Development And Clinical Application Of Up-converting Phosphor Technology-based Lateral-flow Assay For Rapidly Quantitative Detection Of Hepatitis B Surface Antibody

ObjectiveHepatitis B virus(HBV)is one of the most important causes of chronic hepatitis, liver cirrhosis,and hepatocellular carcinoma.It is estimated that there are nearly 300 million persistent carriers of HBV around the world with one-third of them in China. About 45%of the world population is living in the high-risk regions of chronic HBV infection,200 million people are infected with HBV and 1 million of them die from hepatitis B-correlated diseases,such as hepatic failure,hepatic cirrhosis and primary hepatic carcinoma.The identification of HBV as a main cause of hepatitis and development of effective vaccines for hepatitis B prevention are great achievements in the last century for understanding hepatitis.China is a high-prevalent region of HBV, and positive rate of hepatitis B surface antigen(HBsAg)is 9.75%.Although the vaccination is effective,there are still some nonresponders to hepatitis B vaccine.Many factors,including aging,smoking,obesity,gender,and genetic factors were found to contribute to the decreases of immune response,and male nonresponders were more frequent than female.Therefore,it is crucial to check postvaccination HBsAb quantitatively in all vaccinees,especially in vaccinated health care workers(HCWs),which is important to prevent nosocomial transmission of HBV. Quantitative HBsAb detection is also important for those who received hepatitis B immunoglobulin(HBIg)treatment for preventing maternal transmission and physical transmission by accidental exposure.Liver transplant recipients usually need receiving HBIg for preventing hepatitis B.The level of HBIg in the blood should be kept above 100mIU/mL for the effective prevention).Therefore,a quantitative measurement of HBIg in blood should be developed to meet this demand. Up-converting phosphor is a kind of lanthanide-containing,submicrometer-sized, ceramic particle,and its special composition and structure provide a unique optical feature that it can emit visible light when excited by infrared light.This energy-up-converting phenomenon makes UCP a reporter free from photobleaching and interference of sample autofluorescence.Therefore,the high sensitivity and stability are ensured for rapid detection.Combining UCP as a reporter with lateral-flow (LF)assay(UCP technology-based LF[UPT-LF]assay)lays a solid foundation for accurately quantitative detection with high sensitivity.Various UPT-LF strips have been developed,which have demonstrated the usefulness of UCP reporters for the sensitive detection of drugs of abuse,nucleic acids, interferonγ,Schistosoma circulating anodic antigen,Escherichia coli,Yersinia pestis, respiratory syncytial virus,and so on.However,no application of UCP-LF quantitative detection in HBV serum makers is reported up to now.The aim of this study was to develop a UPT-LF assay for quantitative detection of HBsAb and evaluate its feasibility using clinical samples.MethodsTwo pairs of primers of gene coding HBsAg were designed and synthesize,using The Software PCGENE.The target gene was amplified,and PCR products were recovered,purified,and ligated with TA clone of pGEM-Teays.The S gene was derived from sub-clone of plasmid vector,ligated with expression vector pRESET-A, and the ligated products were transformed to competence DH5α.The recombinant clones were selected and identified.The transformed single colony of engineering bacteria was inoculated to LB(containing ampicillin 50μg/ml and alficetin 35μg/ml), cultured at 37℃overnight.The isopropy-β-D-thiogalactoside(IPTG)was added into culture(the final concentration is 1mol/L),then the culture was induced and cultured for 4h.The bacteria were collected and optimal inducing concentration was determined. The expression product was analyzed with Tri-Gly SDS-PAGE.The bulk of engineering bacteria were expressed and primarily and purified.His fusion protein was purified with HisTrap affinity column.Finally,the expression roduct was analyzed through Western blot.The UCT-LF strip consists of 5 parts:sample pad,conjugate pad,analytical membrane wicking pad and laminating card,sample pad,conjugate pad and analytical membrane wicking pad were fixed to laminating card by glue.HBsAg and goat anti-HBsAg were coated on the analytical membrane as the test line and control line, respectively,and UCP-HBsAg conjugate was immobilized on the conjugate pad.A special ending index line that can show a color change was used to indicate the end of immunochrornatographic reaction.The finished strip was put into the plastic cartridge to stabilize overlaps on the strip with the sample pad,the analytical membrane(the test line and control line),and the absorbent pad(the ending index line)corresponding to the sample adding window,the result scanning window,and the ending index window, respectively.For detection of a sample by UPT-LF strip,a mixture of 70-μL serum sample and 70-μL sample-treating buffer was added to the sample adding window.Once the absorbent pad became sodden(approximately 10 min after sample application),the flow of the sample would stop while the ending index line on the end of the absorbent pad turned from white to blue,indicating that the result could be obtained by the UPT-based biosensor.For data collection,the UPT-based biosensor scanned the test line and control line on the analytical membrane.A positive sample gave 2 major peak signals(test and control lines),whereas a negative one showed a single major peak signal(control line only).The peak areas corresponding to the test line and control line were assigned as T(test)and C(control),respectively,and T/C ratio was reported as the detection result.The HBsAg sandwich ELISA was used to detect HBsAb.The result was shown as OD(Optical Density)value with cutoff threshold of 0.105.The Abbott Axsym AUSAB(AUSAB)assay use recombinant HBsAg on microparticles as the solid phase and biotin coupled to recombinant HBsAg as the conjugate.Alkaline phosphatase-conjugated antibiotin antibody will bind to the antigen sandwich.The quantitative range of AUSAB is from 0 to 1000 mIU/mL with the cutoff threshold of 10 mIU/mL.Fifty-two serum samples from healthy people were tested by UPT-LF assay to determine the cutoff threshold.Thirteen samples with standard concentrations from 20 to 900 mIU/mL were tested thrice by each of the 3 assays to estimate their reproducibility and sensitivity,to deduce the standard quantitative equation of UPT-LF assay,and to evaluate the accuracy of quantitative detection by AUSAB assay.Then, 306 clinical serum samples were tested by the 3 assays simultaneously to estimate the sensitivity,specificity,and concordance of the assays. Results1.Cloning and expression of HBsAg geneAfter PCR amplification and agarose gel electrophoresis,we found a 700 bp-size specific fragment without non-specific products.The pGEM-Teasy-HbsAg was digested with EcoR I,a gene fragment coding HbsAg was obtained,which is correspondent with predictive molecular weight.The forword and reverse sequecing results proved that HbsAg expression vector was successfully construted.After HBV-S gene coded products was expressed and purified,the result of SDS-PAGE showed that purity of recombinant-HBV S protein(r-BS)is over 90%,which could be employed as an immunogen.2.Development of UPT-LF assayFifty-two samples from the healthy people were tested to determine the cut-off threshold of UPT-LF assay and T/C ratios that ranged from 0.053 to 0.083 with an average((?))of 0.065 and a standard deviation(s)of 0.009.Thus,the cutoff threshold of UPT-LF assay for HBsAb detection was calculated to be 0.092((?)+3s).We deduced the standard quantitative equation by testing 13 standard samples with concentrations of HBsAb ranging from 20 to 900 mIU/mL.y=327.93m(x)+783.43Thirteen samples with known concentration of HBsAb were also tested thrice by ELISA and AUSAB for comparing reproducibility and sensitivity of these 3 assays. The coefficient of variation of the UPT-LF assay ranged from 3.000 to 25.157(with an average of 15.490),whereas that of ELISA and AUSAB were from 2.446 to 754.983 (with an average of 93.416)and from 0.994 to 48.540(with an average of 27.213), respectively.3.Clinical application of UPT-LF assayThree hundred and six clinical srum samples were detected by the 3 assays.The same results from the 3 assays were gotten in 279 samples with 226 positive and 53 negative,whereas 27 samples showed the discordant results(i.e.,not all assays showed the same results rather some were positive and some were negative).UPT-LF assay exhibited the highest sensitivity(99.18%)among the 3 assays(97.55%for ELISA and 95.51%for AUSAB).Although the specificity of UPT-LF assay(95.08%)was not satisfactory compared with that of AUSAB(100%),the adjusted agreement of UPT-LF assay(97.43%),which stood for the accuracy of a detecting technique,was the highest among the 3 methods.As far as AUSAB assay was concerned,the relative low sensitivity had a negative effect on its performance(with the adjusted agreement of 95.06%).For ELISA,the insufficient sensitivity and specificity and the inability of quantitative detection limited its application.Conclusions1.The prokaryotic expression vector of HbsAg containing 226 amino acids was sucessfully constructed,and expressed in E.coli.The detection and identification results of SDS-PAGE,western blot and ELISA showd that molecular size expression product conform to prediction,which lays a foundation for further study of HbsAg detection.2.The use of UCP as the reporter further improves LF assay sensitivity and specificity and renders it with the quantitative capability.3.A UCT-LF assay for rapidly quantitative detection of HbsAb was successfully developed in this study,it exhibited the highest sensitivity and reproducibility among the 3 assays(UPT-LF,AUSAB and ELISA).Our results promise UPT-LF a feasible assay in clinical applications.