检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

新型布尼亚病毒抗体间接ELISA检测方法的建立北大核心CSCD

目的本研究旨在建立新型布尼亚病毒抗体检测的高特异性、低成本间接ELISA方法。方法通过优化抗原表达、纯化、包被、血清稀释、孵育时间等条件,以Gn的截短蛋白Gn2为包被抗原,开发了一种间接ELISA方法。检测该方法的稳定性、敏感性、特异性、符合率。结果批内和批间变异系数均小于10%,阳性血清临床符合率达76.9%(10/13),阴性血清临床符合率达86.7%(26/30)。结论本研究所建立的间接ELISA方法可用于临床血清样品的检测,为发热伴血小板减少综合征的疾病诊断和监测提供了较为可靠和廉价的检测方法。     

人博卡病毒VP2蛋白单克隆抗体的制备

目的 表达纯化人博卡病毒( human Bocavirus,HBoV) VP2蛋白,采用杂交瘤方法制备抗HBoV VP2蛋白的单克隆抗体.方法 应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB/c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定.结果 表达并纯化获得了重组HBoV VP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1∶4×105.结论 利用HBoV VP2蛋白免疫制备了单克隆抗体,并具有较高的效价.本研究为快速诊断和研究HBoV打下基础.     

柯萨奇B组病毒IgM抗体特性研究

目的 研究柯萨奇B组病毒（CVB）感染后患者急性期CVB－IgM抗体的反应特性。方法 对临床确诊为CVB感染的患者急性期血清用免疫印迹法检测CVB－IgM抗体。结果 患者的急性期血清IgM抗体均仅针对CVB的VP1抗原反应。经ELISA与免疫印迹方法的比较表明：用免疫印迹法检测的CVB－IgM抗体可对CVB各型病毒抗原反应,具有CVB各型病毒之间的交 叉反应性。而同时,在16例ELISA检测反应阴性的健康人血清中未见有CVB的VP1特异性的IgM抗体存在。结论 临床感染CVB的患者急性期,其血清中的特异性的IgM抗体是针对CVB的VP1抗原,而且,此类抗体对各型的CVB抗原具有交叉反应性。     

人博卡病毒VP2蛋白的原核表达及血清学检测方法的建立

目的 获得高表达量的人博卡病毒( HBoV1、HBoV2) VP2蛋白,建立血清学检测方法.方法 按照大肠埃希菌密码子使用偏性,对HBoV1、HBoV2 VP2衣壳蛋白基因编码区序列进行优化合成.将合成的目的基因克隆至表达载体pET30a,构建重组表达质粒pET30a-VP2,转化大肠埃希菌BL21 (DE3),使用IPTG诱导表达并摸索最佳表达条件;粗提取蛋白进行Ni亲和层析纯化后建立间接酶联免疫吸附试验( ELISA),进行临床血清标本筛查,分析人群HBoV1、HBoV2感染情况.结果 优化合成的HBoV1、HBoV2 VP2基因正确连接至pET30a载体,开放阅读框正确,用1 mmol/L IPTG过夜诱导表达(25℃)得到高表达量的VP2蛋白,目的蛋白主要以包涵体的形式存在.粗提取蛋白经NiNTA亲和层析,在pH4.5时获得较理想的纯化蛋白,以其为抗原建立ELISA检查方法,筛查临床血清标本IgG抗体水平,结果显示健康儿童HBoV1阳性率为62.2％,HBoV2阳性率为55.5％,混合感染率为37％.结论 本研究成功将HBoV1、HBoV2 VP2衣壳蛋白基因编码区序列进行优化,得到了较高的蛋白表达量,所建立的ELISA法可以应用于HBoV1、HBoV2人群血清流行病学调查.     

建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒（FMDV）感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白（VP1epi）作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。     

复合前列腺特异性抗原的单克隆抗体制备及化学发光法免疫定量检测试剂研究

目的:获得特异性检测复合前列腺特异性抗原(c-PSA)的单克隆抗体配对,建立基于化学发光c-PSA 免疫定量试剂。方法:利用市购c-PSA 抗原免疫6 周龄雌性BALB/ c 小鼠,间接法ELISA 差异筛选抗c-PSA、游离前列腺特异性抗原(f-PSA)、总前列腺特异型抗原(t-PSA)的抗体,抗体配对后化学发光定量检测血清中c-PSA 蛋白。结果:获得抗体配对1A10/7D6-SAE,经c鄄PSA 标准品及临床血清样本初步筛选,该抗体配对适用于基于化学法发光的免疫反应定量检测试剂的开发;血清阳性样本与阴性样本检测结果显示具有统计学意义(P<0.000 1);阳性样本定量结果与Siemens 公司复合前列腺特异性抗原测定试剂盒(直接化学发光法)的相关系数为0.97;线性范围为0郾1 ~ 100 ng/ ml;检测灵敏度为0.005 ng/ ml。结论:成功筛选获得特异性检测c-PSA 的抗体配对,并开发c鄄PSA 化学发光免疫定量试剂,其检测能力与国际主流试剂相当。     

实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立

目的 采用逆转录结合实时荧光定量PCR技术,建立一种快速、准确、特异甄别单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李氏菌)死活状态的定量方法.方法 根据单增李氏菌hlyA基因序列设计引物和探针;对实时荧光PCR反应体系进行优化后,提取菌体mRNA,通过随机引物进行逆转录反应;产生的cDNA通过实时荧光定量PCR进行鉴定.进一步评价逆转录结合实时荧光定量PCR方法的特异性、灵敏度、重复性后,对20份模拟双盲样本进行检测.结果 本实验所建立的逆转录结合实时荧光定量PCR方法可准确、特异地检测单增李氏菌,其他菌株和失活的单增李氏菌均无阳性结果出现;该方法检测纯菌和模拟样本的灵敏度分别可达到10 CFU/ml和1000CFU/ml;定量检测的批间和批内的变异系数均小于5%;对20份模拟样本进行检测,其中10份含有活性单增李氏菌样本的检测结果均为阳性,其他含有失活单增李氏菌的5份样本和其他致病菌的5份样本检测结果为阴性.结论 本文建立的检测活性单增李氏菌实时荧光定量PCR方法快速、准确,结果可靠,实用性强,可进行定量分析,为食品安全监测和现场流行病学调查提供较好的分析手段和完整的数据.     

肠道病毒71型抗原含量检测方法的建立及初步验证

目的 建立肠道病毒71型(EV71)抗原含量检测方法并进行初步验证,用于疫苗研制及生产过程中抗原含量的检测.方法 以抗EV71单克隆抗体为包被抗体,酶标抗EV71多克隆抗体为检测抗体,建立双抗体夹心ELISA方法,以本室自制EV71抗原参考品为定量标准,建立抗原标准曲线,确定定量限度及最佳定量范围,并对该方法的准确度、精密度和特异性进行初步验证.结果 建立了双抗体夹心检测EV71抗原含量的ELISA方法,定量限度为0.23μg/ml,最佳定量范围为7.32～0.23μg/ml,相关系数为R2=0.9976;准确性较好,回收率在90%～110%之间;精密性较好,变异系数小于10%;除与EV71样品有特异性反应外,与其他样品无交叉反应.结论 建立了检测EV71抗原含量的双抗体夹心ELISA方法并进行了初步验证,为疫苗研制过程中抗原含量测定提供了简便、快捷的检测手段.     

小鼠抗KLH多克隆抗体的制备及TDAR实验间接ELISA定量检测方法的建立北大核心CSCD

目的:通过制备小鼠抗KLH多克隆抗体,优化反应条件建立KLH特异性抗体ELISA定量分析方法,以期提供一种特异性好、灵敏度高及误差低的TDAR实验检测方法。方法:利用盐析沉淀和亲和层析纯化技术分离和纯化小鼠抗KLH特异性多克隆IgG抗体;以纯化后的抗体作为标准品,建立TDAR实验间接ELISA定量检测方法,并对该方法进行线性范围、特异性、变异系数(CV)及检测限验证。结果:以KLH包被浓度为80μg/ml,山羊抗小鼠IgG/HRP酶标二抗的稀释倍率为1∶20000条件进行间接ELISA定量分析的方法学验证,检测线性范围为390.63~25000 ng/ml,R^(2)=0.9848,线性良好;批内和批间CV均<10%;检测限为194.83 ng/ml。结论:通过制备抗KLH多克隆抗体,并优化实验反应条件来建立间接ELISA定量分析方法,其线性范围、特异性及批内、批间CV均满足实验要求,是一种高灵敏度的TDAR实验检测方法。     

鹦鹉热嗜衣原体蛋白酶样活性因子免疫优势区基因重组蛋白的诊断应用研究

目的 检测鹦鹉热嗜衣原体(Chlamydophila psittaci,Cps)蛋白酶样活性因子(chlamydial protease-like activity factor,CPAF)免疫优势区基因在E.coli BL21中高效表达的产物在Cps感染诊断中的应用,为建立Cps感染的快速诊断奠定实验基础.方法 利用生物信息学软件筛选出Cps CPAF免疫优势区基因序列(CPAFm,A196～A450),设计特异性引物,PCR扩增目的基因,将其克隆入pGEX6p-2载体后转化E.coli BL21,利用IPTG诱导表达重组蛋白,Western blot鉴定重组蛋白.以纯化的重组蛋白作为包被抗原,建立血清学诊断的间接ELISA法;同时用商品ELISA试剂盒与建立的间接ELISA法检测180份可疑Cps感染的病鸭血清标本,将检测结果进一步用Western blot方法验证.结果 构建原核重组质粒pGEX6p-2/CpsCPAFm,表达相对分子质量约为54×103的目的蛋白产物.以纯化的重组蛋白为包被抗原建立间接ELISA法检测Cps参考血清,其阴、阳性符合率均为100％;与肺炎嗜农原体(C.pneumoniae,Cpn)、沙眼衣原体(C.trachomatis,Ct)无交叉反应.对180份可疑病鸭血清标本进行检测,与商品Birds Chlamydia Psittaci IgG ELISA试剂盒比较,以Western blot方法作对照,自建的ELISA法符合率均为100％,Birds Chlamydia Psittaci IgG ELISA Kit符合率为77.5％～95.0％.结论 以Cps CPAF免疫优势区(A196～A450)作为诊断抗原,建立血清学诊断的间接ELISA法具有较好的临床诊断价值.     

人博卡病毒VP2蛋白单克隆抗体的制备

目的表达纯化人博卡病毒（human Bocavirus,HBoV）VP2蛋白,采用杂交瘤方法制备抗HBoVVP2蛋白的单克隆抗体。方法应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB／c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定。结果表达并纯化获得了重组HBoVVP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1：4×10^5。结论利用HBoVVP2蛋白免疫制备了单克隆抗体,并具有较高的效价。本研究为快速诊断和研究HBoV打下基础。     

Nanoparticle-based bio-barcode assay for the detection of bluetongue virus

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the outer-core protein VP7 of bluetongue virus (BTV). The target antigen VP7 was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMP) coated with VP7 monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 0.1fg/ml was measured for purified VP7, seven orders of magnitude more sensitive than that of conventional antigen capture ELISA. The BCA demonstrated the same enhanced sensitivity for detecting BTV in serum samples from sheep. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of BTV proteins that could be adapted to measure other proteins.     

Use of enzyme-linked immunosorbent assays with chimeric fusion proteins to titrate antibodies against Epstein-Barr virus nuclear antigen 1.

Two new enzyme-linked immunosorbent assays (ELISAs) with chimeric fusion polypeptides for the detection of human antibodies specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1) are described. One is an indirect ELISA with affinity-purified beta-galactosidase-EBNA-1 fusion protein as the antigen. The other is a "sandwich" assay based on the use of anti-beta-galactosidase antibody to capture beta-galactosidase-EBNA-1 fusion proteins in bacterial extracts. A good correlation was shown between antibody titers determined by the ELISA with the EBNA-1 fusion proteins and those determined by a conventional anticomplement immunofluorescence test which is being widely performed with Raji cells for the purpose of research and clinical diagnosis. The advantage of the ELISAs for seroepidemiologic studies on Epstein-Barr virus was demonstrated by sensitive detection of marginal immunoglobulin G antibody to the EBNA-1 domain in serum samples from patients with infectious mononucleosis.     

Differential IgM response to conformational and linear epitopes of parvovirus B19 VP1 and VP2 structural proteins

The IgM immune response against conformational and linear epitopes of B19 structural proteins VP1 and VP2 was examined in serum samples with a suspect B19 infection to determine the most suitable antigen for use in IgM detection and also to evaluate a possible relationship between the course of B19 infection and the presence of epitope type-specific IgM. The detection of IgM against conformational epitopes was performed by ELISA using undenatured VP1 and VP2 antigens whereas the detection of IgM against linear epitopes was performed by Western blot assays using denatured VP1 and VP2. IgM immune response against VP1 conformational epitopes appeared dominant, being detected in all serum samples positive for specific IgM, whereas IgM against VP2 linear antigen were found less frequently, being identified in less than half of the B19 IgM positive sera. In the examination of the course of infection, IgM against VP1 conformational epitopes appeared in the active phase of B19 infection at the same time and with the same frequency as IgM anti VP2 conformational epitopes and anti linear VP1 epitopes. IgM against VP1 conformational epitopes were seen to be long-lasting because in the recent phase of infection they were still present when other specific IgM were absent. During the active phase of B19 infection, IgM against VP2 linear epitopes were less frequently found than other specific IgM and in the recent phase they underwent a rapid temporal diminution. The data demonstrate that a sensitive B19 IgM test needs to be performed in diagnostic laboratories by ELISA using conformational B19 antigens; Western blot assays can be used only as confirmatory tests using VP1 linear antigens.     

Prokaryote expression of HPeV-1 VP1 protein, production of VP1 polyclonal antibody and the development of an ELISA

The VP1 protein of human parechovirus (HPeV) plays a critical role in receptor binding based on its functional arginine-glycine-aspartic acid (RGD) motif region. Currently, only the neutralisation assay is used for seroepidemiological surveys of HPeVs. In the present study, the VP1 gene of HPeV-1 was cloned into the vector pET28a(+) to express the His-tagged VP1 protein in the bacterium Escherichia coli Rosetta. The recombinant protein was purified from inclusion bodies by Ni(+)-NTA affinity chromatography under denaturing conditions, followed by a refolding process in gradient urea. The identity and antigenicity of the His-tagged protein was confirmed by Western blotting using an anti-His monoclonal antibody and human HPeV-1-positive serum respectively. Polyclonal antibodies against the His-tagged VP1 protein were raised in rabbits by standard procedures, and the reactivity and specificity were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. An indirect ELISA was developed based on the fusion protein VP1, and evaluated in order to facilitate the detection of antibodies in persons who had been infected naturally with HPeVs. A serological survey was performed using the assay amongst children in the Shanghai region of China; the seropositivity rate was found to be about 73%.     

Detection of parvovirus B19 IgG: choice of antigens and serological tests.

Serum samples were analysed for the presence of (a) IgG against VP1+VP2 using recombinant native conformational antigens by ELISA test (b) IgG against VP2 using recombinant native conformational antigens by ELISA test and (c) IgG against VP1 and against VP2 using denatured linear antigens by Western blot. Out of the 446 samples examined, 353 were positive for specific B19 IgG and out of these, 98.6 % proved positive in the ELISA assay using conformational VP1+VP2 antigens, 94.6% proved positive in the ELISA assay using conformational VP2 antigens, 89.5% were positive at the Western blot assay using denatured linear VP1 and VP2 antigens, with all proving positive for linear VP1 and only 29.5% out of the positive samples proving positive for linear VP2. Since all samples positive by Western blot proved positive by ELISA, our data show that recombinant capsids obtained either with VP1+VP2 or with VP2 alone, used in ELISA, are very useful for detecting the immune response against both conformational and native linear epitopes of B19 structural proteins although some sera may have antibodies directed exclusively against VP1+VP2 antigens and few may have antibodies directed exclusively against VP2 antigens alone.     

乙型肝炎病毒前S1抗原检测的临床意义

探讨乙型肝炎病毒前s1抗原（Pre-S1）检测在乙型肝炎病毒诊断中的临床意义。采用酶联免疫吸附试验（ELISA）和荧光定量聚合酶链反应技术（fluorescenee quantitative PCR,FQ-PCR）对650份HBV-M不同阳性模式及40份HBV—M全阴性模式血清标本进行乙型肝炎病毒Pre-S1、乙肝五项和HBV—DNA检测,并对三种检测结果进行统计学分析。在650份HBV—M不同阳性模式标本中,在119份大三阳标本中Pre—S1阳性检出率92．4％,HBV-DNA阳性检出率100％,在186份小三阳标本中Pre—SI阳性检出率42．5％,HBV—DNA阳性检出率63．4％,在21例HBsAg（＋）和HBcAb（＋）阳性组中Pres1阳性检出率47．6％,HBV．DNA阳性检出率66．7％;在297例HBsAb（＋）标本中Pre—S1阳性检出率0．4％,HBV-DNA阳性检出率0％,在268例HBV—DNA阳性的标本中Pre-S1阳性检出率79．3％。在40份HBV—M全阴模式中Pre-S1阳性检出率0％,HBV-DNA阳性检出率0％。Pre—S1在大三阳、小三阳及HBV-DNA阳性组阳性检出率明显高于阴性组（P〈0．01）,Pre—S1检测可补充和完善乙肝“两对半”检测的不足,尤其对HBeAg阴性或变异的HBV感染者能更好的反映病毒的复制状态和传染性。