Screening of specific antigens for SARS clinical diagnosis using a protein microarray.
In this study several SARS-CoV structural proteins and fragments were expressed in E. coli as GST or TRX fusion proteins. They were fabricated on a microarray and tested with sera from SARS patients. Antigenic screening indicated that recombinant GST-N2 fusion protein, the carboxy-terminus 213aa-423aa of N protein, was strongest positive and weakest non-specific compared with others. An indirect antibody ELISA method was developed and clinical positive and negative sera for their antibodies against GST-N2 fusion protein were assayed. 311 out of the 442 sera from clinical SARS inpatients, as well as 229 out of 302 sera from convalescent patients gave positive reactivities; positive rates were 70.4% and 75.8% respectively. Sera from a total of 2726 non-SARS patients and healthy individuals were tested and the false positive rate was only 0.07%. When the sensitivity control sample was diluted 1 : 64, it yielded OD values above the cutoff value. Reported data showed that this was a relatively high degree of sensitivity and specificity for SARS-CoV antibody testing. The data indicate that GST-N2 fusion protein, which was screened by protein microarray, may be a valuable diagnostic antigen for the development of serological assays for SARS. In addition, protein microarray assay presents a higher positive rate and sensitivity (86.1% and 1 : 200) compared with the traditional ELISA screening method, and could provide a rapid, parallel and high-throughput antigen screening platform.
Lu
Dan-Dan
DD
Beijing Institute of Radiation Medicine, Beijing, 100850, People's Republic of China.
Chen
Su-Hong
SH
Zhang
Shi-Meng
SM
Zhang
Min-Li
ML
Zhang
Wei
W
Bo
Xiao-Chen
XC
Wang
Sheng-Qi
SQ
D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000302:isolation & purification; D019149:Bioreactors; D016022:Case-Control Studies; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins; D027361:Organic Anion Transporters / Q000235:genetics; D040081:Protein Array Analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D027361:Organic Anion Transporters; D011993:Recombinant Fusion Proteins; C494631:VAC14 protein, human; C487009:SLCO6A1 protein, human