Constructing A Novel Biochip Method For EBV Serum Antibody Detection

Objectives: To develope an easy accessible biochip for the detection of Epstein-Barr virus(EBV)virus capsid antigen(VCA)Ig M antibody,virus capsid antigen Ig G antibody and nuclear antigen 1 protein(EBNA-1)Ig G antibody in serum.Methods: EBV VCA and EBNA-1 probes immobilized on the solid carrier of the protein biochip;solid support was gold chip with chemical modification of S-S-PEG-COOH,the carboxyl group was activated by EDC and NHS and then fixed with specific antigen as a probe.The surface chemical characteristics of the biochips were identified by atomic force microscope(AFM)and Fourier Translation Infrared Spectroscopy(FTIR).The chip parameters were optimized through the quality control of antigen and antibody binding and serum dilution.In the biological immune detection of antigen probed chip,the relative sensitivity to CLIA standards for comparison was detected,and the antibody blocked by excessive antigen was proceeded to verify the specific binding of serum antigen and antibody.To analyze correlation of the EBV antigen-probed biochip with CLIA-L,two hundred and seventy-four patients’ sera representing various EBV serological profiles were assayed simultaneously;the cut off value was defined as three times of control value,the data were analyzed using chi-square test of SPSS,P-values of 5% or lower was considered to be statistically significant.Finally,a combined biochip was used to detect EBV infection in 14 patients with infectious mononucleosis(IM)and 25 patients with systemic lupus erythematosus(SLE).Results: The results indicating that chemical functional groups were covalently bound on the gold surfaces and formed SAM.Compared with CLIA,the protein chip showed similar sensitivity and specificity,and the detection limit of the EBV antibodies by the biochip was nearly identical to that by CLIA-L(2.91U/ml v.s.3.00U/ml for EBNA-1Ig G,8U/ml vs.10U/ml for EBV-VCA Ig G).The fluorescence values by the biochip displayed a linear correction to the values by the CLIA-L in the cases whose EBNA1-Ig G titres ranged between 6U/ml and 600 U/ml in EBNA1-Ig G and EBV VCA-Ig G titres between 17U/ml and 480U/ml,respectively(R2=0.8246 and R2=0.8128),indicating the biochip method has similar detection results as CLIA-L.Testing of the three serological antibodies in 274 clinical sera,the positive rate tested by the biochip and by the CLIA-L was 69.34% vs.71.89% in EBNA-1 Ig G(P=0.230),73.72% vs.71.17% in EBV-VCA Ig G(P=0.311)and 6.93% vs.4.74% in EBV-VCA Ig M(P=0.210),respectively(P>0.05).Finally,the combined biochip was successfullyused for diagnostic identification of EBV infection in 14 patients with infectious mononucleosis(IM)and 25 patients with systemic lupus erythematosus(SLE).In IM,14 out of 15 cases appeared positive VCA-Ig M antibody,15 out of 15 cases appeared positive VCA-Ig G antibody and 3 out of 15 showed positive response to EBNA-1 Ig G antibody.Conclusion: We designed a valuable S-S-PEG chemical modified gold surface platform and thereby developed a protein biochip,aiming at EBV serological screening,this chip could be used not only for diagnosis of EBV infection in a variety of EBV associated diseases,but also for a large-scale of EBV epidemiological screening in human vulnerable population.The biochip enabled concurrent detection of infectious antibodies against EBV and confirms infection status.It would be a versatile tool for large-scale epidemiological screening according to miniaturization and high throughput.