夹心免疫-PCR检测旋毛虫循环抗原的研究(英文)

目的建立以多抗为捕获抗体和单抗为检测抗体的夹心免疫-PCR检测旋毛虫循环抗原。方法利用杂交瘤技术制备抗旋毛虫单抗;旋毛虫抗原免疫家兔,分离纯化兔血清,制备兔抗旋毛虫多抗。多抗作为捕获抗体包被酶标板,单抗为检测抗体,选择适宜多抗和单抗浓度,建立夹心ELISA方法,再利用亲和素和生物素的高结合力连接起标记了生物素的抗体和DNA片段,将抗原抗体特异反应的免疫技术同无限扩增DNA片段的基因检测技术结合,利用PCR反应对DNA片段的扩增能力,提高检测灵敏度。结果制备了兔抗旋毛虫多抗,间接ELISA法筛选出与多种寄生虫抗原无交叉反应的单抗F4C6,夹心ELISA检测旋毛虫抗原最低浓度为0.05μg/ml,免疫PCR方法检测最低浓度为0.1ng/ml。结论首次建立夹心免疫-PCR检测旋毛虫抗原的方法,检测旋毛虫抗原的灵敏度高于传统ELISA方法104倍。     

多房棘球蚴病特异性诊断抗原Em18的基因克隆、表达和血清学评价

目的　克隆从我国四川省分离的多房棘球绦虫Em18抗原基因片段,表达重组抗原,用于多房棘球蚴病(AE)的诊断,并用患者血清对其诊断价值进行评价。　方法　PCR扩增目的基因片段与质粒载体pET28a(+)连接构建表达质粒,转化大肠埃希菌BL21(DE3)菌株表达重组蛋白,用镍次氮基三乙酸琼脂糖树脂(NI NTAagarose)亲和层析纯化重组抗原,酶联免疫吸附测定(ELISA)评价重组抗原的血清学诊断效果。　结果　获得两个高效表达克隆ReEm181和ReEm182。其中ReEm181与预期序列一致,ReEm182为同一序列,但有27bp的核苷酸缺失。两个克隆表达的融合蛋白相对分子质量(Mr)分别为28000和26000。对101例AE、47例细粒棘球蚴病、30例囊尾蚴病、10例肝癌、9例血吸虫病和40名健康人共237份血清进行ELISA检测 ,结果显示,ReEm181和ReEm182重组抗原对AE血清诊断的敏感性为861%和901%、特异性为934%和941%、阳性预期值为906%和919%、阴性预期值为901%和928%、诊断效率分别为903%和924%;对58例AE患者肝脏病灶大小与重组抗原检测血清平均吸光度(A)的相关性比较分析结果表明,抗体反应水平在病程早期有较好的相关性。　结论　Em18重组抗原对AE具有特异性诊断价值     

弓形弓形虫抗原检测方法的研究

本研究制备了与多种寄生虫抗原无交叉反应的抗弓形虫单抗B6C3和兔抗弓形虫多抗,多抗为捕获抗体,单抗为检测抗体,建立单-多抗夹心ELISA方法。用亲和素连接分别标记生物素的抗体和酶,建立ABC(avidin-biotin-peroxidase complex)-ELISA[D1]方法。同样用亲和素连接标记生物素的抗体和DNA片段,利用PCR反应对DNA片段巨大的扩增能力,提高检测灵敏度,发展单-多抗夹心ELISA为免疫-PCR(I-PCR)检测弓形虫抗原。单-多抗夹心ELISA检测弓形虫抗原最低浓度为0.6μg/ml,ABC-ELISA检测最低浓度为0.075μg/ml。I-PCR检测最低浓度为0.1ng/ml,检测弓形虫抗原灵敏度高于传统ELISA法6000倍,高于ABC-ELISA法750倍。     

基于A1E3及BIC4单克隆抗体检测日本血吸虫循环抗原ELISA法的建立及现场初步应用

目的建立基于AlE3及B1C4单克隆抗体检测日本血吸虫循环抗原的夹心酶联免疫吸附试验（ELISA）,并对其应用情况进行初步评价。方法采用十二烷基磺酸钠一聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹试验（Westernblot-ring）对A1E3及B1C4单克隆抗体进行特性分析,用ELISA法测定B1C4、A1E3单克隆抗体效价。采用棋盘格滴定法确定双单抗夹心ELISA法检测循环抗原的最佳工作浓度。在最佳条件下,分别检测20份急性血吸虫病病人血清、46份慢性m吸虫病病人血清及20份止常人血清,评价其检测敏感性和特异性。用建立的双单抗夹心ELISA法和市售检测血吸虫循环抗原的ELISA试剂盒检测湖北省江陵县IHA阳性血吸虫病病人血清72份,以评估双单抗夹心ELlSA法的检测效能。结果经SDS-PAGE和Westernblotting分析,纯化后的A1E3及B1C4在相对分子质量（M,）88000和52000处各有一条清晰重链,在吖．20000处有一条相同的轻链,且A1E3及B1c4可与可溶性虫卯抗原（SEA）及急性血口发虫病病人血清发牛特异性反应。BlC4单抗效价可达1：10^5,A1E3单抗效价可达1：30000。用B1C4、A1E3双单抗夹心ELISA法检测急、慢性血吸虫病病人粗清阳性率分别为100％和86．9％,检测20份正常人血清特异性为100％。双单抗夹心ELISA法及市售EIJJsA试剂盒检测日本血吸虫循环抗原阳性率分别为45．8％及43．1％。结论成功建立了基于A1E3及B1C4双单抗夹心ELISA法,该法检测日本m吸虫循环抗原具有较高的敏感性及特异性。     

双抗体夹心ELISA法检测HIV-1 p24抗原的初步建立

目的 建立敏感、特异的艾滋病病毒Ⅰ型(HIV-1) p24抗原的酶联免疫吸附试验(ELISA)检测方法,为试剂盒的研制奠定基础.方法 用纯化的基因工程p24表达抗原免疫小鼠及兔子,获得单克隆抗体(单抗)及多克隆抗体(多抗),用单抗包被酶标板,辣根过氧化物酶标记多抗,初步建立HIV-1 p24抗原的ELISA检测方法.进一步与美国杜邦公司的HIV-1 p24抗原检测试剂同时检测88份HIV感染者血清和50份健康献血员血清中的p24抗原,确定其特异性;采用倍比稀释法的p24抗原标准品,测定试剂的灵敏度.结果 两种方法检测88份HIV感染者血清和50份献血员血清中的p24抗原.结果 均为阴性(符合率为100%);自建ELISA法检测HIV-1 p24抗原的灵敏度为100pg/ml.结论 初步建立了检测HIV-1 p24抗原的ELISA法,与国外同类试剂比较,本法的特异性和灵敏度均接近国外同类试剂,有较好的特异性和灵敏度,为进一步研制HIV-1 p24抗原ELISA检测试剂盒打下了基础.     

基于A1E3及B1C4单克隆抗体检测日本血吸虫循环抗原ELISA法的建立及现场初步应用

目的 目的 建立基于A1E3及B1C4单克隆抗体检测日本血吸虫循环抗原的夹心酶联免疫吸附试验 （ELISA）, 并对其 应用情况进行初步评价。 方法 方法 采用十二烷基磺酸钠?聚丙烯酰胺凝胶电泳 （SDS?PAGE） 和免疫印迹试验 （Western blot? ting） 对A1E3及B1C4单克隆抗体进行特性分析, 用ELISA 法测定B1C4、 A1E3单克隆抗体效价。采用棋盘格滴定法确定双 单抗夹心ELISA法检测循环抗原的最佳工作浓度。在最佳条件下, 分别检测20份急性血吸虫病病人血清、 46份慢性血吸 虫病病人血清及20份正常人血清, 评价其检测敏感性和特异性。用建立的双单抗夹心ELISA法和市售检测血吸虫循环抗 原的ELISA试剂盒检测湖北省江陵县IHA阳性血吸虫病病人血清72份, 以评估双单抗夹心ELISA法的检测效能。 结果 结果 经SDS?PAGE和Western blotting分析, 纯化后的A1E3及B1C4在相对分子质量 （Mr ） 88 000和52 000处各有一条清晰重链, 在Mr 20 000处有一条相同的轻链, 且A1E3及B1C4可与可溶性虫卵抗原 （SEA） 及急性血吸虫病病人血清发生特异性反 应。B1C4单抗效价可达1∶105 , A1E3单抗效价可达1∶30 000。用B1C4、 A1E3双单抗夹心ELISA法检测急、 慢性血吸虫病 病人血清阳性率分别为100%和86.9%, 检测20份正常人血清特异性为100%。双单抗夹心ELISA法及市售ELISA试剂盒 检测日本血吸虫循环抗原阳性率分别为45.8%及43.1%。 结论 结论 成功建立了基于A1E3及B1C4双单抗夹心ELISA法, 该 法检测日本血吸虫循环抗原具有较高的敏感性及特异性。     

双抗原夹心ELISA法检测登革病毒感染患者血清中的EDⅢ抗体

目的 建立一种检测登革病毒(dengue virus, DENV)特异性抗体的双抗原夹心ELISA方法?方法 利用毕赤酵母系统表达4个血清型登革病毒包膜蛋白Ⅲ区(envelope protein domain Ⅲ, EDⅢ),并采用改良过碘酸钠法对EDⅢ蛋白标记辣根过氧化物酶(Horseradish peroxidase, HRP),建立一种可同时检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法?并对2014年广州珠江医院门诊和住院确诊的登革热患者血清标本进行检测,并与澳洲Panbio MAC ELISA检测的敏感性进行比较?结果 本研究成功建立了一种检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法,该方法能同时检测到4个血清型登革病毒EDⅢ蛋白免疫的小鼠血清和Ⅰ型登革病毒EDⅢ蛋白免疫的兔血清,而与烟曲霉AF-MP蛋白免疫的小鼠血清和兔血清均无交叉反应?对184份健康人血清标本检测全为阴性,而对168份确诊为登革病毒感染患者血清标本检测结果表明,两种诊断方法检测结果差异无统计学意义,P<0.001(57.1% vs 57.7%),联合NS1抗原检测方法,其敏感性提高到97.6%?结论 双抗原夹心ELISA法检测登革病毒EDⅢ特异性抗体具有高度的特异性,但如果能同时联合抗原检测可明显提高登革热的早期诊断率?     

双抗体夹心ELISA法检测广州管圆线虫循环抗原的研究

目的建立双抗体夹心ELISA法检测广州管圆线虫循环抗原(Cag)。方法采用两株抗广州管圆线虫成虫可溶性抗原单克隆抗体,应用双抗体夹心ELISA法检测广州管圆线虫CAg,并摸索该法的最佳实验条件。对感染大鼠、小鼠、疑似和确诊广州管圆线虫病病例血清进行检测,评价方法的敏感性;对日本血吸虫、肺吸虫、旋毛虫、囊虫病人血清进行交叉反应试验,评价方法的特异性。结果10μg/ml单抗包被,酶标记单抗1∶1 600稀释为最佳工作浓度。利用该法检测不同血清广州管圆线虫循环抗原的敏感性为84.2%~87.2%,特异性为100%。结论建立的双抗体夹心ELISA法检测广州管圆线虫循环抗原具有敏感性高、特异性强和经济、简便易行等优点。     

戊型肝炎病毒抗体双抗原夹心法ELISA的建立与初步应用

目的　建立抗戊型肝炎病毒 (HEV)抗体检测的双抗原夹心ELISA(DS -ELISA) ,并应用于多种动物血清的检测。方法　将大肠杆菌表达的一段HEVORF2区重组抗原分别包被微孔板和进行辣根过氧化物酶 (HRP)标记 ,利用 5份阳性血清和 4 0份阴性血清建立双抗原夹心ELISA ;用 4 0 0份义务献血员血清比较双抗原夹心ELISA与间接法IgG抗体ELISA的符合情况 ;用 3只HEV感染猴系列血清比较双抗原夹心ELISA试剂和Genelabs公司HEVIgG试剂 ;用双抗原夹心ELISA试剂检测新疆地区的部分牛、绵羊、山羊、猪血清和上海地区的部分鸡血清中的HEV抗体。结果　建立了检测HEV抗体的双抗原夹心ELISA方法 ,对 4 0 0份义务献血员血清的检测表明其与间接法IgG抗体ELISA试剂的符合情况良好 ,并且有更高的s/co比值 ;与Genelabs公司HEVIgG试剂的比较表明双抗原夹心ELISA试剂的检出更早 ,尤其是持续时间及强度明显优于Genelabs试剂 ;在所检测的各种动物中均发现了HEV抗体 ,其中猪抗体的阳性率最高 ,表明双抗原夹心ELISA试剂可同时用于不同动物的抗HEV抗体检测。结论　利用大肠杆菌表达的HEV重组抗原建立了双抗原夹心法ELISA ,并可同时用于不同动物的抗HEV抗体检测。     

抗广州管圆线虫成虫单克隆抗体的研制及初步应用

目的制备抗广州管圆线虫成虫单克隆抗体并研究其初步应用。方法用广州管圆线虫成虫可溶性抗原免疫BALB/c小鼠,经融合,筛选分泌高滴度、高特异性单克隆抗体的杂交瘤细胞株。使用所得的单克隆抗体以Western blotting检测广州管圆线虫病患者血清,并建立双抗体夹心ELISA法检测感染大鼠血清。结果获得3株分泌高滴度抗广州管圆线虫成虫可溶性抗原的单克隆抗体杂交瘤细胞株(2A2、3F1、4H2),其分泌的抗体与日本血吸虫、卫氏并殖吸虫、猪囊尾蚴、旋毛虫抗原均不发生交叉反应。3株单克隆抗体均可识别广州管圆线虫成虫可溶性抗原蛋白Mr 15 000,以及患者血清中两种循环抗原Mr24 000和Mr15 000。双抗体夹心ELISA检测阳性率为76.5%。结论制备的抗广州管圆线虫成虫可溶性抗原的杂交瘤细胞株能分泌高滴度、高特异性的单克隆抗体,且在循环抗原的检测方面有应用前景。     

旋毛虫三种抗原作为免疫诊断抗原的比较研究

用旋毛虫成虫可溶性抗原,成虫排泄分泌抗原和肌肉幼虫抗原作为免疫诊断抗原,采用ＥＬＩＳＡ方法检测,观察各自在旋毛虫病免疫不诊断中的敏感性和特异性。结果表明;抗原的敏感性由强到弱依次是肌肉幼虫抗原,成虫排泄分泌抗原和成虫可溶性抗原;特异性由高到低分别是成虫排泄分泌抗原,肌肉幼虫抗原和成虫可溶性抗原。     

The Haakestad Antigen Is Identical with the Hov Antigen

The rare blood group antigen previously referred to as the 'Haakestad antigen' is shown to be identical with the Hov antigen (numerical designation: 700.038). Family studies add AB0 and HLA to the systems already known to segregate independently from Hov. Among 2,021 Danish blood donors, 4 Hov+ persons were found. Anti-Hov activity has been demonstrated in several 'multiple-antibody sera'.     

The D Antigen Characteristic of RoHar Is a Partial D Antigen

The results of testing R_o^Harr cells with panels of monoclonal anti-D suggest that the D antigen (referred to as D^Har) encoded by the haplotype (D)c(e) Rh³³ is a partial D antigen. IgM monoclonal anti-D are more efficient than IgG monoclonal anti-D in detecting D^Har. D^Har expresses some but not all of both epD5 and epD6/7 and appears to lack epD1-epD4, epD8 and epD9.     

New Determinants of Hepatitis B Antigen (Au or HB Antigen)

Abstract. 261 HB antigens were subtyped for d and y. In the healthy carriers, 63 were found to be ad and 75 were ay. In the patients with acute hepatitis, 24 were ad and 33 ay (6 were ad and 2 ay in chronic hepatitis). Among haemodialyzed patients, 50 were ay and only 6 were ad. In 2 of these renal patients, we found a true association of ad and ay (ady). The w and r determinants were detected in 63 of these 261 HB antigens. 61 were w (33 ayw , 26 adw and 2 adyw) , only 2 were w negative (I ayr and 1 adr).
In the course of this study, several abnormalities: reactions of some ay with anti- d sera, peculiar behaviour of an antiserum from Dakar which was responsible for the formation of spurs between some ay (and three ad) over all other HB antigens could not be explained by the w and r determinants. This and other observations (especially selected absorptions) led us to subdivide the ay into three categories (1, 2 and 3) and the ad into two categories (4 and 5). A sixth category consisted of the genuine association of ad and ay (ady).
The observation that ay of category 3 and ad of category 5 behaved similarly in absorption studies and with the antiserum 'Dakar', led us to conclude that the common a determinant was heterogeneous and included 3 subdeterminants: al, a2 and a3 , of decreasing antigenic strength. The five categories observed were interpreted as follows: (1) aly , healthy carriers from Vietnam; (2) a2y , healthy carriers, niainly Caucasians, and all the ay patients; (3) a3y , healthy carriers, mainly Africans; (4) a2d the great majority of ad carriers and patients, and (5) a3d , only 3 cases (see text). These new subdeterminants are discussed and compared with the determinants already described by other investigators.     

The Antigen Duclos

An antibody is described which defines a new high frequency red cell antigen, Duclos, whose expression seems to require the presence of both U and Rh fundamental antigens. Apart from the antibody maker's own red cells the only nonreactive samples were from Rhnull U impaired individuals, one example of which was shown however to yield very slight amounts of antibody through absorption-elution tests. Rhmod U weak cells gave very depressed and Rhnull U positive or Rh common U negative cells moderately depressed reactions. The proposita's red cells had an apparently normal Rh-LW condition but their U antigen was significantly decreased. No further Duclos negative individual was found by screening 8,500 blood donors in the Paris area.     

Antigen Recognition: Early Surface-Receptor Phenomena Induced by Binding of a Tritium-Labeled Antigen

The rate of antigen binding by mouse lymphoid cells has been investigated with polymerized flagellin of Salmonella adelaide that had been biosynthetically labeled with tritium. Autoradiographs of lymphnode cells incubated with the tritiated antigen at 37° showed aggregation of antigen receptors to one cell pole. This was followed 4-5 hr later by the appearance of antigen receptors on the cell surface, at a density severalfold higher than at the time of first contact with the antigen. Antigen-binding cells exposed in vitro to antigen concentrations known to cause high zone tolerance induction failed to form polar antigen caps once they had entered the phase of increased receptor formation. The data suggest that sufficient crosslinking of receptors by the antigen to cause their aggregation triggers the cell to differentiate and to increase its density of antigen receptors. In the presence of antigen concentrations favoring high zone tolerance, receptors may become interlinked to such an extent that they are prevented from aggregation. Such a “frozen” state of the antigen recognition system would render the cell unresponsive to antigenic stimuli.