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1、CLINICAL ANDVACCINEIMMUNOLOGY, Mar. 2007, p. 331333Vol. 14, No. 31556-6811/07/$08.00?0doi:10.1128/CVI.00351-06Copyright 2007, American Society for Microbiology. All Rights Reserved.Recombinant Protein-Based Assays for Detection of Antibodies toSevere Acute Respiratory Syndrome Coronavirus Spike andN
2、ucleocapsid Proteins?Lia M. Haynes,1* Congrong Miao,1Jennifer L. Harcourt,1Joel M. Montgomery,2Mai Quynh Le,7Sergey A. Dryga,5Kurt I. Kamrud,5Bryan Rivers,5Gregory J. Babcock,6Jennifer Betts Oliver,2James A. Comer,2Mary Reynolds,3Timothy M. Uyeki,4Daniel Bausch,2Thomas Ksiazek,2William Thomas,6Harol
3、d Alterson,5Jonathan Smith,5Donna M. Ambrosino,6and Larry J. Anderson1National Centers for Immunization and Respiratory Diseases, Division of Viral Diseases, Respiratory and Gastroenteritis Viruses Branch,1Division of Viral and Rickettsial Diseases, Special Pathogens Branch,2Poxvirus Activity,3and I
4、nfluenza Division,4Centers forDisease Control and Prevention (CDC), 1600 Clifton Rd. NE, Atlanta, Georgia; AlphaVax, Inc., Research Triangle Park,North Carolina5; Massachusetts Biologic Laboratories, University of Massachusetts Medical School, Jamaica Plain,Massachusetts6; and National Institute of
5、Hygiene and Epidemiology, Hanoi, Vietnam7Received 26 September 2006/Returned for modification 31 October 2006/Accepted 8 January 2007Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immuno-globulin G immunoassays were developed and evaluated. Our assays demon
6、strated high sensitivity andspecificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms.These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological andantibody kinetic studies.The 2003 outbreak of severe
7、acute respiratory syndrome(SARS) lasted fewer than 9 months, yet it had a major impacton public health and socioeconomics. Since the end of theSARS outbreak, there have been 17 identified SARS-associ-ated coronavirus (SARS-CoV) infections, 6 from direct labo-ratory exposure, 1 of which resulted in c
8、ommunity transmissionto seven individuals, and 4 sporadic, community-acquired in-fections (9). Since additional cases may occur and could, ifundetected, quickly lead to another global outbreak, it is im-portant to continue to improve our ability to reliably monitorSARS-CoV infections (3, 16, 18).As
9、with other coronaviruses, the spike (S) and nucleocapsid(N) proteins are abundantly expressed during virus infectionand are most effective among the coronavirus structural pro-teins at inducing antibody responses (10, 14, 15, 23). Previousstudies have demonstrated the utility of anti-N and anti-S pr
10、o-teins in the diagnosis of SARS-CoV infections (2, 5, 12, 21). Inthis study, we describe the evaluation and comparison of re-combinant spike and nucleocapsid enzyme-linked immunosor-bent assays (ELISAs) for specifically detecting SARS-CoV in-fection.The recombinant full-length SARS N gene was ampli
11、fiedfrom SARS-CoV RNA (Urbani strain), modified to contain aC-terminal His6tag, and cloned into the Venezuelan equineencephalitis virus replicon vector (17). Baby hamster kidney(BHK) cells were transfected with SARS N replicon RNA byelectroporation. Cells were harvested, and expressed proteinwas pur
12、ified by metal affinity chromatography and analyzed bysilver staining and Western blot analysis for the appropriatelysized (50-kDa) immunoreactive protein (8). The control anti-gen, the nontoxic 50-kDa C-terminal fragment of the botu-linum neurotoxin serotype A (BoNt/HcA), was expressed asdescribed
13、above (7).The soluble codon-optimized SARS-CoV S glycoprotein(170 kDa; amino acids 1 to 1190; S1190) and the controlantigen, truncated angiotensin-converting enzyme 2 (120 kDa;glycosylated; tACE-2), were cloned into pcDNA3.1 Myc/Hisand expressed in HEK-293T/17 cells. The proteins were puri-fied usin
14、g metal-affinity chromatography and analyzed as de-scribed by Babcock et al. (1).Recombinant SARS N and S protein indirect ELISAs weredeveloped using a modified version of the inactivated whole-virus ELISA described by Ksiazek et al. (6). Briefly, ELISAplates (Immulon) were coated with either purifi
15、ed recombi-nant N protein (12.5 ng/well) and the control antigen BoNT/HcA or purified His6/c-myc-tagged recombinant S1190 protein(12.5 ng/well) and the control antigen tACE-2. The plates werewashed and incubated with serum diluted 1:400 in phosphate-buffered saline (PBS) containing skim milk and Twe
16、en 20(PBS-T-M) for 1 h at 37C and washed and incubated againwith horseradish peroxidase-conjugated goat anti-human im-munoglobulin G (IgG) (1:4,000; heavy plus light chainKPL)in PBS-T-M. After washing, substrate ABTS; 2,2?-azi-nobis(3-ethylbenzthiazolinesulfonic acid) was added and theabsorbance rea
17、d at 405 nm with a 490-nm reference filter.ELISA conditions were optimized using antibody-positiveand -negative serum specimens, and the resultant assays werethen evaluated using available serum samples collected from61 patients from Vietnam and Taiwan with laboratory-con-firmed SARS-CoV infection (
18、2 to 150 days postonset of symp-* Corresponding author. Mailing address: Centers for DiseaseControl and Prevention, 1600 Clifton Rd. NE, Mailstop G-18, At-lanta, GA 30333. Phone: (404) 639-4004. Fax: (404) 639-4005.E-mail: loh5cdc.gov.?Published ahead of print on 17 January 2007.331 on March 8, 2015
19、 by DAHLGREN MEDICAL LIBRARYhttp:/cvi.asm.org/Downloaded from toms dpo), from 46 U.S. patients with non-SARS-relatedrespiratory infections, and from 483 healthy U.S. donors withno exposure to SARS-CoV. An additional 10 serum sampleswere collected from non-SARS patients from Vietnam andTaiwan. Specim
20、ens used in this study were exempt from CDCInstitutional Review Board review under 45 CFR 46.101(b)(4)(20). ELISA results from representative serum specimens frompatients with SARS and healthy controls are shown in Fig. 1.The sums of the mean absorbance values for the healthy con-trols and three tim
21、es the standard deviations for the S and Nprotein assays were 0.201 and 0.210, respectively. With thesecutoff values, 7 of the 483 serum samples from healthy donorshad absorbance values above the cutoff value in the N proteinELISA, and 3 samples were above the cutoff for the S proteinELISA, resultin
22、g in specificities of 98.6 and 99.4% for the Nand S protein ELISAs, respectively (Table 1). Control samplesreactive to either the N or S protein did not show reactivity tothe alternate protein and also showed no reactivity against theinactivated SARS-CoV lysate by ELISA. In these samples,reactivity
23、may have been due to nonspecific reactivity or cross-reacting antibodies.To further evaluate the specificity of the assays, availablesera from 46 individuals infected with non-SARS-related res-piratory viruses, including human coronavirus strains 229E(HCoV-229E) (n ? 22 paired specimens) and HCoV-OC
24、43(n ? 17 paired specimens), respiratory syncytial virus (n ? 2),human parainfluenza virus 2 (n ? 1) and 3 (n ? 1), influenzaB virus (n ? 1), adenovirus (n ? 1), and mumps virus (n ? 1),were analyzed. None of the serum samples were positive byeither assay (data not shown). In addition, serum samples
25、 fromnon-SARS patients from Taiwan and Vietnam showed no re-activity by either assay (data not shown).All 61 sera from SARS cases were antibody positive to the Nprotein, and 59 were positive to the S protein. Two acute-phasespecimens (?20 dpo) were weakly reactive to the S protein andfell below the
26、assay cutoff of 0.201. All were also positive byboth immunofluorescence antibody testing and ELISA, usingwhole, gamma-irradiated SARS-CoV as the antigen (data notshown). The sensitivities for the N and S protein assays were100% and 96.7%, respectively.Persistent levels of SARS-CoV IgG have been dete
27、cted inSARS cases for several months and up to 2 years after diseaseonset (4, 11, 12, 19, 21). In this study, serum samples from 48SARS patients from Vietnam and the United States were col-lected 221 to 735 days (44 from days 221 to 250; 4 from days633 to 735) after the onset of illness and tested f
28、or the presenceof SARS-CoV- and N- and S-protein-specific IgG by ELISA.Antibodies (IgG) specific to whole virus, N protein, and Sprotein were detected in 40 (83.3%), 45 (93.8%), and 36 (75%)of the samples tested, respectively. Interestingly, anti-SARS-CoV and S antibodies were detected in three pati
29、ents, two ofwhom also demonstrated a response to the N protein, almost 2years postonset of symptoms (SARS titers ? 1:400 to 1:1,600n ? 3; N protein titer ? 1:400 n ? 2; S protein titer ? 1:400to 1:1,600 n ? 3).Our evaluation of these ELISAs illustrates the value of hav-ing several assay systems to d
30、etect and then confirm a SARS-CoV infection. Although very few serum specimens from un-exposed persons (?1.5%) tested positive for SARS-CoVinfection, the potential for cross-reactivity between SARS-CoV and other coronaviruses, including the known humancoronaviruses HCoV-OC43, HCoV-229E, and recently
31、 identi-fied HKU1 and NL63, remains a concern (12, 13, 22). Whetherthese positive results are due to nonspecific reactivity to therecombinant SARS N protein or to cross-reactivity to otherhuman CoVs requires further study. The use of protein frag-ments or peptides, instead of the whole recombinant N
32、 pro-tein, for antibody detection may resolve the issue of potentialcross-reactivity with proteins of other human CoVs and is thefocus of further study. These false positives could present apublic health dilemma as illustrated in the laboratory evalua-tion of four sporadic cases reported by Liang et
33、 al. (9). In thosecases, it was necessary to conduct confirmatory testing usingseveral different types of assays, because there was concernFIG. 1. Scatter chart of absorbance values with representative sera from both SARS patients and healthy controls for IgG antibodies torecombinant spike protein (
34、A) and recombinant nucleocapsid protein (B). Specimens were obtained from 61 SARS patients at 2 to 150 dayspostonset of symptoms and from 384 healthy U.S. blood donors. Results are plotted as A405values with a 490-nm reference filter. The black lineindicates the cutoff values of 0.201 (A) and 0.210
35、(B) for each assay.TABLE 1. Comparison of sensitivities and specificities ofrecombinant nucleocapsid protein- and spikeprotein-based ELISAsAntigenSARS patientsHealthy donorsNo. with positivereactivity/total%SensitivityNo. with negativereactivity/total%SpecificitySARS-N61/61100476/48398.5SARS-S59/619
36、6.7480/48399.4332NOTESCLIN. VACCINEIMMUNOL. on March 8, 2015 by DAHLGREN MEDICAL LIBRARYhttp:/cvi.asm.org/Downloaded from that infection with non-SARS coronaviruses may induce cross-reacting antibodies (9). Our data from that study suggest thata combination of assays may be needed to confirm the spe
37、ci-ficity of presumed SARS antibodies. Since the costs (i.e., publichealth interventions or outbreak response) of detecting a false-positive result and not detecting a case of SARS-CoV infectionare both high, it is important to have well-characterized detec-tion and confirmatory assays. In the absen
38、ce of virus-specificcontrol measures, e.g., a vaccine or antiviral drug, the key tocontrolling a reemergence of SARS is rapid diagnosis andimplementation of infection control measures, i.e., isolatingcases and identifying and managing contacts to prevent furthertransmission. The development of well-
39、characterized detectionand confirmatory serologic tests is the key to laboratory diag-nostic support should SARS reemerge. These two ELISAs canbe used as components of the SARS diagnostic system. Theseassays can also be used to study the kinetics of the protein-specific SARS antibody response and to
40、 help characterizeSARS immunity and the pathogenesis of disease.We thank Der-Yuan Wang (Bureau of Food and Drug Analysis,Department of Health, Taiwan, Republic of China), Mei-ying W. Yu(CBER/Food and Drug Administration, Bethesda, MD), and Li-ChingHsu (Center for Disease Control, Department of Healt
41、h, Taiwan,Republic of China) for providing a panel of sera from SARS patients.Thanks also to Ann Falsey (University of Rochester School of Medi-cine, Rochester, NY) and Dean Erdman (CDC, Atlanta, GA) forproviding sera from patients with non-SARS-related respiratory infec-tions and Debi Cannon (CDC,
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