【作者】 李鲁平；

【导师】 吕昌龙；

【作者基本信息】 中国医科大学 ， 免疫学， 2009， 博士

【摘要】 目的乙型肝炎病毒(Hepatitis B Virus,HBV)是一种严重危害人类健康的致病因子,是慢性肝炎、肝硬变和肝细胞癌最重要的病因之一。据估计,全球有3亿人是HBV的持续携带者,其中1/3在中国。全球约45%的人口生活在慢性HBV感染的高风险地区,约20亿人感染HBV,每年有100万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝癌。我国属HBV感染高流行区,一般人群的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性率为9.75%。尽管疫苗接种卓有成效,但仍有部分人群对乙肝疫苗无应答。诸多因素,包括衰老、吸烟、肥胖、性别和遗传因子等均与免疫应答的降低相关,男性无应答者的出现频率高于女性。因此,定量检测各类疫苗接种者在接种后的乙型肝炎表面抗体(hepatitis B surface antibody,HBsAb)水平十分必要,特别是检测疫苗接种后的医务工作者,对防止HBV在医院内的传播具有重要意义。对于那些为防止因偶然因素导致母婴传播和接触传播而注射乙肝免疫球蛋白(hepatitis Bimmunoglobulin,HBIg)的个体来说,定量检测HBsAb也十分重要。肝移植接受者为了预防乙肝,通常需要注射HBIg。为了有效预防乙肝的发生,血液中的HBIg水平应当高于100 mIU/mL。因此,开发血液中HBIg的定量检测技术也势在必行。上转发光(up-converting phosphor,UCP)材料是一种含有稀土元素的亚显微陶瓷微粒,它的特殊组成和结构为其提供一个独特的光学特征,既在红外光激发下,可以发射可见光。这种能量上转现象使UCP成为一种能够摆脱荧光焠灭现象和样品自发荧光干扰的示踪物。UCP作为示踪物与免疫层析(lateral-flow,LF)技术结合而成的上转发光免疫层析检测技术(UCP technology-based LF assay,UPT-LF)为高敏感性的、精确的定量检测奠定了坚实的基础。目前已经开发出多种UPT免疫层析试纸,并且在违禁药品监控、核酸、INFγ、血吸虫循环抗原、大肠埃希菌、鼠疫耶尔森菌、呼吸道合胞病毒等的敏感检测中显示了UCP示踪物的作用。但迄今为止,在国内外的相关文献中,尚未发现UCP-LF在HBV血清标志物定量检测方面的应用。本研究的目的是研制一种用于定量检测HBsAb的上转发光免疫层析技术,并通过对临床样品的检测评估其应用的可行性。实验方法利用PCGENE计算机软件,分析已知的HBV基因组序列,结合国内克隆的HBsAg编码区序列设计、合成引物。扩增目的基因的,回收、纯化PCR产物,与pGEM-Teays载体的TA克隆连接。从亚克隆质粒载体中获取S基因,与表达载体pRESET-A的连接,采用DH5α感受态细菌试剂盒转化连接产物,筛选及鉴定重组克隆。挑取转化工程菌单菌落,接种于LB(含氨苄青霉素50μg/ml和氯霉素35μg/ml)液体培养基,37℃振荡培养过夜。培养物中加入异丙基硫代半乳糖昔(IPTG)至终浓度为1 mol/L,37℃振摇培养,诱导培养4h。收集细菌,选择最佳IPTG诱导浓度。Tris-甘氨酸SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,大量表达工程菌,采用His Trap亲和层析柱纯化His融合蛋白,Western blot分析表达产物。UCT免疫层析试纸主要是由五部分组成,包括:样品垫、结合垫、分析膜、吸水垫和粘性底衬。样品垫、结合垫、分析膜和吸水垫通过粘胶固定于粘性底衬上。HBsAg和羊抗HBsAg分别包被在分析膜上作为检测带和质控带。将UCP-HBsAg结合物固化在结合垫上,特殊的终点指示带能显示颜色变化,用以提示免疫层析反应的结束。将制备完成的测试条放入塑料盒式存储器内,样品垫、分析膜(检测带和质控带)、吸收垫(终点指示带)的连接部分重叠,分别对应加样孔、结果扫描窗和终点指示窗。将70μL血清样品和70μL样品处理缓冲夜混合后,加到加样孔中。当样品垫被浸透后(约在加样后10 min),样品的流动将停止,而此时在吸水垫末端的终点指示带将由白色转变为蓝色,提示UPT传感器读取结果。UPT传感器扫描在分析膜上的检测带和质控带,以采集数据。阳性样品将出现2个主要的峰值信号(检测带和质控带),而阴性样品将只出现一个单一的主要峰值信号(只有质控带)。对应检测带和质控带的峰值区域分别命名为T和C,T/C的比率即为检测结果。在本研究中是采用本法检测52份健康献血者的血清样品,以确定其cut-off值。本研究中,同时应用目前常规检测HbsAb的二种方法抗原夹心法ELISA和雅培酶促免疫荧光试验(Abbott Axsym AUSAB assay,AUSAB)检测HBsAb,以估价UPT-LF测定法的特异性和敏感性。抗原夹心法ELISA检测结果以OD值表述,cut-off直为0.105。AUSAB法以结合在微粒子表面的重组HBsAg(ad/ay)作为固相抗原,以连接在重组HBsAg上的生物素为标记物。碱性磷酸酶标记的抗生物素抗体与抗原-生物素复合物结合。AUSAB的定量范围为0～1000 mIU/mL,其cut-off值是10 mIU/mL。为评价实验的重复性和敏感性,推导UPT-LF试验的标准量化方程,对13份标准HBsAb阳性血清样品(已知浓度为20～900 mIU/mL不等)均采用3种方法测试;进而检测306份临床样品,每份样品均采用上述3种方法进行检测,评估其敏感性,特异性和UCT-LF检测结果同另二种检测结果的符合率。实验结果一、乙型肝炎病毒表面抗原基因的克隆与表达HBsAg基因扩增产物经琼脂糖凝胶电泳,可见扩增出一条大小约700bp的特异性片段,无非特异性产物。以限制性内切酶EcoR I酶切质粒pGEM-Teasy-HBsAg,获得HBsAg编码基因目的片段,约700bp,与预期值相符。DNA双向测序证实,证明HBsAg表达载体已成功构建。HBV S基因编码序列产物经大量表达和纯化,SDS-PAGE鉴定显示,重组表达HBV表面抗原S蛋白(r-BS)的纯度在90%以上,可以作为进一步使用的免疫原。二、UPT-LF方法的建立检测52份健康献血者血清样品获得的T/C比率范围为0.053～0.082,(?)=0.065,s=0.009。UPT-LF测定法检测HBsAb的cut-off值确定为0.092((?)+3s)。通过测试13份标准样品(已知HBsAb浓度为20～900 mIU/mL不等),推导出标准量化方程。方程式如下:y=327.93ln(x)+783.43为比较不同测定法的重复性和敏感性,对13份已知HBsAb浓度的样品同时进行了ELISA和AUSAB法的检测。UPT-LF方法的变异系数为3.000～25.157((?)=15.490),而ELISA和AUSAB方法的变异系数分别为2.446～754.983((?)=93.416)和0.994～48.540(x=27.213)。三、UPT-LF方法的临床应用本实验同时采用上述三种测定法对306份临床血清样品进行了检测,其中279份样品在三种方法检测中获得相同结果:226例阳性,53例阴性,符合率为91.2%。其余27份样品的三种检测结果不相符合(即对某一样品的检测结果,有的方法呈阳性,有的方法呈阴性)。按统一后的标准(245例真实阳性,61例真实阴性)评价三种方法的表现。UPT-LF在三种方法中的敏感性最高(99.18%),ELISA的敏感性为97.55%,AUSAB为95.51%。虽然与AUSAB(特异性100%)相比,UPT-LF方法的特异性(95.08%)不令人满意,但该法的调整一致性(代表检测技术的精确性)为97.43%,在三种方法中位居首位。对AUSAB方法而言,相对较低的敏感性是其在操作中的负面效应(调整一致性为95.06%)。ELISA方法因敏感性和特异性的欠缺,以及不能进行定量检测而限制了它的应用。结论1、本实验成功构建了对应226个氨基酸的HBsAg原核表达质粒重组体并在大肠杆菌中进行了表达。表达产物经SDS-PAGE、western blot及ELISA检测和鉴定,分子量大小与预期相符,为进一步对HBsAb检测方法的研究奠定了基础。2、UCP作为示踪物进一步增强了免疫层析技术的敏感性和特异性,并赋予其定量检测能力。3、成功建立了用于HbsAb快速定量检测的UPT-LF方法,该技术在三种检测方法(UPT-LF、AUSAB和ELISA)中敏感性最强和重复性最好。并经临床样品检测结果证明UPT-LF的实际应用的可行性。更多还原

【Abstract】 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.更多还原