Sensitive and Specific Enzyme-Linked Immunosorbent Assay Using Chemiluminescence for Detection of Severe Acute Respiratory Syndrome Viral Infection▿
Identifieur interne : 000657 ( Pmc/Corpus ); précédent : 000656; suivant : 000658Sensitive and Specific Enzyme-Linked Immunosorbent Assay Using Chemiluminescence for Detection of Severe Acute Respiratory Syndrome Viral Infection▿
Auteurs : Kotaro Fujimoto ; Kwok-Hung Chan ; Kazuhiko Takeda ; Kam-Fai Lo ; Raymond H. K. Leung ; Takashi OkamotoSource :
- Journal of Clinical Microbiology [ 0095-1137 ] ; 2007.
Abstract
Here we report the development of a more-sensitive immunoassay for severe acute respiratory syndrome (SARS) based on an enzyme-linked immunosorbent assay using chemiluminescence (CLEIA) to detect the viral nucleocapsid (N) antigen in nasopharyngeal aspirate (NPA) from patients infected with SARS coronavirus (CoV). The CLEIA was established with an optical combination of monoclonal antibodies (MAbs) against SARS CoV N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MAbs of the CLEIA reacted to the carboxyl-terminal and amino-terminal peptides of the N protein, respectively. The CLEIA was capable of detecting recombinant N protein at 1.56 pg/ml and viral N protein in SARS CoV cell culture lysates at 0.087 of 50% tissue culture infective doses/ml. The CLEIA showed no cross-reactivities to recombinant N proteins of common human CoV (229E, OC43, and NL63) or lysates of cells infected with 229E and OC43. In addition, an evaluation with 18 SARS-positive NPA samples, all confirmed SARS positive by quantitative PCR and antibodies to SARS CoV, revealed that all (18/18) were found positive by the CLEIA; thus, the sensitivity of detection was 100%. When we tested 20 SARS-negative NPA samples, the CLEIA was shown to have high specificity (100%). The sensitivity of our novel SARS CLEIA was significantly higher than the previous EIA and comparable to the other methods using reverse transcription-PCR.
Url:
DOI: 10.1128/JCM.01006-07
PubMed: 18032623
PubMed Central: 2224272
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PMC:2224272Le document en format XML
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<author><name sortKey="Fujimoto, Kotaro" sort="Fujimoto, Kotaro" uniqKey="Fujimoto K" first="Kotaro" last="Fujimoto">Kotaro Fujimoto</name>
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<author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok-Hung" last="Chan">Kwok-Hung Chan</name>
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<author><name sortKey="Takeda, Kazuhiko" sort="Takeda, Kazuhiko" uniqKey="Takeda K" first="Kazuhiko" last="Takeda">Kazuhiko Takeda</name>
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<author><name sortKey="Lo, Kam Fai" sort="Lo, Kam Fai" uniqKey="Lo K" first="Kam-Fai" last="Lo">Kam-Fai Lo</name>
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<author><name sortKey="Leung, Raymond H K" sort="Leung, Raymond H K" uniqKey="Leung R" first="Raymond H. K." last="Leung">Raymond H. K. Leung</name>
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<author><name sortKey="Okamoto, Takashi" sort="Okamoto, Takashi" uniqKey="Okamoto T" first="Takashi" last="Okamoto">Takashi Okamoto</name>
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<author><name sortKey="Takeda, Kazuhiko" sort="Takeda, Kazuhiko" uniqKey="Takeda K" first="Kazuhiko" last="Takeda">Kazuhiko Takeda</name>
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<author><name sortKey="Lo, Kam Fai" sort="Lo, Kam Fai" uniqKey="Lo K" first="Kam-Fai" last="Lo">Kam-Fai Lo</name>
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<author><name sortKey="Okamoto, Takashi" sort="Okamoto, Takashi" uniqKey="Okamoto T" first="Takashi" last="Okamoto">Takashi Okamoto</name>
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<series><title level="j">Journal of Clinical Microbiology</title>
<idno type="ISSN">0095-1137</idno>
<idno type="eISSN">1098-660X</idno>
<imprint><date when="2007">2007</date>
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<front><div type="abstract" xml:lang="en"><p>Here we report the development of a more-sensitive immunoassay for severe acute respiratory syndrome (SARS) based on an enzyme-linked immunosorbent assay using chemiluminescence (CLEIA) to detect the viral nucleocapsid (N) antigen in nasopharyngeal aspirate (NPA) from patients infected with SARS coronavirus (CoV). The CLEIA was established with an optical combination of monoclonal antibodies (MAbs) against SARS CoV N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MAbs of the CLEIA reacted to the carboxyl-terminal and amino-terminal peptides of the N protein, respectively. The CLEIA was capable of detecting recombinant N protein at 1.56 pg/ml and viral N protein in SARS CoV cell culture lysates at 0.087 of 50% tissue culture infective doses/ml. The CLEIA showed no cross-reactivities to recombinant N proteins of common human CoV (229E, OC43, and NL63) or lysates of cells infected with 229E and OC43. In addition, an evaluation with 18 SARS-positive NPA samples, all confirmed SARS positive by quantitative PCR and antibodies to SARS CoV, revealed that all (18/18) were found positive by the CLEIA; thus, the sensitivity of detection was 100%. When we tested 20 SARS-negative NPA samples, the CLEIA was shown to have high specificity (100%). The sensitivity of our novel SARS CLEIA was significantly higher than the previous EIA and comparable to the other methods using reverse transcription-PCR.</p>
</div>
</front>
</TEI>
<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Clin Microbiol</journal-id>
<journal-title>Journal of Clinical Microbiology</journal-title>
<issn pub-type="ppub">0095-1137</issn>
<issn pub-type="epub">1098-660X</issn>
<publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">18032623</article-id>
<article-id pub-id-type="pmc">2224272</article-id>
<article-id pub-id-type="publisher-id">1006-07</article-id>
<article-id pub-id-type="doi">10.1128/JCM.01006-07</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Virology</subject>
</subj-group>
</article-categories>
<title-group><article-title>Sensitive and Specific Enzyme-Linked Immunosorbent Assay Using Chemiluminescence for Detection of Severe Acute Respiratory Syndrome Viral Infection<xref ref-type="fn" rid="fn1">▿</xref>
</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Fujimoto</surname>
<given-names>Kotaro</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="aff" rid="aff1">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Chan</surname>
<given-names>Kwok-Hung</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Takeda</surname>
<given-names>Kazuhiko</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lo</surname>
<given-names>Kam-Fai</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Leung</surname>
<given-names>Raymond H. K.</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Okamoto</surname>
<given-names>Takashi</given-names>
</name>
<xref ref-type="aff" rid="aff1">2</xref>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
</contrib-group>
<aff id="aff1">Research and Development Division, Sysmex Co. Ltd., Kobe, Japan,<label>1</label>
Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan,<label>2</label>
Department of Microbiology, The University of Hong Kong and Queen Mary Hospital, Hong Kong<label>3</label>
</aff>
<author-notes><fn id="cor1"><label>*</label>
<p>Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan. Phone: 81-52-853-8205. Fax: 81-52-859-1235. E-mail: <email>tokamoto@med.nagoya-cu.ac.jp</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><month>1</month>
<year>2008</year>
</pub-date>
<pub-date pub-type="epub"><day>21</day>
<month>11</month>
<year>2007</year>
</pub-date>
<volume>46</volume>
<issue>1</issue>
<fpage>302</fpage>
<lpage>310</lpage>
<history><date date-type="received"><day>15</day>
<month>5</month>
<year>2007</year>
</date>
<date date-type="rev-recd"><day>16</day>
<month>7</month>
<year>2007</year>
</date>
<date date-type="accepted"><day>25</day>
<month>10</month>
<year>2007</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2008, American Society for Microbiology</copyright-statement>
</permissions>
<self-uri xlink:title="pdf" xlink:href="zjm00108000302.pdf"></self-uri>
<abstract><p>Here we report the development of a more-sensitive immunoassay for severe acute respiratory syndrome (SARS) based on an enzyme-linked immunosorbent assay using chemiluminescence (CLEIA) to detect the viral nucleocapsid (N) antigen in nasopharyngeal aspirate (NPA) from patients infected with SARS coronavirus (CoV). The CLEIA was established with an optical combination of monoclonal antibodies (MAbs) against SARS CoV N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MAbs of the CLEIA reacted to the carboxyl-terminal and amino-terminal peptides of the N protein, respectively. The CLEIA was capable of detecting recombinant N protein at 1.56 pg/ml and viral N protein in SARS CoV cell culture lysates at 0.087 of 50% tissue culture infective doses/ml. The CLEIA showed no cross-reactivities to recombinant N proteins of common human CoV (229E, OC43, and NL63) or lysates of cells infected with 229E and OC43. In addition, an evaluation with 18 SARS-positive NPA samples, all confirmed SARS positive by quantitative PCR and antibodies to SARS CoV, revealed that all (18/18) were found positive by the CLEIA; thus, the sensitivity of detection was 100%. When we tested 20 SARS-negative NPA samples, the CLEIA was shown to have high specificity (100%). The sensitivity of our novel SARS CLEIA was significantly higher than the previous EIA and comparable to the other methods using reverse transcription-PCR.</p>
</abstract>
</article-meta>
</front>
<floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label>
<caption><p>Purified recombinant N protein expressed in <italic>E. coli</italic>
. (A) Results of SDS-PAGE (5 to 20% gradient gel) for recombinant N protein. (B) Results of Western blot analysis for recombinant N protein that reacted with anti-SARS CoV nucleocapsid MAb (catalog no. 654; IMGENEX Co. Ltd.). Lane 1, molecular mass marker; lane 2, purified recombinant His<sub>6</sub>
-tagged N protein.</p>
</caption>
<graphic xlink:href="zjm0010878260001"></graphic>
</fig>
<fig position="float" id="f2"><label>FIG. 2.</label>
<caption><p>Immunoreactivities of MAbs. Immunoreactivity of anti-SARS CoV N protein MAbs. The immunoreactivity of each MAb to full-length recombinant His<sub>6</sub>
-tagged N protein is shown: •, I-16; ▪, I-23; ▴, II-3; ⧫, II-12; and ○, II-21. The reactivities of serially diluted MAbs were measured in duplicates on a microtiter plate coated with full-length recombinant N protein. The mean value for 0 μg/ml of MAb was 0.042. Results show the means of net values (the mean value for each concentration of MAb/the mean value for 0 μg/ml of MAb).</p>
</caption>
<graphic xlink:href="zjm0010878260002"></graphic>
</fig>
<fig position="float" id="f3"><label>FIG. 3.</label>
<caption><p>Western blot detection of MAb reactivity to SARS CoV N protein. (Coomassie stain panel) Coomassie blue staining of purified recombinant SARS CoV N protein. The recombinant viral protein was loaded at 1.0 μg/lane and analyzed on a 5 to 20% SDS-PAGE and stained by Coomassie blue dye. (MAb I-16, MAb I-23, MAb II-3, MAb II-12, and MAb II-21 panels) Western blot detection of recombinant SARS CoV N protein by MAbs. Recombinant N proteins were transferred to polyvinylidene difluoride membrane after SDS-PAGE, and each membrane was reacted to different MAbs (I-16, I-23, II-3, II-12, and II-21). Lane 1, full-length N protein was applied; lane 2, truncated N protein (NΔ283-422) was applied; and lane 3, truncated N protein of SARS CoV (NΔ1-141) was applied. ○, completely stained and partially degraded N protein (full length); •, completely stained N protein ΔN283-422; ▴, completely stained and partially degraded N protein (NΔ1-141); *, anti-N MAb reacted to complete and partially degraded N protein (full length); **, anti-N protein MAb reacted to complete N protein (NΔ283-422); ***, anti-N protein MAb reacted to complete and partially degraded N protein (NΔ1-141).</p>
</caption>
<graphic xlink:href="zjm0010878260003"></graphic>
</fig>
<fig position="float" id="f4"><label>FIG. 4.</label>
<caption><p>Evaluation of sandwich ELISA system with various MAbs. (A) Evaluation of sandwich ELISA by color detection system using 25 different combinations of anti-SARS CoV N protein MAbs. Twenty nanograms of recombinant full-length N protein was measured by different combination of MAbs with sandwich ELISA systems. Asterisks indicate the best five results among the 25 ELISAs with different combinations of MAbs. *1, ELISA with I-23/II-12 (capture MAb/detection MAb); *2, ELISA with II-21/II-3; *3, ELISA with II-12/I-23; *4, ELISA with II-21/I-23; and *5, ELISA with I-16/I-23. (B) Standard antigen ELISA curves determined with purified recombinant N protein by sandwich ELISA (CLEIA) using five kinds of MAb pairs (capture MAb/detecting MAb): •, I-23/II-12; ▪, II-21/I-23; ▴, II-12/I-23; ⧫, II-21/II-3; and ○, I-16/I-23. Serial dilutions of recombinant N protein were measured in triplicate by CLEIA. Results show the means of triplicate results. Error bars indicate the range of standard deviations.</p>
</caption>
<graphic xlink:href="zjm0010878260004"></graphic>
</fig>
<fig position="float" id="f5"><label>FIG. 5.</label>
<caption><p>Sensitivity of CLEIA in detecting SARS CoV N antigen. (A) Limiting dilution experiment with cultured SARS CoV (HKU-39849) for detecting SARS CoV N antigen by CLEIA using I-23 as capture MAb and II-12 as detecting MAb. Twofold dilutions of cell culture lysates of SARS CoV were measured in triplicate by the CLEIA. Results show mean values of the triplicates. Error bars express the range of 2× standard deviation values. The limit of detection of CLEIA is 0.087 TCID<sub>50</sub>
/ml (a coarse dotted line), which is the minimum concentration (TCID<sub>50</sub>
/ml) of cultured SARS CoV (HKU-39849) giving the signal value of the mean minus 2.0 standard deviations, which is higher than that of the mean plus 2.0 standard deviations of 0.0 TCID<sub>50</sub>
/ml (a fine dotted line). (B) Limiting dilution experiment with cell culture lysates of common human CoV (229E [▪] or OC43 [▴]). Serial dilutions of recombinant N protein were measured in triplicate by the CLEIA. Results show mean values of the triplicates. Error bars indicate 2× standard deviation values.</p>
</caption>
<graphic xlink:href="zjm0010878260005"></graphic>
</fig>
<fig position="float" id="f6"><label>FIG. 6.</label>
<caption><p>Correlation between CLEIA and Q-PCR in detecting SARS CoV N antigen using clinical samples obtained from SARS patients. In CLEIA, I-23 and II-12 MAbs are used as capture and detecting Abs, respectively. For the Q-PCR detection of SARS CoV RNA, Q-PCR was performed as previously reported (<xref ref-type="bibr" rid="r32">32</xref>
). Eighteen clinical NPA samples from 18 SARS-positive patients (•) and 20 SARS-negative NPA samples (○) were analyzed. RNA was prepared from each NPA sample, and the SARS CoV RNA concentration was quantitatively evaluated by Q-PCR. The amount of SARS CoV N protein was measured by CLEIA as described above. Results show the means of triplicates for each specimen. Cutoff value of CLEIA, 718 RLU (as measured by the mean of negative specimens plus 3 standard deviations). *, the results with SARS-positive NPA with the minimum concentration of viral RNA by Q-PCR (757 copies/ml); ¶, the four dots in the ellipse drawn with a dotted line indicate the chemiluminescence intensity for four NPA samples which exceeded the maximum limit of detection by luminometer.</p>
</caption>
<graphic xlink:href="zjm0010878260006"></graphic>
</fig>
<fig position="float" id="f7"><label>FIG. 7.</label>
<caption><p>N protein detection and the difference between the days from onset and age groups of SARS patients. (A) Chemiluminescence intensity of CLEIA with NPA from SARS patients who were classified into four groups by their days from the onset of symptoms (4 to 5, 6 to 10, 11 to 15, and 16 to 18 days). *, the CLEIA signal of patient P16 in the early stage (4 days from onset of symptoms) whose viral load is 2.43E + 07 copies/ml. (B) Chemiluminescence intensity of CLEIA with NPA from SARS patients who were classified into four groups by their ages (18 to 26, 32 to 39, 43 to 49, and 55 to 72 years). Chemiluminescence intensity (RLU) of the NPA from each SARS patient is expressed in its proper lane of panel A or B. Bars and numbers in each diagram indicate mean values of signals with specimens in each group. (C) Chemiluminescence intensity of CLEIA with NPA from SARS patients who were classified into four groups by a combination of their age and their days from the onset of symptoms. Group A, NPA from patients 18 to 39 years old and at 4 to 10 days from the onset of symptoms; group B, NPA from patients 44 to 57 years old and at 4 to 10 days from the onset of symptoms; group C, NPA from patients 24 to 35 years old and at 11 to 17 days from the onset of symptoms; group D, NPA from patients 43 to 71 years old and at 12 to 18 days from the onset of symptoms.</p>
</caption>
<graphic xlink:href="zjm0010878260007"></graphic>
</fig>
<table-wrap position="float" id="t1"><label>TABLE 1.</label>
<caption><p>SARS patients from whom NPA specimens were collected</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Patient</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Sex<xref ref-type="table-fn" rid="t1fn1"><italic>a</italic>
</xref>
</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Age (yr)</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Days from onset of symptoms</th>
</tr>
</thead>
<tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">P1</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">44</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">9</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P2</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">39</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">10</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P3</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">39</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">8</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P4</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">49</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">8</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P5</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">35</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">12</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P6</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">26</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">11</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P7</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">24</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">12</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P8</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">71</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">18</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P9</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">57</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">4</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P10</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">58</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">12</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P11</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">34</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">17</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P14</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">43</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">15</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P15</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">35</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">7</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P16</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">34</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">4</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P17</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">32</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">6</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P18</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">55</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">5</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P19</td>
<td colspan="1" rowspan="1" align="center" valign="top">F</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">34</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">8</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">P20</td>
<td colspan="1" rowspan="1" align="center" valign="top">M</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">18</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">8</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Mean</td>
<td colspan="1" rowspan="1" align="center" valign="top"></td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">40.4</td>
<td colspan="1" rowspan="1" align="char" char="." valign="top">9.7</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t1fn1"><label>a</label>
<p>M, male; F, female.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap position="float" id="t2"><label>TABLE 2.</label>
<caption><p>Primers for synthesizing cDNA of SARS CoV N protein</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Primer<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic>
</xref>
</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Sequence</th>
</tr>
</thead>
<tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">S1</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-ATGAGCGATAACGGCCCGCAGAGCAACCAGCGTAGCGCGCCGCGTATTACCTTTGGCGGCCCGACCGATAGCACCGATAACAACCAGAACGGCGGCCG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S2</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GTTTACCGCGCTGACCCAGCATGGCAAAGAAGAACTGCGTTTTCCGCGTGGCCAGGGCGTGCCGATTAACACCAACAGCGGCCCGGATGATCAGATTGGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S3</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CGCGTTGGTATTTTTATTATCTGGGCACCGGCCCGGAAGCGAGCCTGCCGTATGGCGCGAACAAAGAAGGCATTGTGTGGGTGGCGACCGAAGGCGCGCTG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S4</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CAGCTGCCGCAGGGCACCACCCTGCCGAAAGGCTTTTATGCGGAAGGCAGCCGTGGCGGCAGCCAGGCGAGCAGCCGTAGCAGCAGCCGTAGCCGTGGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A1</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GCTGGGTCAGCGCGGTAAACCAGCTCGCGGTGTTGTTCGGCAGGCCCTGCGGACGACGCTGTTTCGGACGCGCGCCGTTACGGCCGCCGTTCTGGTTGTTATC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A2</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GATAATAAAAATACCAACGCGGGCTCAGTTCTTTCATTTTGCCATCGCCGCCACGCACACGACGGGTCGCACGACGATAATAGCCAATCTGATCATCCGGGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A3</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GTGGTGCCCTGCGGCAGCTGCAGCACGGTCGCCGCGTTGTTGTTCGGGTTACGGGTGCCAATATGATCTTTCGGGGTGTTCAGCGCGCCTTCGGTCGCCAC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A4</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CAGCGCGGTTTCGCCGCCGCCGCTCGCCATACGCGCCGGGCTGTTGCCACGGCTGCTGCCCGGGGTGCTGTTACGGCTGTTGCCACGGCTACGGCTGCTGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S5</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-ACAGCCGTAACAGCACCCCGGGCAGCAGCCGTGGCAACAGCCCGGCGCGTATGGCGAGCGGCGGCGGCGAAACCGCGCTGGCGCTGCTGCTGCTGGATCGTCTG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S6</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-AAAGCGCGGCGGAAGCGAGCAAAAAACCGCGTCAGAAACGTACCGCGACCAAACAGTATAACGTGACCCAGGCGTTTGGCCGTCGTGGCCCGGAACAGACCCAGGG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S7</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GCGCAGTTTGCGCCGAGCGCGAGCGCGTTTTTTGGCATGAGCCGTATTGGCATGGAAGTGACCCCGAGCGGCACCTGGCTGACCTATCATGGCGCGATTAAAC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">S8</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-ACCTTTCCGCCGACCGAACCGAAAAAAGATAAAAAAAAAAAAACCGATGAAGCGCAGCCGCTGCCGCAGCGTCAGAAAAAACAGCCGACCGTGACCCTGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A5</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GCTCGCTTCCGCCGCGCTTTTTTTGGTCACGGTCTGGCCCTGCTGCTGCTGGCCTTTGCCGCTCACTTTGCTTTCCAGCTGGTTCAGACGATCTAGCAGCAGCA-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A6</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CGCGCTCGGCGCAAACTGCGCAATCTGCGGCCAATGTTTATAATCGGTGCCCTGACGAATCAGATCCTGATCGCCAAAGTTGCCCTGGGTCTGTTCCGGGCC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A7</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GGTTCGGTCGGCGGAAAGGTTTTATACGCATCAATATGTTTGTTCAGCAGAATCACGTTATCTTTAAACTGCGGATCTTTATCATCCAGTTTAATCGCGCCATGATAG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">A8</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-TTACGCCTGGGTGCTATCCGCGCTCGCGCCGCTCATGCTGTTCTGCAGCTGACGGCTAAAATCATCCATATCCGCCGCCGGCAGCAGGGTCACGGTCGGCTG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">NS</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CCCGGATCCATGAGCGATAACGGCCCGCA-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">NA</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CCCACAGCCGTAACAGCACCCCG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">CS</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CCCGGATCCCAGCGCGGTTTCGCCGCCGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">CA</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-CCCTTACGCCTGGGTGCTATCCG-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">N2/3</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GGATCCCTGTTCCGGGCCACGACGGC-3′</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">C2/3</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′-GGATCCACCCCGAAAGATCATATTGG-3′</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t2fn1"><label>a</label>
<p>S1, S2, S3, S4, A1, A2, A3, and A4: primer set of the 1st PCR for the N-terminal cDNA fragment of SARS CoV N protein from the 1st to 220th amino acid residues. S5, S6, S7, S8, A5, A6, A7, and A8: primer set of the 1st PCR for the C-terminal cDNA fragment of SARS CoV N protein from the 215th to 422nd amino acid residues. NS and NA: primer set of the 2nd PCR for the N-terminal cDNA fragment of SARS CoV N protein from 1st to 220th amino acid residues. CS and CA: primer set of the 2nd PCR for the C-terminal cDNA fragment of SARS CoV N protein from the 215th to 422nd amino acid residues. NS and CA: primer set of PCR for the full-length cDNA fragment of SARS CoV N protein from the 1st to 422nd amino acid residues. NS and N2/3: primer set of PCR for the N-terminal cDNA fragment of SARS CoV N protein from the 1st to 282nd amino acid residues. N2/3 and CA: primer set of PCR for the C-terminal cDNA fragment of SARS CoV N protein from the 143rd to 422nd amino acid residues.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap position="float" id="t3"><label>TABLE 3.</label>
<caption><p>Sensitivity of CLEIA for detecting recombinant SARS CoV N protein</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="center" valign="middle">Capture MAb/detecting MAb</th>
<th colspan="4" rowspan="1" align="center" valign="bottom">Sensitivity (RLU ± SD) with the following concns of SARS CoV recombinant N protein (pg/ml)<xref ref-type="table-fn" rid="t3fn1"><italic>a</italic>
</xref>
<hr></hr>
</th>
</tr>
<tr><th colspan="1" rowspan="1" align="center" valign="bottom">0</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">1.56</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">3.13</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">6.25</th>
</tr>
</thead>
<tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">I-16/I-23</td>
<td colspan="1" rowspan="1" align="center" valign="top">375 ± 154</td>
<td colspan="1" rowspan="1" align="center" valign="top">532 ± 118</td>
<td colspan="1" rowspan="1" align="center" valign="top">1,219 ± 344<xref ref-type="table-fn" rid="t3fn2"><italic>b</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">3,297 ± 991<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">II-21/I-23</td>
<td colspan="1" rowspan="1" align="center" valign="top">1,858 ± 401</td>
<td colspan="1" rowspan="1" align="center" valign="top">2,412 ± 96<xref ref-type="table-fn" rid="t3fn2"><italic>b</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">4,327 ± 322<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">7,531 ± 141<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">II-12/I-23</td>
<td colspan="1" rowspan="1" align="center" valign="top">1,306 ± 548</td>
<td colspan="1" rowspan="1" align="center" valign="top">2,592 ± 874</td>
<td colspan="1" rowspan="1" align="center" valign="top">3,563 ± 205<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">7,321 ± 116<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">II-21/II-3</td>
<td colspan="1" rowspan="1" align="center" valign="top">3,963 ± 473</td>
<td colspan="1" rowspan="1" align="center" valign="top">6,164 ± 465<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">9,266 ± 521<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">13,906 ± 618<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">I-23/II-12</td>
<td colspan="1" rowspan="1" align="center" valign="top">782 ± 283</td>
<td colspan="1" rowspan="1" align="center" valign="top">2,040 ± 66<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">4,561 ± 252<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
<td colspan="1" rowspan="1" align="center" valign="top">10,859 ± 183<xref ref-type="table-fn" rid="t3fn3"><italic>c</italic>
</xref>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t3fn1"><label>a</label>
<p>All data are expressed as means ± SD (<italic>n</italic>
= 3).</p>
</fn>
<fn id="t3fn2"><label>b</label>
<p>The value of the mean minus 1.0 SD is higher than that of the mean plus 1.0 SD of the control (0 pg/ml).</p>
</fn>
<fn id="t3fn3"><label>c</label>
<p>The value of the mean minus 2.0 SD is higher than that of the mean plus 2.0 SD of the control (0 pg/ml).</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-wrap>
</pmc>
</record>
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