HCV免疫学诊断抗原与链亲和素融合蛋白的制备及应用研究

目的 制备带有链亲和素（SA）标签的丙型肝炎病毒（HCV）融合蛋白,并初步探讨其在抗-HCV ELISA检测中的应用.方法 构建了HCV诊断抗原与链亲和素融合蛋白重组表达质粒pET-11d-C44P-SA,在大肠埃希菌BL21（DE3）中表达,并用Western Blot进行鉴定,表达的融合蛋白经镍金属螯合（Ni-NTA）层析柱进行纯化,透析复性后分别包被生物素化以及普通酶联板,进行人血清抗-HCV检测.结果 成功构建了带有SA标签的重组表达质粒,其在大肠埃希菌BL21（DE3）中实现高效表达,制备的融合蛋白纯度可达90%以上.建立ELISA法进行人血清抗-HCV检测显示：融合蛋白包被生物素化酶联板检测灵敏度与特异性明显高于普通酶联板.结论 构建的融合蛋白作为包被抗原,结合利用SA标签与生物素化酶联板,明显提高了抗-HCV检出的灵敏度与特异性,该策略为进一步提高抗-HCV检测试剂的质量打下了基础.     

探讨三种检测方法在丙型肝炎诊断中的应用价值

目的探讨ELISA法检测丙型肝炎病毒核心抗原(HCVcAg)、病毒抗体(抗-HCV)及RT-PCR法检测丙型肝炎病毒RNA(HCV-RNA)3种方法在丙型肝炎诊断的应用价值。方法采用HCVcAg ELISA试剂盒,抗-HCV ELISA试剂盒及HCV-RNA PCR试剂盒,对临床200例疑似丙肝病毒感染的样本进行HCVcAg、抗-HCV和HCV-RNA检测。结果 HCVcAg阳性检出率为42%;HCV-RNA阳性检出率最高,为61%;抗-HCV阳性检出率为52%。Kappa检验示3种检测方法结果阳性吻合度基本一致。HCVcAg阳性检出率随着HCV病毒含量的升高而升高。结论 3种方法中,RT-PCR检测HCV-RNA仍是判断丙肝感染最准确方法。HCV核心抗原检测可以有效缩短窗口期,联合运用抗-HCV和HCVcAg或抗-HCV和HCV-RNA,能有效降低单独使用抗-HCV检测的漏检风险。HCVcAg可作为HCV抗体常规检测的补充指标,提高检出率。     

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。     

丙型肝炎病毒抗原检测方法的建立

目的以特异性单克隆抗体为基础建立丙型肝炎病毒(HCV)抗原检测的酶联免疫吸附(ELISA)法,探索从血浆或血清中检测HCV抗原的可能性.方法利用我们制备的抗-HCV核心及NS3区单克隆抗体(McAbs),进行多种交叉组合模式的分析,确立实验室模式,并测定348份义务献血员血样,确定此方法的Cutofff值,并分析146份抗-HCV阳性及225份抗-HCV阴性血浆,阳性结果用套式PCR试剂盒确证.结果构建了以抗-HCV核心区单抗C39及NS3区单抗C7-6为包被抗体,以C39及NS3区C7-57为标记抗体的夹心ELISA检测模型,以C7为抗原,其检出灵敏度为5ng/ml,其Cutoff值为阴性对照均值±0.25.146份抗-HCV阳性样本中,11份为抗原反应阳性,225份抗-HCV阴性样本中,16份为抗原阳性,这些阳性样本经PCR检测后,23份为HCVRNA阳性.结论用单抗构建的HCV抗原检测方法分别从抗-HCV阳性及阴性样本中检测出HCV抗原反应阳性样本,并经PCR确证,表明直接从血浆样本中检测HCV抗原是可能的,这将对于HCV和基础研究及控制HCV的传播有重要意义.     

游离丙型肝炎病毒核心抗原检测的临床价值探讨

目的:探讨游离丙型肝炎病毒(HCV)核心抗原检测在HCV感染诊断中的价值。方法:采用荧光定量PCR法检测HCV-RNA、ELISA法同步检测抗-HCV和游离HCV核心抗原。结果:191例HCV感染者HCV-RNA的检出率为71·2%(136/191);抗-HCV的检出率为97·4%(186/191);游离HCV核心抗原的检出率为33·0%(63/191)。其中有2例经抗病毒治疗的患者HCV-RNA和抗-HCV检测均阴性,但游离HCV核心抗原检测阳性;另有1例患者抗-HCV阴性,而游离HCV核心抗原阳性,经HCV-RNA证实为HCV感染。27例非HCV感染者HCV-RNA、抗-HCV和游离HCV核心抗原检测结果均为阴性。结论:HCV核心抗原检测作为抗-HCV检验的补充试验对HCV感染的诊断具有重要价值。     

丙型肝炎病毒不同基因型NS3蛋白的抗原异质性分析

目的探讨不同基因型丙型肝炎病毒(HCV)NS3蛋白的抗原特性及其用于抗-HCV检测的意义。方法分别构建和表达含有HCV1型和6型NS3基因片段的重组质粒和重组蛋白，以EIJSA法和Western blot分析不同基因型重组蛋白与已知抗-HCV阳性血清的抗原反应性。结果HCV1型和6型NS3重组蛋白氨基酸序列的同源性为83．2％；85份抗-HCV阳性血清以此不同基因型HCV NS3单片段抗原检测，阳性检出率分别为61．2％(1型)和58．8％(6型)，其中有7份标本以NS3-1型抗原检测阴性，但可被NS3-6型抗原检出，反之，有9份血清以NS3-6型重组蛋白检测为阴性，而NS3．1型检测阳性；54份大学生体检血清和39份阴性质控血清以此两种抗原检测均为阴性。结论HCV1型和6型NS3重组蛋白存在抗原异质性，在发展HCV抗体检测试剂时需考虑加入不同基因型的NS3抗原。     

三种检测SARS抗体试剂盒的临床使用效果比较

目的 :对重组双抗原夹心法ELISA、全病毒间接法ELISA和免疫荧光分析法 3种SARS抗体诊断试剂盒进行临床应用效果评价 ,比较不同试剂的使用效果。方法 :使用 3种试剂检测了 2 5 7例临床确诊SARS患者的血清标本 2 79例和其他非SARS患者标本 2 4例、健康体检者血清标本 80份。结果 :临床确诊SARS患者发病 1～ 2 0d的病例中双抗原夹心法ELISA检出率最高 ,间接免疫荧光法与其接近 ,全病毒间接法ELISA检出率最低。在发病 2 0d后 ,三者检出率接近 (94 %左右 ) ,3种方法的符合率在 97%以上。在 10 4例非SARS病例中 ,双抗原夹心法ELISA和免疫荧光法均未见出阳性结果 ,全病毒间接法ELISA检出阳性结果 3例 ,假阳性率 2 .9%。结论 :检测SARS抗体双抗原夹心法ELISA试剂盒、免疫荧光试剂盒具有类似的灵敏度和特异性 ,其灵敏度和特异性高于全病毒间接法ELISA。     

双抗体间接夹心ELISA法检测乳腺癌患者外周血MUC1蛋白水平的评价

目的优化双抗体间接夹心ELISA试剂盒,并探讨其在乳腺癌患者中检测MUC1黏蛋白水平的应用价值。方法用基因重组MUC1-GST和MUC1-MBP融和蛋白免疫家兔和大鼠,获得抗MUC1血清,并对其纯化,获得纯化的家兔抗人及大鼠抗人MUC1多克隆抗体;经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗人MUC1抗体作为检测抗体的双抗体间接夹心试剂盒,敏感度可达到0.2 ng/ml。结果应用建立的试剂盒对40例乳腺癌,18例乳腺良性疾病和120健康对照者血清中MUC1蛋白水平的进行检测,检测结果绘制ROC曲线,分析得出以2.75 ng/ml为乳腺癌患者与乳腺良性疾病患者的临界值,以1.86 ng/ml为乳腺疾病与正常人为临界值,检测结果表明本研究对乳腺癌诊断的阳性率高达97.5%,乳腺良性疾病的阳性率为66.7%,正常人特异性为96.7%。对于乳腺癌同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测,其检出率为3.33%,特异度为100%。绘制ROC曲线对比显示,本研究所建立的双抗体夹心ELISA方法对乳腺癌诊断的准确度明显高于CA15-3试剂盒。结论本研究成功建立了特异性强,灵敏度良好的双抗体间接夹心ELISA试剂盒,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。     

HIV-1/2抗体和P24抗原联合检测试剂盒的研制与评价

目的研制HIV-1／2抗体和P24抗原联合检测酶免疫试剂盒并评价其实用性。方法联合使用基因工程HIVI／2型抗原和抗HIVP24单克隆抗体包被酶联反应板，以辣根过氧化物酶标记的HIVI／2型抗原和生物素化的兔抗HIVP24抗体作为标记物，研制了联合检测HIVI／2抗体和P24抗原的ELISA诊断试剂，并对其特异性、敏感性、稳定性等进行评价和临床考评。结果检测P24抗原质控品的灵敏度可达0．2ng／ml；与雅培公司试剂比较检测78份AIDS患者血清和85份正常人血清、对照检测中国药品生物制品检定所研制的HIV参比血清，特异度和灵敏度均为100％。临床考核检测12051份各种血清，灵敏度为100％（543／543），特异度为99．48％（11448／11508）。试剂在37℃放置6d后，试验结果无明显差异。结论本试剂盒具备特异度强、敏感度高、稳定性好、操作简便等优点，可以一步检出HIV特异性抗体和HIVP24抗原，缩短了HIV感染的检测窗口期，适用于HIV感染的实验室诊断和流行病学调查。     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

Polystyrene, poly-L-lysine and nylon as adsorptive surfaces for the binding of whole cells of Mycobacterium tuberculosis H37 RV to ELISA plates

Several methods of coating whole cells of Mycobacterium tuberculosis H37 RV to ELISA microtitre plates were compared with the aim of developing an ELISA screening assay for murine monoclonal antibodies in culture supernatants and human antibodies in patient sera. Undercoats of nylon or poly-L-lysine were compared to polystyrene as adsorptive surfaces for the bacteria, the effect of increased ionic strength and iclusion of SDS in the coating buffer measured, and methanol (70%) and glutaraldehyde (5%) investigated for their efficiency as fixatives of the bacterial monolayers. The results suggest PBS as a satisfactory coating buffer for the bacterial cells on polystyrene, and 70% methanol the preferred fixative for the dried antigen-coated plates.     

用重组结构区抗原检测抗丙型肝炎病毒IgM抗体方法？…

目的 检测抗丙型肝炎病毒（ＨＣＶ）结构区蛋白ＩｇＭ抗体。方法 采用丙型肝炎病毒Ｃ，Ｅ１、Ｅ２区重组抗原混合包被和分别包被酶标板；用兔抗人γ链血清处理人血清标本，再用固相包被羊抗兔抗体吸附兔抗人γ链－人ＩｇＧ复合物，建立了抗－ＨＣＶＩｇＭ检测方法。结果 对７６例现型肝炎病人血清进行抗－ＨＣＶ ＩｇＭ检测，同时与逆转录－巢式聚合酶链反应（ＲＴ＋ＰＣＲ）检测结果进行比较，再会得具有相关性（Ｐ〈０．００５     

HCV感染后NS5区抗体的动态观察与检测意义

本文报道用ＨＣＶＮＳ５区两段抗原性较好的合成肽研制的ＥＬＩＳＡ试剂盒及ＵＢＩＨＣＶＮＳ５区抗体检测ＥＬＩＳＡ试剂盒观察１４例ＨＣＶ感染者ＮＳ５区抗体的动态变化，证实ＮＳ５区抗体如同ＮＳ４区抗体一样比Ｃ及ＮＳ３区抗体出现晚。ＮＳ５区抗体总体检出率近似于ＮＳ４区抗体；１．５５％的血清为单独ＮＳ５区抗体阳性；存在ＮＳ５区抗体与其他区抗体滴度有互补作用的标本等提示ＮＳ５区抗体仍有一定的诊断价值。采用ＵＢＩＮＳ５区抗体检测试剂盒发现，９５．６５％（２２／２３）ＵＢＩ试剂盒诊断为ＮＳ５区抗体阳性的标本中含ＨＣＶＲＮＡ，６／１４ＨＣｖ感染者用ＵＢＩ试剂盒检出的抗体出现在ＡＬＴ再次异常升高或剧烈升高（高于参考值的３倍以上）前后，４／１４的感染者抗体出现于ＡＬＴ首次升高前后（其余４／１４的感染者未检出抗ＮＳ５抗体），因此ＵＢＩ抗ＨＣＶＮＳ５抗体诊断试剂盒检测的抗体似与疾病的活动有关。     

人禽流感酶联免疫诊断方法的建立及其初步应用

目的建立人禽流感H5抗体的酶联免疫检测方法（ELISA）。方法用ELISA法检测疑似高致病性禽流感患者及正常人群血清标本中H5IgG抗体。结果23例疑似高致病性禽流感患者血清样品中有4例H5IsG抗体阳性，阳性率为17．40％，与血凝抑制试验（HI）和微量中和试验（NI）相比较，三者符和率为100％。234例正常人群血清H5 IgG抗体检测均为阴性。结论建立的ELISA方法可用于高致病性人禽流感的血清样品初步筛查。     

Evaluation of the biological activity of human growth hormone by enzyme linked immuno-receptor assay (ELIRA) method

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R(2) = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R(2) = 0.95 to 0.97). Finally, the coating of microwells by 250 μg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R(2)?=?0.985) and lower detection limit about 2 ng/ml of bioactive hGH.     

Evaluation of the Biological Activity of Human Growth Hormone by Enzyme Linked Immuno-Receptor Assay (ELIRA) Method

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R² = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R² = 0.95 to 0.97). Finally, the coating of microwells by 250 μg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R²?=?0.985) and lower detection limit about 2 ng/ml of bioactive hGH.     

Reducing agents interfere with the detection of lettuce necrotic yellows virus in infected plants by immunoblotting with monoclonal antibodies

A procedure is described for the detection of lettuce necrotic yellows virus (LNYV) nucleocapsid protein (N) or envelope glycoprotein (G) by immuno-blotting with their respective monoclonal antibodies. The antigens can be detected in 1-10 mg of fresh tissue from systemically infected Nicotiana glutinosa leaves showing prominent symptoms. The composition of the extraction buffer played a crucial role in the recovery of antigenically active proteins. Procedures involving tissue extraction in the presence of the reducing agents 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) failed to detect either the N or G antigen. For optimum detection of the N or G antigens, leaf tissue was ground with 10 mM phosphate buffer, pH 7.6 (PB), containing 3% (w/v) sodium dodecyl sulphate (SDS) and 1 mM of the protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK). The extract was then mixed with an equal volume of dissociation buffer containing 2-ME before electrophoresis and immunoblotting.     

弓形虫多表位基因重组抗原在弓形虫免疫检测中的应用

目的评价弓形虫多表位基因重组抗原在弓形虫感染诊断中的效果，探索多表位抗原在弓形虫免疫检测中的应用前景。方法以Ni—NTAAgarose和割胶电渗两步纯化法。获取弓形虫多表位基因的原核表达纯化产物rMAG，以适宜浓度的rMAG包被ELISA微孔板，分别检测弓形虫急性和慢性感染血清，评价试剂盒检测的敏感性和特异性。结果经Ni—NTAAgarose和割胶纯化，得到了纯度为95．86％的弓形虫多表位重组可溶性抗原。以3μg／ml的抗原浓度包被ELISA微孔板，对兔和小鼠的急慢性弓形虫感染血清进行了检测。结果检测小鼠血清141份，其中慢性感染弓形虫小鼠血清117份、正常小鼠血清24份，检测体系的敏感性为88．88％，特异性为91．67％，一致性为89．36％。检测兔血清24份，其中急性感染弓形虫兔血清18份，正常兔血清6份，阳性血清检出率为94．4％，总一致性为91．5％。结论以弓形虫多表位重组抗原构建的ELISA试剂盒既可以检测弓形虫急性感染血清．又可以检测弓形虫慢性感染血清．具有一定的应用前景。     

Enzyme-linked immunosorbent assay for screening monoclonal antibody production: Use of intact cells as antigen

An enzyme-linked immunosorbent assay (ELISA) was developed for screening production of monoclonal antibodies with specificity for surface membrane components on human mononuclear cells. Whole cells used as antigen were dessicated under vaccum in flexible polyvinyl chloride plates or in rigid plates coated with protein-detergent solution. Rabbit or goat anti-mouse IgG conjugated with peroxidase was used as indicator after affinity column purification. Under these conditions, the sensitivity of ELISA proved comparable to other binding techniques or microcytoxicity and allowed rapid, reproducible, and efficient detection of antibody producing hybridomas.     

Efficient infection of human natural killer cells with an EBV/retroviral hybrid vector

Enzyme-linked immunosorbent assay (ELISA) has been considered extremely useful for the detection of markers of allergenic substances in food, because it is simple, offers a suitable sensitivity, and is useful in providing quantitative results. Allergenic protein present in processed food can be denatured or altered, hindering therefore their possibility to be extracted and detected. This paper reports the development of an ELISA method that can be used for the determination of allergenic proteins in buffer solutions containing SDS, a surfactant, and 2-mercaptoethanol, a reducing agent. Measurement by ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol has been made possible by using an antibody prepared through immunization with an antigen denatured with SDS and 2-mercaptoethanol. This ELISA technique can be used to measure proteins in food that have been denatured by various manufacturing processes. An example is egg white albumin, which is susceptible to heat denaturation and has been difficult to recover from food in the past. Its recovery was improved 10- to 100-fold by the new ELISA method as compared with previous methods. This means that allergenic substances in food can now be detected quantitatively. This method can be very useful in allergy prevention and control strategies.