Update on SARS diagnostic assays in China

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Update on SARS diagnostic assays in China

• Youchun Wang MD PhD
• Department of Cell Biology
• National Institute for the Control of
  Pharmaceutical and Biological Products

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Assays to detect SARS virus antibody

• Immunofluorescence Assay(IFA)
• Indirect ELISA
• Sandwich ELISA under development
• Protein chips under development

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The expression of nucleic protein of SARS virus
Provided by professor Bi Shenli, Institute of
Virology, CDC
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Expression part of spike protein
Provided by professor Bi Shenli, Institute of
Virology, CDC
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Establishing reference panel of antibody

• Collecting about 100 samples from different
  population except SARS patients
• Collecting about 100 samples from SARS patients
  at different phase
• All the samples were tested with different
  antibody assays
• The candidate panel were selected from the
  samples above according the results

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The composition of lab reference panel for IgM
antibody
Samples Number
Negative samples 20 Positive samples 11 Diluted samples 6(12164) Samples for CV 1
Some negative samples are positive for
rheumatoid factor, some positive for measles
virus antibody, some positive for flu virus
antibody and other positive for HEV
antibody. Among positive samples, some are weak
positive, some are strong positive and others
middle positive. The diluted samples are positive
for IgM antibody only.
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Detecting results with different assays (IgM)
Assays Negative Positive dilution CV (15)
A 20/20 11/11 14 B 20/20 11/11 14 lt15 C 20/20 11/11 132 lt15
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The composition of lab reference panel for IgG or
total antibody
Samples Number
Negative samples 20 Positive samples 18 Diluted samples 7(181512) Samples for CV 1
Some negative samples are positive for
rheumatoid factor, some positive for measles
virus antibody, some positive for flu virus
antibody and other positive for HEV
antibody. Among positive samples, some are weak
positive, some are strong positive and others
middle positive. The diluted samples are positive
for IgG antibody only.
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Detecting results with different assays(IgG or
total antibody)
Assays Negative Positive dilution CV (15)
A 20/20 18/18 132 B 20/20 18/18 1128 lt15 C 20/20 14/18 132 lt15 D 20/20 18/18 1256 lt15 E 20/20 10/18 lt18
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Detecting results of SARS virus antibody
Days after Number Positive rate Positive rate Positive rate onset for IgG for IgM for total
1-5 18 0 0 0 6-10 26 0 11.5 7.7 11-15 16 43.8 56.3 87.5 16-20 14 92.9 100 100 21-25 10 60 80 100 26-30 16 100 100 100 Total 100 42 50 56
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IgM and IgG response in series sera of SARS
patients
Patients days after IgM IgG onset Patients days after IgM IgG onset
A 6 - - 10 - 14 - B 7 - - 15 30 42 C 2 - - 12 - 25 D 7 - - 13 28
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Specificity of different antibody assays
Samples Number IgG IgM Total
Hepatitis A 70 0 2.9 2.9 Hepatitis B 30 0 0 0 Hepatitis C 30 0 0 0 Measles 10 0 0 0 Mumps 6 0 0 0 Blood donors 15 0 0 0 Contacted with SARS patients 26 3.8 0 3.8
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SARS RNA diagnostics under development

• DNA chips
• Several institutes try to develop
• Can detect several respiratory viruses
  simultaneously
• Real time RT-PCR
• Several institutes or manufactures try to
  develop
• Most of them use Taqman technology
• Develop qualitative and quantitative
  assays

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Taqman Probe
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Principle of quantitative analysis
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Establishing the reference panel of SARS virus RNA

• Collecting respiratory virus and other virus
  positive samples
• Collecting SARS virus RNA positive samples from
  patients and cell culture
• Extracting SARS virus RNA and the purified RNA
  was kept in special solution which is similar to
  serum
• The candidate panel was detected using different
  RT-PCR assays and calibrated with WHO standard

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Curve of WHO SARS virus standard
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Calibrating results of positive
samples (P1-P6)
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Calibrating results of positive
samples (P7-P10)
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The composition of SARS virus RNA reference panel

• 9 negative samples
• including HCV RNA, HIV RNA, Measles virus
  RNA, Mumps virus RNA, Rubella virus,
  Respiratory syncytial virus, Japanese
  encephalitis virus, Muries hepatitis virus
• 10 positive samples
• P1-P6 samples used as quantitative analysis
• P3 P6 samples used as linearity analysis
• P6 sample used as evaluation of CV
  (Coefficient of variation)
• P7-P9 samples used as sensitivity

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Detecting results of Artus RT-PCR using different
extraction methods
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Detecting results of one RT-PCR using different
extraction methods
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Detecting results of one RT-PCR using different
extraction methods
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Conclusion

• Lab reference panel for IgM, IgG, total antibody
  and virus RNA have been established
• The virus RNA panel has been calibrated with WHO
  standard
• The purified virus RNA used in panel cannot
  affect efficiency of extraction and detection by
  different assays
• Those panels can differentiate the quality of
  different assays
• IgM antibody may be useful marker for early
  diagnosis

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Acknowledgements Li xiouhua, Meng
Shufang, Lin Lin, Zhang Chuntao From department
of cell biology, NICPBP Mao Panyong from
Infectious Hospital Bi Shengli, Xu Wenbo from
institute of virology, CDC
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Thanks