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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Preparation and characterization of SARS in-house reference antiserum</title><author><name sortKey="Zhang, Jiansan" sort="Zhang, Jiansan" uniqKey="Zhang J" first="Jiansan" last="Zhang">Jiansan Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Liu, Yuxuan" sort="Liu, Yuxuan" uniqKey="Liu Y" first="Yuxuan" last="Liu">Yuxuan Liu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Hu, Liangxiang" sort="Hu, Liangxiang" uniqKey="Hu L" first="Liangxiang" last="Hu">Liangxiang Hu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gao, Qiang" sort="Gao, Qiang" uniqKey="Gao Q" first="Qiang" last="Gao">Qiang Gao</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Zhenshan" sort="Zhang, Zhenshan" uniqKey="Zhang Z" first="Zhenshan" last="Zhang">Zhenshan Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Xiaomei" sort="Zhang, Xiaomei" uniqKey="Zhang X" first="Xiaomei" last="Zhang">Xiaomei Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Chen, Jiangting" sort="Chen, Jiangting" uniqKey="Chen J" first="Jiangting" last="Chen">Jiangting Chen</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gong, Xuejie" sort="Gong, Xuejie" uniqKey="Gong X" first="Xuejie" last="Gong">Xuejie Gong</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Song, Lifei" sort="Song, Lifei" uniqKey="Song L" first="Lifei" last="Song">Lifei Song</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Liu, Yufen" sort="Liu, Yufen" uniqKey="Liu Y" first="Yufen" last="Liu">Yufen Liu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Li, Jing" sort="Li, Jing" uniqKey="Li J" first="Jing" last="Li">Jing Li</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Li, Shufen" sort="Li, Shufen" uniqKey="Li S" first="Shufen" last="Li">Shufen Li</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Huang, Jinfeng" sort="Huang, Jinfeng" uniqKey="Huang J" first="Jinfeng" last="Huang">Jinfeng Huang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Ning, Ye" sort="Ning, Ye" uniqKey="Ning Y" first="Ye" last="Ning">Ye Ning</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gao, Hong" sort="Gao, Hong" uniqKey="Gao H" first="Hong" last="Gao">Hong Gao</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="aff2">The Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, PR China</nlm:aff></affiliation></author><author><name sortKey="Dong, Xiaoping" sort="Dong, Xiaoping" uniqKey="Dong X" first="Xiaoping" last="Dong">Xiaoping Dong</name><affiliation><nlm:aff id="aff3">Institute for Virology, Chinese Center for Disease Control and Prevention, Beijing 100052, PR China</nlm:aff></affiliation></author><author><name sortKey="Wei, Jianguo" sort="Wei, Jianguo" uniqKey="Wei J" first="Jianguo" last="Wei">Jianguo Wei</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Dong, Guanmu" sort="Dong, Guanmu" uniqKey="Dong G" first="Guanmu" last="Dong">Guanmu Dong</name><affiliation><nlm:aff id="aff4">National Institute for the Control of Pharmaceutical and Biological Products, Tiantan Xili 2, Beijing 100050, PR China</nlm:aff></affiliation></author><author><name sortKey="Yin, Weidong" sort="Yin, Weidong" uniqKey="Yin W" first="Weidong" last="Yin">Weidong Yin</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">16269206</idno><idno type="pmc">7115399</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115399</idno><idno type="RBID">PMC:7115399</idno><idno type="doi">10.1016/j.vaccine.2004.12.032</idno><date when="2005">2005</date><idno type="wicri:Area/Pmc/Corpus">001527</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001527</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Preparation and characterization of SARS in-house reference antiserum</title><author><name sortKey="Zhang, Jiansan" sort="Zhang, Jiansan" uniqKey="Zhang J" first="Jiansan" last="Zhang">Jiansan Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Liu, Yuxuan" sort="Liu, Yuxuan" uniqKey="Liu Y" first="Yuxuan" last="Liu">Yuxuan Liu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Hu, Liangxiang" sort="Hu, Liangxiang" uniqKey="Hu L" first="Liangxiang" last="Hu">Liangxiang Hu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gao, Qiang" sort="Gao, Qiang" uniqKey="Gao Q" first="Qiang" last="Gao">Qiang Gao</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Zhenshan" sort="Zhang, Zhenshan" uniqKey="Zhang Z" first="Zhenshan" last="Zhang">Zhenshan Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Xiaomei" sort="Zhang, Xiaomei" uniqKey="Zhang X" first="Xiaomei" last="Zhang">Xiaomei Zhang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Chen, Jiangting" sort="Chen, Jiangting" uniqKey="Chen J" first="Jiangting" last="Chen">Jiangting Chen</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gong, Xuejie" sort="Gong, Xuejie" uniqKey="Gong X" first="Xuejie" last="Gong">Xuejie Gong</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Song, Lifei" sort="Song, Lifei" uniqKey="Song L" first="Lifei" last="Song">Lifei Song</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Liu, Yufen" sort="Liu, Yufen" uniqKey="Liu Y" first="Yufen" last="Liu">Yufen Liu</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Li, Jing" sort="Li, Jing" uniqKey="Li J" first="Jing" last="Li">Jing Li</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Li, Shufen" sort="Li, Shufen" uniqKey="Li S" first="Shufen" last="Li">Shufen Li</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Huang, Jinfeng" sort="Huang, Jinfeng" uniqKey="Huang J" first="Jinfeng" last="Huang">Jinfeng Huang</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Ning, Ye" sort="Ning, Ye" uniqKey="Ning Y" first="Ye" last="Ning">Ye Ning</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Gao, Hong" sort="Gao, Hong" uniqKey="Gao H" first="Hong" last="Gao">Hong Gao</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="aff2">The Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, PR China</nlm:aff></affiliation></author><author><name sortKey="Dong, Xiaoping" sort="Dong, Xiaoping" uniqKey="Dong X" first="Xiaoping" last="Dong">Xiaoping Dong</name><affiliation><nlm:aff id="aff3">Institute for Virology, Chinese Center for Disease Control and Prevention, Beijing 100052, PR China</nlm:aff></affiliation></author><author><name sortKey="Wei, Jianguo" sort="Wei, Jianguo" uniqKey="Wei J" first="Jianguo" last="Wei">Jianguo Wei</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author><author><name sortKey="Dong, Guanmu" sort="Dong, Guanmu" uniqKey="Dong G" first="Guanmu" last="Dong">Guanmu Dong</name><affiliation><nlm:aff id="aff4">National Institute for the Control of Pharmaceutical and Biological Products, Tiantan Xili 2, Beijing 100050, PR China</nlm:aff></affiliation></author><author><name sortKey="Yin, Weidong" sort="Yin, Weidong" uniqKey="Yin W" first="Weidong" last="Yin">Weidong Yin</name><affiliation><nlm:aff id="aff1">Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</nlm:aff></affiliation></author></analytic><series><title level="j">Vaccine</title><idno type="ISSN">0264-410X</idno><idno type="eISSN">1873-2518</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>A reference antiserum for SARS is in urgent need in the development of SARS vaccine and other serological test of SARS research. Convalescent serum was collected from clinical confirmed patient. ELISA, Western-blotting and neutralization assay detected specific antibody against SARS coronavirus. This antiserum was prepared as in-house reference antiserum, freeze-dried and sealed in ampoules. The potency of this reference antiserum is defined to be 52.7 U after extensive calibration. Further, collaborative studies for the evaluation of this serum are needed in order to satisfy the requirements for international reference antiserum.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="May, J C" uniqKey="May J">J.C. May</name></author><author><name sortKey="Grim, E" uniqKey="Grim E">E. Grim</name></author><author><name sortKey="Wheeler, R M" uniqKey="Wheeler R">R.M. Wheeler</name></author></analytic></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Vaccine</journal-id><journal-id journal-id-type="iso-abbrev">Vaccine</journal-id><journal-title-group><journal-title>Vaccine</journal-title></journal-title-group><issn pub-type="ppub">0264-410X</issn><issn pub-type="epub">1873-2518</issn><publisher><publisher-name>Elsevier Ltd.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16269206</article-id><article-id pub-id-type="pmc">7115399</article-id><article-id pub-id-type="publisher-id">S0264-410X(05)00301-4</article-id><article-id pub-id-type="doi">10.1016/j.vaccine.2004.12.032</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Preparation and characterization of SARS in-house reference antiserum</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Jiansan</given-names></name><email>ZhangJS@Sinovac.com</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Yuxuan</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Hu</surname><given-names>Liangxiang</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Qiang</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Zhenshan</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Xiaomei</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Jiangting</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Gong</surname><given-names>Xuejie</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Lifei</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Yufen</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Jing</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Shufen</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Jinfeng</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Ning</surname><given-names>Ye</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Hong</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Qin</surname><given-names>Chuan</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Dong</surname><given-names>Xiaoping</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Wei</surname><given-names>Jianguo</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Dong</surname><given-names>Guanmu</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Yin</surname><given-names>Weidong</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Sinovac Biotech Co. Ltd., No. 39, Shangdi Xi Road, Haidian District, Beijing 100085, PR China</aff><aff id="aff2"><label>b</label>The Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, PR China</aff><aff id="aff3"><label>c</label>Institute for Virology, Chinese Center for Disease Control and Prevention, Beijing 100052, PR China</aff><aff id="aff4"><label>d</label>National Institute for the Control of Pharmaceutical and Biological Products, Tiantan Xili 2, Beijing 100050, PR China</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Fax: +86 10 62966910. <email>ZhangJS@Sinovac.com</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>16</day><month>3</month><year>2005</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>1</day><month>12</month><year>2005</year></pub-date><pub-date pub-type="epub"><day>16</day><month>3</month><year>2005</year></pub-date><volume>23</volume><issue>48</issue><fpage>5666</fpage><lpage>5669</lpage><history><date date-type="received"><day>24</day><month>2</month><year>2004</year></date><date date-type="rev-recd"><day>24</day><month>9</month><year>2004</year></date><date date-type="accepted"><day>24</day><month>12</month><year>2004</year></date></history><permissions><copyright-statement>Copyright © 2005 Elsevier Ltd. All rights reserved.</copyright-statement><copyright-year>2005</copyright-year><copyright-holder>Elsevier Ltd</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>A reference antiserum for SARS is in urgent need in the development of SARS vaccine and other serological test of SARS research. Convalescent serum was collected from clinical confirmed patient. ELISA, Western-blotting and neutralization assay detected specific antibody against SARS coronavirus. This antiserum was prepared as in-house reference antiserum, freeze-dried and sealed in ampoules. The potency of this reference antiserum is defined to be 52.7 U after extensive calibration. Further, collaborative studies for the evaluation of this serum are needed in order to satisfy the requirements for international reference antiserum.</p></abstract><kwd-group><title>Keywords</title><kwd>SARS</kwd><kwd>Reference antiserum</kwd><kwd>Neutralization antibody potency</kwd></kwd-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>SARS is a severe acute respiratory infective disease. A safe and effective vaccine would be an important complement to the existing SARS control strategies <xref rid="bib1" ref-type="bibr">[1]</xref>. Neutralization antibodies against SARS virus in immunized animals are measured by potential serological assays. A well-characterized SARS reference antiserum is of great necessity to provide reliable standardized evaluation and quality control for the assay, and moreover to instruct the development of inactivated vaccine against the SARS virus.</p><p>The current report summarizes preparation, characterization and calibration of a SARS reference antiserum, according to the WHO biological reference requirements <xref rid="bib2" ref-type="bibr">[2]</xref>.</p></sec><sec><label>2</label><title>Materials and methods</title><sec><label>2.1</label><title>Viral strains and virus manipulation</title><p>Viral strains: SARS viral strains Sino1, Sino2, Sino3 and Sino6 were isolated from pharyngeal swabs of clinically confirmed SARS patients at Peking Union Hospital.</p><p>SARS virus was propagated on Vero cells in a culture flask cultivated at 37 °C until 75% cells showed cytopathic effect. After three freeze–thaw cycles, virus was harvested and well characterized. The batch of virus was stored at −80 °C as reference virus (manuscript in preparation) for neutralization assay.</p><p>After harvest, the SARS virus was inactivated and the total protein was purified by centrifugation, ultrafiltration and gel filtration. The gel filtration elutent was used for Western blot assay.</p></sec><sec><label>2.2</label><title>Cell line</title><p>Vero cells, a line of African green monkey kidney cells, were obtained from ATCC (American Type Culture Collection). Vero cells were grown at 37 °C in minimum essential medium (MEM), containing 10% (w/v) fetal calf serum and 1% (w/v) glutamine.</p></sec><sec><label>2.3</label><title>Serum</title><p>A panel of positive sera, collected from 20 patients in the convalescent stage of SARS, was heat inactivated at 56 °C for 30 min and stored at −20 °C. In order to verify the inactivation efficiency, Vero cells were inoculated with the inactivated serum and cultivated for three generations. No cytopathic effect was detected. In addition, a panel of serum samples was collected from 10 healthy people for negative control.</p></sec><sec><label>2.4</label><title>Titration of infective virus</title><p>Vero cells were cultivated in 96-well plates at 37 °C to form confluent monolayer. Cells were inoculated with 200 μl per well of viral suspension that was reconstituted and diluted serially in tenfold steps in MEM containing 2% new-born calf serum and 50 μg/ml penicillin (pH 7.2), with each dilution filling 8-well. The 96-well plates were incubated at 37 °C in 5% CO<sub>2</sub> incubator. Virus titre was expressed as cell culture infectious dose (CCID), which is the dilution of virus causing half of the cultured cells to produce CPE (cytopathic effect).</p></sec><sec><label>2.5</label><title>Neutralization assay <xref rid="bib3" ref-type="bibr">[3]</xref>, <xref rid="bib4" ref-type="bibr">[4]</xref></title><p>Heat inactivated sera (two-fold serial dilution from 1:2 to 1:2048, 100 μl for each dilution) were added to equal volume of virus (diluted to 100CCID<sub>50</sub>/μl) and incubated for 1h at 37 °C before being added to confluent monolayers of Vero cells in 96-well plates and incubated at 37 °C in 5% CO<sub>2</sub> incubator for 1–4 days. During the incubation period the cell cultures were checked for development of CPE.</p></sec><sec><label>2.6</label><title>Western blot assay</title><p>Proteins from SDS-PAGE gels were transferred onto nitrocellulose membrane. For immunostaining of the transferred proteins, the blocked membrane was incubated with convalescent serum. Anti-human antibody conjugated with HRP (horseradish peroxidase, Sigma) was used as the second antibody, and the color developing substrate was DAB.</p></sec><sec><label>2.7</label><title>ELISA</title><p>The convalescent serum of SARS was identified and confirmed by a commercially obtained enzyme-linked immunosorbent assay (ELISA) Kit (Beijing WanTai). The antigenic protein employed in the ELISA kit was part of the recombinant N protein of the SARS CoV (the polypeptide product of the third ORF of the N protein coding region) expressed in an <italic>Escherichia coli</italic> strain. The purified SARS virus protein was immobilized on a 96-well plate. The antiserum against SARS virus was added to the coated wells and incubated. HRP-labelled SARS antigen was then added to react with the bound antiserum. TMB was used for color development.</p></sec><sec><label>2.8</label><title>Preparation of reference antiserum</title><p>The serum STS-D-Zhang-05 was mixed thoroughly and filled into ampoules (0.5 ml/ampoule). The serum was freeze-dried and the ampoules were sealed.</p></sec><sec><label>2.9</label><title>Moisture content</title><p>The moisture content of freeze-dried reference antiserum was detected by Karl Fischer method described as previously <xref rid="bib5" ref-type="bibr">[5]</xref>. The moisture content of at least three ampoules was tested separately.</p></sec><sec><label>2.10</label><title>Stability test</title><p>The stability of the reference antiserum was tested by an accelerated degradation test using temperature at −20, 37 and 56 °C, for 7, 14, 21 and 28 days of storage, respectively. Potency was determined by neutralization assay. All samples were monitored against the −20 °C stored samples.</p></sec></sec><sec><label>3</label><title>Results</title><sec><label>3.1</label><title>Identification of SARS corona virus</title><p>The viruses were electron microscopically visualized, and appeared to have clearly Corona virus typical characteristics. Cytopathic changes could be seen when the viruses were inoculated on Vero cells, which are susceptible to SARS virus infection. The two virus strains, Sino1 and Sino3, which we used extensively for our research have been sequenced (they have more than 99% similarity with the available SARS CoV virus sequences) and have been accepted by GenBank with the accession number of <ext-link ext-link-type="gen" xlink:href="AY485277">AY485277</ext-link> (Sino1) and <ext-link ext-link-type="gen" xlink:href="AY485278">AY485278</ext-link> (Sino3). The virus could neutralize convalescent sera. All the above evidence has proved that the virus we used is SARS corona virus.</p></sec><sec><label>3.2</label><title>Epidemiological identification</title><p>The antiserum was from a male SARS patient with clear epidemiological history and was confirmed clinically. This male patient was identified as a SARS probable case according to the WHO criteria and was confirmed to be SARS case according to the diagnosis criteria of the Chinese Ministry of Health. He had been infected SARS through contact with a SARS patient at Zhangjiakou No. 2 Hospital affiliated with Zhangjiakou Medical Institute. There were two other patients who had had close contact with him. They also showed SARS symptoms such as fever, cough and dyspnea and pulmonary could be detected by X-ray check. They were also confirmed to be SARS probable cases according to the WHO criteria and were confirmed to be SARS cases according to the diagnosis criteria of the Chinese Ministry of Health. All patients were interviewed to ascertain their contacts with each other.</p></sec><sec><label>3.3</label><title>Serological assays</title><p>Convalescent serum was collected from the patient in 3.2, and was named STS-D-Zhang-05 after inactivation process. Antibody specificity of the serum was identified by ELISA, Western blot assay, and neutralization assay.</p><p>The convalescent sera were collected from 20 SARS patients (probable cases according the WHO criteria) in the Inner Mogolia Autonomous Region (NeiMengGu, thus the three letter initial code Nei), the cities of Beijing and Zhang Jiakou (thus the three letter initial codes Jing and Zhang, respectively). The collection was arranged by Chinese Ministry of Health and China CDC. Sera were collected 1–5 months post the onset of the symptoms.</p><p>Nineteen sera from the above 20 sera tested gave positive results and all negative ones proved negative. Results are given in <xref rid="tbl1" ref-type="table">Table 1</xref>. The serum STS-D-Zhang-05 had strong positive reaction, confirming SARS antibody specificity. The serum volume of STS-D-Zhang-05 is sufficient for reference preparation and its neutralization potency is close to the potency GMT (1:54) of all the 19 positive sera.<table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Neutralization test results for 20 convalescent sera from SARS patients</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Serum code</th><th align="left">NT potency</th><th align="left">Serum code</th><th align="left">NT potency</th><th align="left">Serum code</th><th align="left">NT potency</th></tr></thead><tbody><tr><td align="left">STS-D-Nei-01</td><td align="left">1:25</td><td align="left">STS-D-Jing-06</td><td align="left">1:203</td><td align="left">STS-D-Jing-09</td><td align="left">1:51</td></tr><tr><td align="left">STS-D-Nei-02</td><td align="left">1:32</td><td align="left">STS-D-Jing-07</td><td align="left">1:64</td><td align="left">STS-D-Jing-10</td><td align="left">1:51</td></tr><tr><td align="left">STS-D-Nei-03</td><td align="left">1:128</td><td align="left">STS-D-Zhang-01</td><td align="left">1:64</td><td align="left">STS-D-Jing-17</td><td align="left">1:16</td></tr><tr><td align="left">STS-D-Nei-04</td><td align="left">1:80</td><td align="left">STS-D-Zhang-02<xref rid="tbl1fn1" ref-type="table-fn">a</xref></td><td align="left"><1:8</td><td align="left">STS-D-Jing-25</td><td align="left">1:32</td></tr><tr><td align="left">STS-D-Nei-05</td><td align="left">1:64</td><td align="left">STS-D-Zhang-03</td><td align="left">1:128</td><td align="left">STS-D-Jing-26</td><td align="left">1:16</td></tr><tr><td align="left">STS-D-Nei-06</td><td align="left">1:51</td><td align="left">STS-D-Zhang-04</td><td align="left">1:80</td><td align="left">STS-D-Jing-27</td><td align="left">1:51</td></tr><tr><td align="left">STS-D-Jing-05</td><td align="left">1:64</td><td align="left">STS-D-Zhang-05</td><td align="left">1:51</td><td></td><td></td></tr></tbody></table><table-wrap-foot><fn><p>The potency GMT of all the 19 sera is 1:54.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fn1"><label>a</label><p>This is the negative serum.</p></fn></table-wrap-foot></table-wrap></p><p>Western blot assay was further performed to identify the SARS antibody in the serum STS-D-Zhang-05. A band of 48 kDa corresponding to the SARS N protein (Nucleocapsid protein) was detected, suggesting the existence of specific anti-SARS antibody. No bands were detected for the negative control serum. The result is shown in <xref rid="fig1" ref-type="fig">Fig. 1</xref>.<fig id="fig1"><label>Fig. 1</label><caption><p>Western blot assay. Lane a, pre-stained protein marker (New England BioLabs), the bands from the upmost to the downmost corresponds to 83 kDa, 62 kDa, 48 kDa, 33 kDa, 25 kDa, 17 kDa and 7 kDa, respectively. Lane b, total SARS virus proteins (about two micrograms were applied). The antiserum STS-D-Zhang-05 was used as the primary antibody.</p></caption><graphic xlink:href="gr1"></graphic></fig></p><p>Neutralization assay demonstrated that the serum is capable of neutralizing four SARS-CoV strains (<xref rid="tbl2" ref-type="table">Table 2</xref>).<table-wrap position="float" id="tbl2"><label>Table 2</label><caption><p>Neutralization potency of the serum STS-D-Zhang-05 for four SARS CoV strains</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">SARS CoV viral strain</th><th align="left">Neutralization potency</th></tr></thead><tbody><tr><td align="left">Sino1</td><td align="left">1:64</td></tr><tr><td align="left">Sino2</td><td align="left">1:32</td></tr><tr><td align="left">Sino3</td><td align="left">1:16</td></tr><tr><td align="left">Sino6</td><td align="left">1:64</td></tr></tbody></table></table-wrap></p></sec><sec><label>3.4</label><title>Preparation of SARS reference antiserum</title><p>After the above identification, STS-D-Zhang-05 was designated to be SARS reference antiserum candidate. The serum, which had a volume of about 100 ml, was filled into ampoules, freeze-dried, sealed, and tested for residual moisture following the WHO requirements <xref rid="bib2" ref-type="bibr">[2]</xref>. In total 163 ampoules were obtained. This batch of reference antiserum, assigned a lot number of 20031009, was stored at −20 °C and was in the charge of a special keeper.</p></sec><sec><label>3.5</label><title>Calibration of SARS reference antiserum</title><p>Neutralization assay was used to calibrate the reference antiserum. Repetitive titrations were performed by different persons at three different times. Statistic analysis identified the range and GMT (geometric mean titre) of the antibody potency. Results are given in <xref rid="tbl3" ref-type="table">Table 3</xref>, <xref rid="tbl4" ref-type="table">Table 4</xref>.<table-wrap position="float" id="tbl3"><label>Table 3</label><caption><p>Results from titration by different persons at different times</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Test</th><th align="left">Number of experiments</th><th align="left">GMT</th></tr></thead><tbody><tr><td align="left">A</td><td align="char">11</td><td align="left">1:52.3</td></tr><tr><td align="left">B</td><td align="char">4</td><td align="left">1:50.6</td></tr><tr><td align="left">C</td><td align="char">6</td><td align="left">1:54.8</td></tr></tbody></table></table-wrap><table-wrap position="float" id="tbl4"><label>Table 4</label><caption><p>Results from titration by the same person at the same time (intra-test) or at different times (inter-test)</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Test number<xref rid="tbl4fn1" ref-type="table-fn">a</xref></th><th colspan="2" align="left">Result<hr></hr></th></tr><tr><th></th><th align="left">Intra-test</th><th align="left">Inter-test</th></tr></thead><tbody><tr><td align="left">1</td><td align="left">1: 80</td><td align="left">1: 51</td></tr><tr><td align="left">2</td><td align="left">1: 44</td><td align="left">1: 40</td></tr><tr><td align="left">3</td><td align="left">1: 64</td><td align="left">1: 64</td></tr><tr><td align="left">4</td><td align="left">–</td><td align="left">1: 64</td></tr><tr><td align="left">5</td><td align="left">–</td><td align="left">1: 51</td></tr><tr><td align="left">6</td><td align="left">–</td><td align="left">1: 51</td></tr><tr><td align="left">7</td><td align="left">–</td><td align="left">1: 32</td></tr><tr><td align="left">8</td><td align="left">–</td><td align="left">1: 51</td></tr><tr><td align="left">Geometric mean</td><td align="left">1: 60.8</td><td align="left">1: 49.4</td></tr><tr><td align="left">Coefficient of variance</td><td align="left">0.288</td><td align="left">0.214</td></tr></tbody></table><table-wrap-foot><fn id="tbl4fn1"><label>a</label><p>Test number indicates independent measurements.</p></fn></table-wrap-foot></table-wrap></p></sec><sec><label>3.6</label><title>Potency definition of SARS reference antiserum</title><p>The potency of SARS reference antiserum is defined to be 52.7 U. The geometric mean titre is 1:52.7, which is derived from 21 independent experiments. The 95% confidence interval was found to lie between 1:47.6 and 1:62.2. The Inter-laboratory GCV is 29%.</p></sec><sec><label>3.7</label><title>Moisture content</title><p>The moisture of the freeze-dried SARS reference antiserum was determined to be 0.5%.</p></sec><sec><label>3.8</label><title>Stability test on SARS reference antiserum</title><p>Results on the stability of SARS reference antiserum at 37 and 56 °C are given as <xref rid="tbl5" ref-type="table">Table 5</xref>. More experiments on stability are still in progress.<table-wrap position="float" id="tbl5"><label>Table 5</label><caption><p>Results on the stability of SARS reference antiserum<xref rid="tbl5fn1" ref-type="table-fn">a</xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th></th><th colspan="6" align="left">Experimental duration (day)<hr></hr></th></tr><tr><th></th><th align="left">0</th><th align="left">7</th><th align="left">14</th><th align="left">21</th><th align="left">28</th><th align="left">35</th></tr></thead><tbody><tr><td colspan="7" align="left">Neutralization Antibody Potency at different temperatures</td></tr><tr><td align="left"> −20 °C</td><td align="left">1:65</td><td align="left">1:45</td><td align="left">1:45</td><td align="left">1:45</td><td align="left">1:45</td><td align="left">1:45</td></tr><tr><td align="left"> 37 °C</td><td align="left">1:65</td><td align="left">1:51</td><td align="left">1:32<xref rid="tbl5fn1" ref-type="table-fn">a</xref></td><td align="left">1:51</td><td align="left">1:51</td><td align="left">1:51</td></tr><tr><td align="left"> 56 °C</td><td align="left">1:65</td><td align="left">1:23</td><td align="left">1:23</td><td align="left">1:16</td><td align="left">1:16</td><td align="left">1:13</td></tr></tbody></table><table-wrap-foot><fn id="tbl5fn1"><label>a</label><p>The results in this table are from single test with no repetition, and there might be deviation in results, as the neutralizing activity on day 14 at 37 °C is lower than that on days 21, 28 and 35.</p></fn></table-wrap-foot></table-wrap></p><p>Results in <xref rid="tbl5" ref-type="table">Table 5</xref> show that neutralization antibody potency has not decreased substantially after SARS reference antiserum was kept at 37 and 56 °C for 0–35 days. Therefore, the storage condition for SARS reference antiserum is designated to be −20 °C in the dark, and the time period before expiration is primarily designated to be 1 year.</p></sec></sec><sec><label>4</label><title>Discussion</title><p>The world has to be prepared to face a possible resurgence of local SARS epidemic. Although progress has been made worldwide to develop a safe and effective human vaccine for SARS prevention, there still lacks international references for and quality control and expressing test results. A recent WHO SARS laboratory workshop has agreed on the urgent need for quality control, standardization of test protocols and reagents, the need for verification of initial cases/clusters in non-epidemic periods, and the establishment of international reference and verification laboratories <xref rid="bib6" ref-type="bibr">[6]</xref>. Antibody capable of neutralizing the virus has been discovered in the convalescent serum from SARS patients, suggesting its role in the immunity to SARS infection. In order to fulfill the needs for standardization, we have been working on a potential reference antiserum in an effort to search for a vaccine to combat SARS.</p><p>A SARS reference antiserum, which will serve as a reference in different serological methods in immunized animals, has been successfully prepared, tested and calibrated.</p><p>Qualitative tests including ELISA and Western blot assay and neutralization assay have made it clear that the reference antiserum has satisfied the basic criteria for references. According to the titration results, the neutralization antibody unit of the reference antiserum is 52.7 U. Primary results on stability showed that neutralization antibody potency of the reference antiserum has not decreased substantially up to 3 weeks. The stability test is still in progress.</p><p>This newly developed reference antiserum is so far only for in-house use. For acceptance to become an international reference candidate, more has to be done to comply with the WHO regulations on references, such as collaborative titration of the serum and methodology verification.</p></sec></body><back><ref-list><title>References</title><ref id="bib1"><label>1</label><mixed-citation publication-type="other">WHO: Global Search for SARS vaccine gains momentum. <ext-link ext-link-type="uri" xlink:href="http://www.who.int/mediacentre/releases/2003/pr83/en">http://www.who.int/mediacentre/releases/2003/pr83/en</ext-link>.</mixed-citation></ref><ref id="bib2"><label>2</label><mixed-citation publication-type="other">Guidelines for the preparation, characterization and establishment of international and other standards and reference reagents for biological substance reagents for biological substances. World Health Organization, Technical report series no. 800, Annex 4, 1990.</mixed-citation></ref><ref id="bib3"><label>3</label><mixed-citation publication-type="other">WHO: Poliovirus neutralization antibody assay. Bull WHO 1997;70:669.</mixed-citation></ref><ref id="bib4"><label>4</label><mixed-citation publication-type="other">WHO: Manual for the virological investigation of polio. WHO/EPI/GEN/97.01, 1997, p. 44–51.</mixed-citation></ref><ref id="bib5"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>May</surname><given-names>J.C.</given-names></name><name><surname>Grim</surname><given-names>E.</given-names></name><name><surname>Wheeler</surname><given-names>R.M.</given-names></name></person-group><article-title>Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer, gravimetric and thermogravimetric methodologies</article-title><source>J Biol Stand</source><volume>10</volume><year>1982</year><fpage>249</fpage><lpage>259</lpage><pub-id pub-id-type="pmid">7130203</pub-id></element-citation></ref><ref id="bib6"><label>6</label><mixed-citation publication-type="other">WHO: Summary of the discussion and recommendation of the SARS laboratory workshop, WHO, 22 October 2003. <ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/sars/en">http://www.who.int/csr/sars/en</ext-link>.</mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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