Mass spectra</term><term>Mass spectrometry</term><term>Membrane</term><term>Negative control</term><term>Negative control peptide</term><term>Next step</term><term>Nitrocellulose immunoblots</term><term>Nmole amounts</term><term>Nucleoprotein</term><term>Open triangles</term><term>Otvos</term><term>Paper dots</term><term>Peptide</term><term>Peptide amount</term><term>Peptide array</term><term>Peptide dots</term><term>Peptide fragments</term><term>Peptide libraries</term><term>Peptide spot</term><term>Peptide spots</term><term>Peptide synthesis</term><term>Rabies</term><term>Rabies virus</term><term>Rabies virus nucleoprotein</term><term>Same medium</term><term>Simultaneous synthesis</term><term>Solid carrier</term><term>Solid line</term><term>Solid phase</term><term>Solution controls</term><term>Solution techniques</term><term>Splenocyte culture</term><term>Splenocytes</term><term>Spot size</term><term>Standard assay</term><term>Standard deviation</term><term>Stimulation assay</term><term>Stimulation assays</term><term>Stimulatory activity</term><term>Synthetic peptides</term><term>Target peptides</term><term>Test samples</term><term>Trifluoroacetic acid</term><term>Triplicate assay data</term><term>True reaction</term><term>Tryptic fragments</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>peptide</json:string><json:string>epitope</json:string><json:string>assay</json:string><json:string>hybridoma</json:string><json:string>otvos</json:string><json:string>immunological</json:string><json:string>ertl</json:string><json:string>immunological methods</json:string><json:string>peptide spot</json:string><json:string>cellulose membrane</json:string><json:string>amino</json:string><json:string>splenocytes</json:string><json:string>peptide spots</json:string><json:string>nucleoprotein</json:string><json:string>deprotection</json:string><json:string>synthetic peptides</json:string><json:string>stimulation assay</json:string><json:string>amino acids</json:string><json:string>peptide synthesis</json:string><json:string>rabies</json:string><json:string>mass 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steps</json:string></teeft></keywords><author><json:item><name>Laszlo Otvos, Jr.</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item><json:item><name>Anne Marie Pease</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item><json:item><name>Krisztina Bokonyi</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item><json:item><name>Wynetta Giles-Davis</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item><json:item><name>Mark E Rogers</name><affiliations><json:string>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</json:string></affiliations></json:item><json:item><name>Paul A Hintz</name><affiliations><json:string>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</json:string></affiliations></json:item><json:item><name>Ralf Hoffmann</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item><json:item><name>Hildegund C.J Ertl</name><affiliations><json:string>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Antigen processing</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Membrane</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Mass spectrometry</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Multiple synthesis</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Solid phase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Stimulation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>IL, interleukin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MHC, major histocompatibility complex</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>NMP, N-methyl-pyrrolidone</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DMF, dimethyl formamide</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TFA, trifluoroacetic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RP-HPLC, reversed-phase high performance liquid chromatography</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ERA, Evelyn–Rokitnicki–Abelseth</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DMEM, Dulbecco's modified Eagles medium</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FBS, fetal bovine serum</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>LC-MS, liquid chromatography-coupled mass spectroscopy</value></json:item></subject><arkIstex>ark:/67375/6H6-RFM3NK18-S</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.</abstract><qualityIndicators><refBibsNative>true</refBibsNative><abstractWordCount>208</abstractWordCount><abstractCharCount>1442</abstractCharCount><keywordCount>16</keywordCount><score>9.496</score><pdfWordCount>5997</pdfWordCount><pdfCharCount>37010</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>540 x 712 pts</pdfPageSize></qualityIndicators><title>In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title><pmid><json:string>10648860</json:string></pmid><pii><json:string>S0022-1759(99)00194-5</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Immunological Methods</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0022-1759</json:string></issn><pii><json:string>S0022-1759(00)X0089-0</json:string></pii><volume>233</volume><issue>1–2</issue><pages><first>95</first><last>105</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string></date><geogName></geogName><orgName><json:string>NIH grant GM45011</json:string></orgName><orgName_funder><json:string>NIH grant GM45011</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Ellen Heber-Katz</json:string><json:string>L. OtÕos</json:string></persName><placeName><json:string>Dusseldorf</json:string><json:string>Germany</json:string><json:string>Lipka</json:string><json:string>Chester</json:string><json:string>PA</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Otvos et al., 1996</json:string><json:string>Rammensee et al., 1993</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-RFM3NK18-S</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - immunology</json:string><json:string>2 - biochemical research methods</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - clinical medicine</json:string><json:string>3 - immunology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Immunology</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Immunology and Allergy</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - immunologie fondamentale</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0022-1759(99)00194-5</json:string></doi><id>C7833F77814D3441F6B65AF955DC4908B86719AF</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2000 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2000</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Fig. 1: Response of T-cells to cellulose-bound synthetic peptides and to solution controls. The solid-phase bound peptides covered the entire length of the rabies virus nucleoprotein sequence. The paper dots, containing approximately 200 nmole (roughly 0.4 mg) peptides were folded, and placed into the wells of the assay plate. For clarity, the label for the X-axis lists only the first and every sixth following antigens. The complete list of the antigens is as follows (left to right; those antigens that are also labeled in the figure are printed in bold letters): medium only; solution controls: 5 μg Evelyn–Rokitnicki–Abelseth (ERA) virus; 1 μg ERA virus; 5 μg peptide 31D, 1 μg peptide 31D; 0.2 μg peptide 31D. Peptide dots (amino acid numbering in parentheses): 1D (1–15); 2D (11–25); 3D (21–35); 4D (31–45); 5D (41–55); 6D (51–65); 7D (61–75); 8D (71–85); 9D (81–95); 10D (91–105); 11D (101–115) 12D (111–125); 13D (121–135); 14D (131–145); 15D (141–155); 16D (151–165); 17D (161–175); 18D (171–185); 19D (181–195); 20D (191–205); 21D (201–215); 22D (211–225); 23D (221–235); 24D (231–245); 25D (241–255); 26D (251–265); 27D (261–275); V12b1 (269–283); V12b2 (279–293); V12b3 (288–303); V12b4 (299–303); V12b (313–338); 28D (343–358); 39D (351–368); V10c (369–383); 30D (394–408); 31D (404–418); 32D (414–428); 33D (424–438); 34D (434–450). The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 2: Determination of the lowest amount of cellulose-bound peptide 31D to elicit a measurable T-cell response. The spots with the highest amounts of peptides (0.4 mg) were synthesized identically to those that were used to generate Fig. 1. The spots containing decreasing amounts of peptides were made by serially diluting the activated amino acid derivatives. All spots remained approximately 7–8 mm in diameter. The uncoupled amino groups on the membrane were endcapped by acetylation (the capacity of the membrane was in the vicinity of the amino acid load of the spots containing the highest amount of peptides). The solid line (closed circles) represents the spots containing peptide 31D. As a negative control for the 9C5.D8.H T-cell hybridoma we used peptide 3D, another major immunodominant epitope of the rabies virus nucleoprotein (broken line and open triangles). The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 3: Dose–response relationships of the T-cell stimulation with peptide 31D applied in solution. The total peptide amounts in the vials are provided for direct comparison with the cellulose-bound peptide amounts needed to elicit comparable T-cell responses (Fig. 2). The solid line (closed circles) represents the test peptide 31D; the broken line (open triangles) represents the negative control peptide 3D. The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 4: High performance liquid chromatogram of peptide 31D (A), the splenocyte–medium mixture only (B) and the same medium with a 31D peptide spot incubated for 1 h (C). Spectra B and C appear to be very similar. Although peptide 31D is eluted near a peak from the splenocyte–medium mixture, mass spectra recorded from chromatogram C failed to identify peptide 31D or its fragments and indicated that the chromatographic differences between B and C were not due to the release of any anticipated peptide.</note><note type="content">Table 1: T-cell stimulation of peptide 31D spots and appropriate controls The data represent the means of triplicate wells. Condition “a”: a 24-h splenocyte culture containing a peptide 31D spot together with the 9C5.D8.H hybridoma (standard solid-phase assay). Condition “b”: the splenocytes were cultured with a 31D spot for 24 h, the membrane was removed and the 9C5.D8.H hybridoma was added to the well. Condition “c”: the splenocytes were cultured alone for 24 h; the 31D spot, which had been removed from condition “b”, was added together with the 9C5.D8.H hybridoma.</note><note type="content">Table 2: Tryptic fragments of peptide 31D detected by electrospray mass spectrometry after 6 h digestion of a peptide spot As expected from an efficient trypsin digestion, only those peptide fragments could be detected which did not contain any additional trypsin site (no partial digestion products were detected; n.d.)</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title><author xml:id="author-0000"><persName><forename type="first">Laszlo</forename><surname>Otvos, Jr.</surname></persName><note type="correspondence"><p>Corresponding author. Tel.: +1-215-898-3772; fax: +1-215-898-5821; e-mail: otvos@wistar.upenn.edu</p></note><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Anne Marie</forename><surname>Pease</surname></persName><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Krisztina</forename><surname>Bokonyi</surname></persName><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Wynetta</forename><surname>Giles-Davis</surname></persName><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Mark E</forename><surname>Rogers</surname></persName><affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Paul A</forename><surname>Hintz</surname></persName><affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Ralf</forename><surname>Hoffmann</surname></persName><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Hildegund C.J</forename><surname>Ertl</surname></persName><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation></author><idno type="istex">C7833F77814D3441F6B65AF955DC4908B86719AF</idno><idno type="ark">ark:/67375/6H6-RFM3NK18-S</idno><idno type="DOI">10.1016/S0022-1759(99)00194-5</idno><idno type="PII">S0022-1759(99)00194-5</idno></analytic><monogr><title level="j">Journal of Immunological Methods</title><title level="j" type="abbrev">JIM</title><idno type="pISSN">0022-1759</idno><idno type="PII">S0022-1759(00)X0089-0</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">233</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="95">95</biblScope><biblScope unit="page" to="105">105</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Antigen processing</term></item><item><term>Membrane</term></item><item><term>Mass spectrometry</term></item><item><term>Multiple synthesis</term></item><item><term>Solid phase</term></item><item><term>Stimulation</term></item></list></keywords></textClass><textClass><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>IL, interleukin</term></item><item><term>MHC, major histocompatibility complex</term></item><item><term>NMP, N-methyl-pyrrolidone</term></item><item><term>DMF, dimethyl formamide</term></item><item><term>TFA, trifluoroacetic acid</term></item><item><term>RP-HPLC, reversed-phase high performance liquid chromatography</term></item><item><term>ERA, Evelyn–Rokitnicki–Abelseth</term></item><item><term>DMEM, Dulbecco's modified Eagles medium</term></item><item><term>FBS, fetal bovine serum</term></item><item><term>LC-MS, liquid chromatography-coupled mass spectroscopy</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999-08-16">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>JIM</jid><aid>8491</aid><ce:pii>S0022-1759(99)00194-5</ce:pii><ce:doi>10.1016/S0022-1759(99)00194-5</ce:doi><ce:copyright year="2000" type="full-transfer">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</ce:title><ce:author-group><ce:author><ce:given-name>Laszlo</ce:given-name><ce:surname>Otvos</ce:surname><ce:suffix>Jr.</ce:suffix><ce:cross-ref refid="AFF1">a</ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>Anne Marie</ce:given-name><ce:surname>Pease</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:given-name>Krisztina</ce:given-name><ce:surname>Bokonyi</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:given-name>Wynetta</ce:given-name><ce:surname>Giles-Davis</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:given-name>Mark E</ce:given-name><ce:surname>Rogers</ce:surname><ce:cross-ref refid="AFF2">b</ce:cross-ref></ce:author><ce:author><ce:given-name>Paul A</ce:given-name><ce:surname>Hintz</ce:surname><ce:cross-ref refid="AFF2">b</ce:cross-ref></ce:author><ce:author><ce:given-name>Ralf</ce:given-name><ce:surname>Hoffmann</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref><ce:cross-ref refid="AFF3">c</ce:cross-ref></ce:author><ce:author><ce:given-name>Hildegund C.J</ce:given-name><ce:surname>Ertl</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>BMFZ, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +1-215-898-3772; fax: +1-215-898-5821; e-mail: otvos@wistar.upenn.edu</ce:text></ce:correspondence></ce:author-group><ce:date-received day="23" month="3" year="1999"></ce:date-received><ce:date-revised day="16" month="8" year="1999"></ce:date-revised><ce:date-accepted day="4" month="10" year="1999"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Antigen processing</ce:text></ce:keyword><ce:keyword><ce:text>Membrane</ce:text></ce:keyword><ce:keyword><ce:text>Mass spectrometry</ce:text></ce:keyword><ce:keyword><ce:text>Multiple synthesis</ce:text></ce:keyword><ce:keyword><ce:text>Solid phase</ce:text></ce:keyword><ce:keyword><ce:text>Stimulation</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>IL, interleukin</ce:text></ce:keyword><ce:keyword><ce:text>MHC, major histocompatibility complex</ce:text></ce:keyword><ce:keyword><ce:text>NMP, <ce:italic>N</ce:italic>-methyl-pyrrolidone</ce:text></ce:keyword><ce:keyword><ce:text>DMF, dimethyl formamide</ce:text></ce:keyword><ce:keyword><ce:text>TFA, trifluoroacetic acid</ce:text></ce:keyword><ce:keyword><ce:text>RP-HPLC, reversed-phase high performance liquid chromatography</ce:text></ce:keyword><ce:keyword><ce:text>ERA, Evelyn–Rokitnicki–Abelseth</ce:text></ce:keyword><ce:keyword><ce:text>DMEM, Dulbecco's modified Eagles medium</ce:text></ce:keyword><ce:keyword><ce:text>FBS, fetal bovine serum</ce:text></ce:keyword><ce:keyword><ce:text>LC-MS, liquid chromatography-coupled mass spectroscopy</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen</title></titleInfo><name type="personal"><namePart type="given">Laszlo</namePart><namePart type="family">Otvos, Jr.</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><description>Corresponding author. Tel.: +1-215-898-3772; fax: +1-215-898-5821; e-mail: otvos@wistar.upenn.edu</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Anne Marie</namePart><namePart type="family">Pease</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Krisztina</namePart><namePart type="family">Bokonyi</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Wynetta</namePart><namePart type="family">Giles-Davis</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mark E</namePart><namePart type="family">Rogers</namePart><affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Paul A</namePart><namePart type="family">Hintz</namePart><affiliation>M-Scan, 606 Brandywine Parkway, West Chester, PA 19380, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Ralf</namePart><namePart type="family">Hoffmann</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hildegund C.J</namePart><namePart type="family">Ertl</namePart><affiliation>The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">1999-08-16</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.</abstract><note type="content">Fig. 1: Response of T-cells to cellulose-bound synthetic peptides and to solution controls. The solid-phase bound peptides covered the entire length of the rabies virus nucleoprotein sequence. The paper dots, containing approximately 200 nmole (roughly 0.4 mg) peptides were folded, and placed into the wells of the assay plate. For clarity, the label for the X-axis lists only the first and every sixth following antigens. The complete list of the antigens is as follows (left to right; those antigens that are also labeled in the figure are printed in bold letters): medium only; solution controls: 5 μg Evelyn–Rokitnicki–Abelseth (ERA) virus; 1 μg ERA virus; 5 μg peptide 31D, 1 μg peptide 31D; 0.2 μg peptide 31D. Peptide dots (amino acid numbering in parentheses): 1D (1–15); 2D (11–25); 3D (21–35); 4D (31–45); 5D (41–55); 6D (51–65); 7D (61–75); 8D (71–85); 9D (81–95); 10D (91–105); 11D (101–115) 12D (111–125); 13D (121–135); 14D (131–145); 15D (141–155); 16D (151–165); 17D (161–175); 18D (171–185); 19D (181–195); 20D (191–205); 21D (201–215); 22D (211–225); 23D (221–235); 24D (231–245); 25D (241–255); 26D (251–265); 27D (261–275); V12b1 (269–283); V12b2 (279–293); V12b3 (288–303); V12b4 (299–303); V12b (313–338); 28D (343–358); 39D (351–368); V10c (369–383); 30D (394–408); 31D (404–418); 32D (414–428); 33D (424–438); 34D (434–450). The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 2: Determination of the lowest amount of cellulose-bound peptide 31D to elicit a measurable T-cell response. The spots with the highest amounts of peptides (0.4 mg) were synthesized identically to those that were used to generate Fig. 1. The spots containing decreasing amounts of peptides were made by serially diluting the activated amino acid derivatives. All spots remained approximately 7–8 mm in diameter. The uncoupled amino groups on the membrane were endcapped by acetylation (the capacity of the membrane was in the vicinity of the amino acid load of the spots containing the highest amount of peptides). The solid line (closed circles) represents the spots containing peptide 31D. As a negative control for the 9C5.D8.H T-cell hybridoma we used peptide 3D, another major immunodominant epitope of the rabies virus nucleoprotein (broken line and open triangles). The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 3: Dose–response relationships of the T-cell stimulation with peptide 31D applied in solution. The total peptide amounts in the vials are provided for direct comparison with the cellulose-bound peptide amounts needed to elicit comparable T-cell responses (Fig. 2). The solid line (closed circles) represents the test peptide 31D; the broken line (open triangles) represents the negative control peptide 3D. The error bars indicate the standard deviation of triplicate assay data.</note><note type="content">Fig. 4: High performance liquid chromatogram of peptide 31D (A), the splenocyte–medium mixture only (B) and the same medium with a 31D peptide spot incubated for 1 h (C). Spectra B and C appear to be very similar. Although peptide 31D is eluted near a peak from the splenocyte–medium mixture, mass spectra recorded from chromatogram C failed to identify peptide 31D or its fragments and indicated that the chromatographic differences between B and C were not due to the release of any anticipated peptide.</note><note type="content">Table 1: T-cell stimulation of peptide 31D spots and appropriate controls The data represent the means of triplicate wells. Condition “a”: a 24-h splenocyte culture containing a peptide 31D spot together with the 9C5.D8.H hybridoma (standard solid-phase assay). Condition “b”: the splenocytes were cultured with a 31D spot for 24 h, the membrane was removed and the 9C5.D8.H hybridoma was added to the well. Condition “c”: the splenocytes were cultured alone for 24 h; the 31D spot, which had been removed from condition “b”, was added together with the 9C5.D8.H hybridoma.</note><note type="content">Table 2: Tryptic fragments of peptide 31D detected by electrospray mass spectrometry after 6 h digestion of a peptide spot As expected from an efficient trypsin digestion, only those peptide fragments could be detected which did not contain any additional trypsin site (no partial digestion products were detected; n.d.)</note><subject><genre>Keywords</genre><topic>Antigen processing</topic><topic>Membrane</topic><topic>Mass spectrometry</topic><topic>Multiple synthesis</topic><topic>Solid phase</topic><topic>Stimulation</topic></subject><subject><genre>Abbreviations</genre><topic>IL, interleukin</topic><topic>MHC, major histocompatibility complex</topic><topic>NMP, N-methyl-pyrrolidone</topic><topic>DMF, dimethyl formamide</topic><topic>TFA, trifluoroacetic acid</topic><topic>RP-HPLC, reversed-phase high performance liquid chromatography</topic><topic>ERA, Evelyn–Rokitnicki–Abelseth</topic><topic>DMEM, Dulbecco's modified Eagles medium</topic><topic>FBS, fetal bovine serum</topic><topic>LC-MS, liquid chromatography-coupled mass spectroscopy</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Immunological Methods</title></titleInfo><titleInfo type="abbreviated"><title>JIM</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0022-1759</identifier><identifier type="PII">S0022-1759(00)X0089-0</identifier><part><date>2000</date><detail type="volume"><number>233</number><caption>vol.</caption></detail><detail type="issue"><number>1–2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>194</end></extent><extent unit="pages"><start>95</start><end>105</end></extent></part></relatedItem><identifier type="istex">C7833F77814D3441F6B65AF955DC4908B86719AF</identifier><identifier type="ark">ark:/67375/6H6-RFM3NK18-S</identifier><identifier type="DOI">10.1016/S0022-1759(99)00194-5</identifier><identifier type="PII">S0022-1759(99)00194-5</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©2000</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RFM3NK18-S/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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