Development of a novel fast immunoassay for HBV genotyping and study on the clinical significance of the quantitative hepatitis B core antibody level

乙型肝炎病毒（HepatitisBvirus,HBV）感染是一个严重的，世界范围内的公共卫生问题，可以导致肝炎，肝硬化（LC）和肝细胞癌（HCC），每年可以引起100万以上的人死亡。在HBV感染的临床诊断中，不同的HBV相关标志物指示了病毒产生，病毒多态性以及宿主免疫反应三个方面，从而全面了解HBV感染者的疾病状态，预测自然转归和治疗应答，监测疾病进展和治疗效果，对HBV感染者的管理和治疗起到关键的指导作用。目前在指示病毒产生的标志物方面，HBsAg，HBVDNA和HBeAg的检测手段相对成熟，商品化试剂检测性能优秀，已广泛应用于临床，然而指示病毒多态性的HBV基因型的检测受到方法学的限制难以...The hepatitis B virus (HBV) is a serious, worldwide public health problem because HBV infection can cause hepatitis, liver cirrhosis (LC) and hepatocellular carcinoma (HCC), resulting in more than one million deaths annually. In the clinical diagnostic of HBV infection, different HBV related biomarkers reflect the production of virus, virus polymorphism, and the host immune response, in order to f...学位：理学博士院系专业：生命科学学院_细胞生物学学号：2162011015395

Application of a novel domestic hypersensitive HBsAg reagent in blood screening

目的对1种新型国产超敏HbSAg试剂在血液筛查中的应用进行评估。方法用考核试剂WT ClEIA分别检测WHO标准品、HbV阴性的血清、各种HbV亚型及HbSAg突变株标本以及无偿献血者标本,并与参比试剂做比较。结果 WT ClEIA对HbSAg阴性标本的检测特异性为99.81%(95%CI:99.57%~99.93%);对WHO标准品检测的分析灵敏度可达0.012 Iu/Ml,高于参比试剂HEPAnOSTIkA HbSAg ulTrA的0.05 Iu/Ml和AbbOTT MurEX V3的0.1 Iu/Ml;对HbSAg突变株及不同亚型的检出率也高于2种参比试剂;对近5 000份标本的检测结果与参比试剂AbbOTT MurEX V3有高度的符合率(99.60%)。结论该试剂检测性能良好,在血液筛查中有较好的应用前景。Objective To study the application of a novel domestic hypersensitive HBsAg reagent in blood screening.Methods The reagent for evaluation named WT CLEIA together with reference reagents were used to test WHO standard,HBV negative samples and specimens with various HBV subtypes and HBsAg mutants.Results The specificity of WT CLEIA was 99.81%(95% CI: 99.57% ～ 99.93%).The analytical sensitivity of WT CLEIA was 0.012 IU / ml,which was higher than Hepanostika HBsAg Ultra(0.05 IU / mL) and Abbott Murex V3(0.1 IU / mL);WT CLEIA also had a higher detection rate of HBsAg mutants and subtypes than these 2 reference reagents.In addition,WT CLEIA had a high accordant rate with Murex V3 in blood donors HBsAg screening.Conclusion WT CLEIA has a good performance in HBsAg screening and would have a good prospect of application in blood screening.福建省自然科学基金面上项目(2011D007); 厦门市输血医学重点专科建设项目(2012-2014

Development and application of CK-MB specific monoclonal antibodies

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(mAb)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB; 定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-; MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评; 价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对mAb,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏; 试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK; -BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930; 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检; 测结果符合率高。The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB); specific monoclonal antibodies (mAb), and characterize the monoclonal; antibody and further development of quantitative detection assay for; CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then; monoclonal antibodies were prepared according to conventional hybridoma; technique and screened by indirect and capture ELISA method. To identify; the epitopes and evaluate the classification, purchased creatine kinase; isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes,; with immunoblotting and synthetic CK-MM and CK-BB in different linear; epitope. A double antibody sandwich ELISA was applied to screen the mAb; pairs for CK-MB detection, and the quantitative detection assay for; CK-MB was developed. We used 74 cases of clinical specimens for; comparison of our assay with Roche's CK-MB assay. We successfully; developed 22 strains of hybridoms against CK-MB, these mAbs can be; divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross; monoclonal antibody and CK-MB specific reaction with partial; conformational monoclonal antibody, and CK-MB quantitative detection; assay was developed by using partial conformational monoclonal antibody.; The correlation coefficient factor r of our reagent and Roche's was; 0.930 9. This study established a screening method for CK-MB partial; conformational specific monoclonal antibody, and these monoclonal; antibodies were analyzed and an established quantitative detection assay; was developed. The new assay had a high concordance with Roche's.厦门市科技计

Analysis of pre-S/S gene in occult hepatitis B virus infection from blood donors in Dalian,China

目的了解大连地区无偿献血者隐匿性肝炎乙型病毒感染(ObI)的情况和PrE-S/S区基因的变异情况。方法对大连市血液中心2010年12月2日-2013年5月31日的无偿献血者血液标本进行常规ElISA(HbS Ag、抗-HCV、抗-HIV和抗-TP)和HIV/HbV/HCV联合nAT筛查,对于单独核酸检测反应性的献血者加以跟踪或回溯,结合乙型肝炎血清学标志物的试验、鉴别试验、病毒定量试验和半巢式PCr来确定ObI,同时对ObI的PrE-S/S区基因序列与对照组(HbS Ag+序列,gEnbAnk)做比对分析。结果共筛查158 232份血液标本,确定了其中的69份ObI,流行率为1∶2 293(69/158 232)。41例ObI获得PrE-S/S区基因序列:b型6例、C型34例、d型1例;与对照组相比,ObI在S区的氨基酸序列的变异明显(Pb=0.013;PC=0.003),主要变异位点为b型的V14g/A、y161f/S、V168A、P217l和C型的E2g/A/V、T118r/k/A/M、P127T/l/H/S、E164d/g、l175S、S174n。结论大连地区献血者ObI在HbV基因组S区的氨基酸序列存在多个位点的变异,这些变异与ObI的产生存在某种关系,且这种关系受基因型的影响。Objective To understand the prevalence of occult infection of hepatitis B virus(OBI) and the mutations of pre-S / S gene from blood donors in Dalian.Methods From December 2nd,2010 to May 31 st,2013,samples from blood donors in Dalian Blood Center were screened with enzyme-linked immunosorbent assay(ELISA) for HBs Ag,anti-HCV,antiHIV and anti-TP and triplex nucleic acid detection(NAT) for HIV RNA,HBV DNA and HCV RNA.A follow-up was conducted on donors who were tested negative by ELISA but reactive by NAT.OBI was determined by testing the specimens from the follow-up or archive with serological markers of hepatitis B virus,identification examinations,quantitative and semi-nested PCR.The sequences of pre-S / S gene of OBIs and the control group were analyzed(HBs Ag +,Genbank).Results158 232 blood samples were screened,69 of which were OBIs.The prevalence rate of OBIs was 1 ∶ 2 293(69 /158 232).The pre-S / S gene sequences were analyzed in forty-one cases of OBI:6 were type B,34 were type C,and 1 was type D.Comparing the amino acid sequences between the experimental and control groups,there were statistical significance in the S region of OBI(PB= 0.013;PC= 0.003).The main mutation sites of amino acids for type B were V14 G / A,Y161 F / S,V168 A and P217 L while for type C were E2 G / A / V,T118 R / K / A / M,P127 T / L / H / S,E164 D / G,L175 S and S174 N.Conclusion There were multiple mutation loci on the amino acid sequence of OBI from blood donors in Dalian.A particular relationship existed between these mutations in S region from HBV genome and the mechanism of OBI,which was influenced by genotypes.大连市科技计划指导性项目(大卫科发[2013]50号); 大连市医学重点学科优秀卫生专业技术人才培养资助项目(大卫科发[2013]383号

A New Method and Device for Fast HBV Genotyping in Point-of-Care Diagnostics

介绍一种现场快速型HBV(乙型肝炎病毒)基因分型方法及装置,包括生物传感器与手持式荧光检测仪。为了提高检测灵敏度与准确性,采用荧光标记法。荧光检; 测仪采用简化方法来快速读取侧向流试纸上各测试线及质控线的信号幅值,基于预先建立的HBV基因分型模式匹配模型,实现快速分型。对48份HBV血清样本; 进行检测,实验结果表明,基于荧光检测仪及侧向流试纸的分型检测结果与传统核酸分析方法的分型检测结果一致,分型准确率达100%。与传统方法相比,现场; 快速型基因分型方法及装置可降低HBV分型检测的复杂度,缩短检测时间,降低检测成本,对乙肝个体化用药与个性化治疗具有重要意义。[无可用摘要]国家自然科学基金; 北京化工大学微系统与可穿戴医疗设备及生物传感技术创新研究团队资助项目; 福建省重点科技项