【病毒外文文獻】2005 Differential Sensitivities of Severe Acute Respiratory Syndrome (SARS) Coronavirus Spike Polypeptide Enzyme-Linked

JOURNAL OF CLINICAL MICROBIOLOGY July 2005 p 3054 3058 Vol 43 No 7 0095 1137 05 08 00H110010 doi 10 1128 JCM 43 7 3054 3058 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Differential Sensitivities of Severe Acute Respiratory Syndrome SARS Coronavirus Spike Polypeptide Enzyme Linked Immunosorbent Assay ELISA and SARS Coronavirus Nucleocapsid Protein ELISA for Serodiagnosis of SARS Coronavirus Pneumonia Patrick C Y Woo 1 2 Susanna K P Lau 1 2 Beatrice H L Wong 1 Hoi wah Tsoi 1 Ami M Y Fung 1 Richard Y T Kao 1 2 Kwok hung Chan 1 J S Malik Peiris 1 2 and Kwok yung Yuen 1 2 Department of Microbiology 1 and Research Centre of Infection and Immunology 2 Faculty of Medicine The University of Hong Kong Hong Kong Received 3 November 2004 Returned for modification 24 February 2005 Accepted 6 April 2005 The use of recombinant severe acute respiratory syndrome coronavirus SARS CoV nucleocapsid protein N enzyme linked immunosorbent assay ELISA based antibody and antigen tests for diagnosis of SARS CoV infections have been widely reported However no recombinant SARS CoV spike protein S based ELISA is currently available In this article we describe the problems and solutions of setting up the recombinant SARS CoV S based ELISA for antibody detection The SARS CoV S based immunoglobulin M IgM and IgG ELISAs were evaluated and compared with the corresponding N based ELISA for serodiagnosis of SARS CoV pneumonia using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS CoV pneumonia patients in Hong Kong Results obtained by the recombinant S rS based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers followed by addition of different regeneration buffer identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection The specificities of the S based ELISA for IgG and IgM detection were 98 6 and 93 9 with corresponding sensitivities of 58 9 and 74 7 respectively The sensitivity of the rN IgG ELISA 94 7 is significantly higher than that of the rS IgG ELISA P 0 001 whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA 55 2 P 0 01 An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS CoV pneumonia Severe acute respiratory syndrome SARS caused by the SARS coronavirus SARS CoV is a new emerging disease that has affected 30 countries with more than 8 000 cases causing more than 750 deaths 5 6 11 15 17 For laboratory diagnosis of SARS CoV pneumonia isolation of the virus from clinical specimens is insensitive and requires biosafety level 3 laboratory facilities while detection of viral RNA using reverse transcription PCR can only achieve a sensitivity of 50 to 79 depending on the type and number of clinical specimens col lected and the protocol used 26 At the moment the most widely used methods for serodiagnosis of SARS CoV infection in clinical microbiology laboratories are antibody detection in acute and convalescent phase sera by indirect immunofluo rescence assay and enzyme linked immunosorbent assay ELISA using cell culture extracts 11 16 However antibody detection by indirect immunofluorescence assay using cell cul ture extracts may be less reproducible more difficult to stan dardize and more labor intensive than ELISA based antibody detection tests using recombinant antigens Furthermore pro ducing the infected cell lines for coating the ELISA plates and the slides for indirect immunofluorescence requires cultivation of the SARS CoV for which biosafety level 3 laboratory facil ities are required Such facilities are not available in most clinical microbiology laboratories ELISA based antibody detection tests using recombinant antigens are well known to offer higher levels of reproducibility are easy to standardize and less labor intensive than antibody detection by indirect immunofluorescence assay and ELISA using cell culture extract and do not require cultivation of the SARS CoV 1 2 21 27 We have reported the use of recom binant SARS CoV nucleocapsid protein N ELISA based an tibody and antigen tests for diagnosis of SARS CoV infections 4 12 22 25 Others have also used similar approaches for serodiagnosis of SARS CoV pneumonia 13 18 20 Recently one group employed recombinant nucleocapsid spike fusion protein expressed in insect Sf9 cells as the antigen in an immunofluorescence assay for detection of SARS CoV anti bodies 8 Although recombinant N rN immunoglobulin G IgG ELISA achieved a sensitivity of 94 3 for serodiagnosis of SARS CoV pneumonia a sensitivity of only 59 4 can be achieved for the IgM ELISA 23 Since the spike protein S another immunogenic protein of SARS CoV virus is located on the surface of the viral particles and therefore potentially Corresponding author Mailing address Department of Microbi ology The University of Hong Kong University Pathology Building Queen Mary Hospital Pokfulam Hong Kong Phone 852 28554892 Fax 852 28551241 E mail hkumicro hkucc hku hk These authors contributed the same to the manuscript 3054 on May 31 2015 by OAKLAND UNIV http jcm asm org Downloaded from more accessible to the immune system rS based ELISA may offer higher sensitivities than rN based ELISA Paradoxically in one report it was noted that the S based antibody test appeared to have lower sensitivity than the N based antibody test by Western blot analysis 10 However the sample size was relatively small and only N based ELISA was subse quently developed In other reports the authors have used pooled S and N peptide based ELISA for serosurveillance studies 3 9 Currently there is no S based ELISA available for serodiagnosis of SARS CoV infections In this article we describe the problems and solutions of setting up the recombinant SARS CoV S based ELISA for antibody detection The SARS CoV S based IgM and IgG ELISA were evaluated and compared to the corresponding N based ELISA for serodiagnosis of SARS CoV pneumonia MATERIALS AND METHODS rN based IgM and IgG ELISA for SARS CoV pneumonia Cloning and puri fication of His 6 tagged rN have been reported previously 22 23 Sera from 148 healthy blood donors who donated blood 3 years ago were used to set up the baseline of the ELISA 22 Cloning and purification of His 6 tagged rS from Escherichia coli The cloning and purification of His 6 tagged rS have been reported previously 22 Briefly to produce a plasmid for protein expression primers LPW742 5H11032 CGCGGATCC GAGTGACCTTGACCGGTGC 3H11032 and LPW931 5H11032 CGGGGTACCTTAAC GTAATAAAGAAACTGTATG 3H11032 were used to amplify the gene encoding amino acid residues 14 to 667 of the S of the SARS CoV by reverse transcription PCR This portion of the S was used because it contains the receptor binding domain within the S1 domain that is highly immunogenic whereas the complete S was not expressible in E coli The PCR product was cloned into the BamHI and KpnI sites of vector pQE 31 QIAGEN Hilden Germany The resulting clone was digested by PstI and the PstI fragment which contained the gene encoding amino acid residues 250 to 667 of the S was cloned into expression vector pQE 30 QIAGEN Hilden Germany in frame and downstream of the series of six histidine residues The His 6 tagged rS was expressed and purified from the insoluble fraction using the Ni 2H11001 loaded HiTrap Chelating System Amersham Pharmacia according to the manufacturer s instructions Development of rS based IgG ELISA by different methods of S regeneration The ELISA based IgG antibody test was performed using the regenerated S prepared by dialysis using decreasing concentrations of urea or direct addition of different coating buffers 1 M N acetylglucosamine 4 M urea 4 M urea with 1 M sarcosine 4 M urea with 2 M sarcosine 4 M urea with 3 M sarcosine or 4 M urea with 4 M sarcosine followed by addition of different regeneration buffers 4 M urea 0 1 M Tris HCl 1 mM EDTA pH 8 with 10 glycerol 30 glycerol 50 glycerol 1 M sarcosine 2 M sarcosine 3 M sarcosine 4 M sarcosine 0 5 M ammonium sulfate 1 M ammonium sulfate 0 5 M N acetyl glucosamine 1 M N acetyl glucosamine or 1 M glucose or no regeneration buffer Each well of a MaxiSorp Nunc immuno 96 MicroWell Plate Nalge Nunc International Roch ester N Y was coated with 10 ng of purified His 6 tagged rS prepared by dialysis determined by box titration using different dilutions of His 6 tagged rS as the coating antigen and pooled sera from two SARS CoV pneumonia patients pos itive for antibody against the SARS CoV or direct addition of different coating buffers and incubated at 4 C for 16 h The wells coated with regenerated S prepared by dialysis were blocked in phosphate buffered saline with 5 skim milk whereas different regeneration buffers were added to the wells coated with S prepared in different coating buffers and incubated at 37 C for 1 h before being blocked in phosphate buffered saline with 5 skim milk Diluted 1 20 human sera pooled from 10 healthy blood donors and two SARS CoV pneumonia patients positive for antibody against the SARS CoV by indirect immunofluo rescence 16 respectively were added to the wells of the His 6 tagged rS coated plates in a total volume of 100 H9262l and incubated at 37 C for 2 h After being washed five times with washing buffer 100 H9262l of diluted horseradish peroxidase conjugated goat anti human IgG 1 4 000 antibodies Zymed Laboratories Inc South San Francisco CA was added to the wells and incubated at 37 C for 1 h After being washed five times with washing buffer 100 H9262l diluted 3 3H11032 5 5H11032 tetramethylbenzidine Zymed Laboratories Inc was added to each well and incubated at room temperature for 15 min One hundred microliters of 0 3 M H 2 SO 4 was added and the absorbance at 450 nm of each well was measured Each sample was tested in duplicate and the mean absorbance for each serum was calculated Evaluation of rS based IgG and IgM ELISA for SARS CoV pneumonia Sera from the 148 healthy blood donors who donated blood 3 years ago all negative for IgG antibodies against the SARS CoV detected by our indirect immunoflu orescence assay and 95 SARS CoV pneumonia patients positive for IgG anti bodies against the SARS CoV detected by our indirect immunofluorescence assay 16 were used for the evaluation of the rS based IgG and IgM ELISA Serum samples positive for IgG antibodies against SARS CoV by indirect im munofluorescence assay from the 95 SARS CoV pneumonia patients were taken at a median of 25 days range 12 to 43 days from the onset of symptoms The rS based IgG ELISA was performed as described above using 60 ng of S per well in coating buffer with 4 M urea and 1 M sarcosine for plate coating no regen eration buffer and serum dilution at 1 80 For the rS based IgM ELISA the conditions for the IgG ELISA was used with 200 ng of rS per well serum dilution of 1 80 and diluted horseradish peroxidase conjugate goat anti human IgM 1 10 000 antibodies Biosource International CA Comparison of rS based and rN based IgG and IgM ELISAs for SARS CoV pneumonia The same sera of the 95 SARS CoV pneumonia patients positive for IgG antibodies against the SARS CoV were tested using the N based IgG and IgM ELISA for SARS CoV pneumonia by our previously described method 23 The sensitivities of the rN based IgG and IgM ELISA and rS based IgG and IgM ELISA for SARS CoV pneumonia were compared by the McNemars test RESULTS Development of rS based IgG ELISA by different methods of S regeneration and evaluation of rS based IgG and IgM ELISA for SARS CoV pneumonia Results obtained by the rS based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addi tion of different coating buffers followed by addition of differ ent regeneration buffer identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most opti mal conditions for subsequent experiments as determined by examining the maximum optical density values at 450 nm OD 450 of titrations of the positive and negative pools of sera Box titration was carried out with different dilutions of His 6 tagged rS as the coating antigen and pooled sera from two SARS CoV pneumonia patients positive for antibody against the SARS CoV The results identified 60 ng and 200 ng of purified His 6 tagged rS per ELISA well as the ideal amount for plate coating for IgG and IgM detection respectively To establish the baseline for the IgG and IgM ELISA serum samples all tested negative by the indirect immunofluores cence assay from 148 healthy blood donors who donated blood 3 years ago were tested by the rS based IgG and IgM ELISA For the 148 specimens from healthy blood donors the mean ELISA OD 450 values for IgM and IgG detection were 0 059 and 0 235 respectively with standard deviations of 0 041 and 0 122 Absorbance values of 0 141 and 0 479 were selected as the cutoff values that equaled the sum of the mean values from the healthy control and two times the standard devia tions Fig 1 Using these cutoff values two of the sera ob tained from the 148 healthy blood donors had OD 450 values of H110220 479 by the IgG ELISA and nine had OD 450 values of H110220 141 by the IgM ELISA Fig 1 The specificities of the IgG and IgM ELISA were 98 6 and 93 9 respectively The mean OD 450 values IgG and IgM for the sera obtained from the 95 SARS CoV pneumonia patients positive for IgG antibodies against the SARS CoV detected by our indirect immunofluorescence assay were 0 690 and 0 339 with stan dard deviations of 0 494 and 0 347 Fifty six sera had OD 450 values of H110220 479 by the IgG ELISA and 71 had OD 450 values VOL 43 2005 SPIKE AND NUCLEOCAPSID PROTEIN ELISA FOR SARS 3055 on May 31 2015 by OAKLAND UNIV http jcm asm org Downloaded from of H110220 141 by the IgM ELISA Fig 1 The sensitivities of the IgG and IgM ELISA using the indirect immunofluorescence assay as the gold standard were hence 58 9 and 74 7 respectively Comparison of rS based and rN based IgG and IgM ELISAs for SARS CoV pneumonia The same 95 serum sam ples of the SARS CoV pneumonia patients positive for IgG antibodies against the SARS CoV were tested using the N based IgG and IgM ELISA for SARS CoV pneumonia Ninety 94 7 and 53 55 8 of the 95 serum samples were positive by the rN based IgG and IgM ELISA respectively The sen sitivity of the rN IgG ELISA was significantly higher than that of the rS IgG ELISA PH11021 0 001 whereas the sensitivity of the rS IgM ELISA was significantly higher than that of the rN IgM ELISA P H11021 0 01 The sensitivities of the rS based ELISA and rN based ELISA for detection of IgG and IgM in serum samples ob tained from patients at different periods after disease onset are shown in Table 1 For IgG detection the sensitivity of the rN ELISA was significantly higher than the rS based ELISA for serum samples obtained from patients at 16 to 20 21 to 25 and 26 to 30 days after disease onset P H11021 0 005 H110210 001 and H11021 0 05 respectively For IgM detection the sensitivity of the rS based ELISA was significantly higher than the rN based ELISA at 21 to 25 days after disease onset P H11021 0 05 Performance of combination of rN based and rS based ELISAs The results for IgG and IgM detection in the 95 serum samples from patients with SARS CoV pneumonia when the rN based ELISA and the rS based ELISA were combined is shown in Table 2 For IgG detection there was no significant difference between the sensitivity of the two ELISAs combined 97 and that of the rN based ELISA 95 but the sensi tivity of the two ELISAs combined and that of the rN based ELISA were significantly higher than that of the rS based ELISA 59 P H11021 0 001 in both comparisons For IgM de tection the sensitivity of the two ELISAs combined 84 and that of the rS based ELISA 75 were significantly higher than that of the rN based ELISA 55 P H11021 0 001 and P H11021 0 01 respectively but there was no significant difference be tween the sensitivity of the two ELISAs combined and that of the rS based ELISA FIG 1 Evaluation of sensitivity and specificity of rS based IgG A and IgM B antibody ELISA for SARS CoV pneumonia Serum specimens were obtained from 95 patients with SARS CoV pneumonia and control serum specimens were obtained from 148 healthy blood donors The test results were plotted as OD 450 values The cutoff line for positive diagnosis is drawn at a value that equals the sum of the mean value and two times the standard deviation for the healthy blood donors TABLE 1 Differential sensitivities of rS and rN based ELISA for detection of IgG and IgM in serum samples obtained at different periods after disease onset Days after disease onset No of serum samples No positive for IgG by ELISA No positive for IgM by ELISA N based S based N based S based 11 15 2 2 100 0 0 1 50 1 50 16 20 17 15 88 7 41 11 65 13 76 21 25 31 31 100 18 58 15 48 24 77 26 30 27 24 89 16 59 16 59 21 78 31 35 7 7 100 6 86 3 43 4 57 36 40 5 5 100 4 80 4 80 4 80 41 45 6 6 100 5 83 3 50 4 67 3056 WOO ET AL J CLIN MICROBIOL on May 31 2015 by OAKLAND UNIV http jcm asm org Downloaded from DISCUSSION In this study we report the development of the rS based ELISA for serodiagnosis of SARS CoV pneumonia Although previous studies have been able to detect S specific antibodies in patients with SARS by Western blot analysis or immunoflu orescence assay 10 19 there are no published data on the availability of S based ELISA for high throughput analysis of antibodies against SARS CoV S This is likely due to the dif ficulties in stably expressing and purifying the S which is gly cosylated with high mannose and or hybrid oligosaccharides The detection of S by immunoassays using human convales cent phase sera was found to be difficult by some researchers who even suggested that the protein may not be strongly im munogenic 7 When we first performed the ELISA for IgG detection using regenerated S prepared by the traditional method of dialysis with decreasing concentrations of urea a significant amount of S was lost during dialysis Moreover 600 ng of rS was required for coating the ELISA plate to obtain a reasonable absorbance value for sera obtained from patients with SARS CoV pneumonia data not shown When 10 ng of rS was used for coating the ELISA plates only a very low absorbance value could be achieved Since it was very time consuming labor intensive and expensive to produce such a large amount of rS for coating ELISA plates various other methods that were reported in the literature for regeneration of resolubilized denatured proteins were examined for their usefulness in the regeneration of the S In a previous study 4 M urea with 4 M sarcosine was observed to be useful for regeneration of lysozyme an enzyme well known to be difficult to regenerate after denaturation 14 In the present study we found that 4 M urea with 1 M sarcosine for plate coating and no regeneration buffer were the most optimal conditions for the ELISA ratio of OD 450 for positive control to OD 450 for negative control H11005 3 85 better than using 4 M urea alone or with 2 3 or 4 M sarcosine ratios of OD 450 for positive control to OD 450 for negative control H11005 2 15 3 25 3 13 and 2 76 respectively The rN based IgG ELISA is more sensitive than the rS based IgG ELISA but the rS based IgM ELISA is more sen sitive than the rN based IgM ELISA for SARS CoV pneumo nia We have reported that the rN based IgG ELISA was useful for serodiagnosis of SARS CoV pneumonia especially during an epidemic of SARS As the prevalence of background seropositivity in the general population increases due to SARS CoV pneumonia and nonpneumonic SARS CoV infec tions single IgG readings may not be useful for serodiagnosis of SARS CoV pneumonia In such circumstances detection of IgM antibody or a rise in IgG titers in serial samples may be the method of