两种抗原检测弓形虫病IgM的效果

目的观察弓形虫P30重组抗原及可溶性抗原检测弓形虫病IgM的效果。方法建立斑点免疫金渗滤法（DIGFA）。采用2种抗原分别包被在两条硝酸纤维素膜（NC）上,与待检血清中的相应IgM抗体结合,通过金标记兔抗人IgM直接显色,以检测弓形虫病IgM。2种抗原的DIGFA分别检测弓形虫IgM抗体阳性、类风湿、支原体肺炎、血吸虫病人和正常人血清。结果两种抗原建立的DIGFA检测试剂共检测5种血清样本166份,其中弓形虫感染IgM抗体阳性病人血清38份,弓形虫P30DIGFA敏感性为92.1%（35/38）,可溶性抗原DIGFA敏感性为81.6%（31/38）,差异无统计学意义（P〉0.05）。正常人群对照血清63份,检测全部阴性,特异性为100%。检测类风湿病人血清30份、血吸虫病人血清25份及支原体肺炎病人血清10份,仅类风湿病人血清出现阳性交叉反应,其中弓形虫P30DIGFA阳性2例,可溶性抗原DIGFA阳性3例。结论弓形虫P30重组抗原检测弓形虫病人血清IgM的DIGFA试剂可用于临床早期或急性期弓形虫病的诊断。     

弓形虫抗体免疫印迹试剂盒的研制

目的 研制敏感、特异的检测弓形虫IgM和IgG抗体免疫印迹试剂盒。 方法 收集人工感染RH株弓形虫速殖子昆明系小鼠的腹腔液，提取弓形虫胞质蛋白，采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离弓形虫可溶性抗原，并经电泳转印至硝酸纤维膜，以无毒灵敏的四甲基联苯胺（TMB）为底物分别检测30份弓形虫IgM和28份IgG阳性血清，40份健康人血清。通过比较抗原制备方法和使用剂量、封闭剂、洗涤和稀释剂、工作浓度、作用时间以及反应带出现率，选择最佳实验条件，以敏感性、特异性、Youden指数以及稳定性作为试剂盒评价标准。 结果 用免疫印迹试剂盒检测30份弓形虫IgM和28份IgG阳性血清，敏感性分别为90.0%（27/30）和85.7%（24/28），40份健康对照者血清的弓形虫IgM和IgG抗体均为阴性，特异性均为100%，Youden指数分别为0.9和0.86。试剂盒于4℃ 保存约6个月的检测结果一致。 结论 该免疫印迹试剂盒敏感性和特异性均较高，且操作简便、快速。     

滴金免疫渗滤法快速诊断肺吸虫病的研究

目的寻找一种快速诊断肺吸虫病的方法.方法以肺吸虫成虫浸出液为抗原,金标抗人IgG为显示剂检测肺吸虫抗体(DIGFA).结果检测肺吸虫病人血清41份,阳性符合率为100%;检测非流行区健康人血清51份,流行区健康人血清30份,阴性符合率100%;检测班氏丝虫病人血清20份,均为阴性;检测日本血吸虫病人血清20份,其中2例呈阳性.用DIGFA与ELISA同时检测肺吸虫病人血清41份,非流行区健康人血清51份及流行区健康人血清30份,两种方法的阳性与阴性符合率均为100%.结论DIGFA检测人体肺吸虫抗体敏感性高,特异性强,检测时无需特定的仪器,操作简便、快速,适于基层单位和现场应用.     

组分抗原斑点金标免疫渗滤法检测血吸虫短程抗体的研究

目的建立一种可用于检测血吸虫病人短程抗体的斑点金标免疫渗滤法（dot immuno-gold filtration assay,DIG-FA）。方法将血吸虫可溶性虫卵组分抗原（107～121 kDa）包被于混纤膜上,以胶体金标记的抗人IgG为二抗,建立组分抗原-DIGFA。用该法检测慢性血吸虫病人、健康人、卫氏并殖吸虫病人和华支睾吸虫病人血清。同时与血吸虫可溶性虫卵抗原（SEA）-DIGFA比较。结果检测54份慢性血吸虫病人血清,组分抗原-DIGFA敏感性为94.4%,SEA-DIGFA敏感性为96.3%,两种方法检测50份正常人血清的特异性均为100%。检测19份卫氏并殖吸虫病人血清和30份华支睾吸虫病人血清,SEA-DIGFA敏感性分别为26.3%和10.0%,组分抗原-DIGFA均未检出阳性反应。结论应用组分抗原-DIGFA检测血吸虫短程抗体具有较高的敏感性和特异性,且与其他吸虫病几乎无交叉反应,特异性优于SEA-DIGFA。     

滴金免疫渗滤法快速诊断肺吸虫病的研究

目的 寻找一种快速诊断肺吸虫病的方法。方法 以肺吸虫成虫浸出液为抗原，金标抗人IgG为显示剂检测肺吸虫抗体（DIGFA）。结果 检测肺吸虫病人血清41份，阳性符合率为100％；检测非流行区健康人血清51份，流行区健康人血清30份，阴性符合率100％；检测班氏丝虫病人血清20份，均为阴性；检测日本血吸虫病人血清20份，其中2例呈阳性。用DIGFA与ELISA同时检测肺吸虫病人血清41份，非流行区健康人血清51份及流行区健康人血清30份，两种方法的阳性与阴性符合率均为100％。结论 DIGFA检测人体肺吸虫抗体敏感性高，特异性强，检测时无需特定的仪器，操作简便、快速，适于基层单位和现场应用。     

弓形虫主要表面抗原P30两个不同片段的重组表达、纯化及应用

目的将刚地弓形虫（简称弓形虫）主要表面抗原P30基因两个不同片段重组表达，纯化所获重组蛋白用于弓形虫病免疫学诊断。方法根据弓形虫P30基因的全长序列，用PCR法从弓形虫RH株基因组DNA中扩增出P30基因的两个不同片段，并构建相应克隆进行诱导表达，表达蛋白以western blot鉴定。用Amylase Resin以亲和层析法纯化所表达的蛋白。以弓形虫RH株感染家兔，用粗抗原和纯化重组抗原以ELISA法比较检测各种寄生虫病患者、感染家兔血清及疟原虫感染小鼠血清。结果1．成功构建相应克隆，并成功诱导表达，表达产物经纯化后获得高纯度日的蛋白。2．重组抗原与粗抗原相比。具有相同的敏感性和更高的特异性。结论成功获得弓形虫主要表面抗原P30的2个表达产物。以此重组抗原与粗抗原对比检测弓形虫相应抗体，结果证明重组抗原特异性高于粗抗原。     

快速检测美洲钩虫特异抗体胶体金免疫层析试条方法的建立与效果评价

目的建立简便、快速检测美洲钩虫特异抗体胶体金免疫层析试条方法并评价其检测效能。方法将美洲钩虫成虫置液氮中研磨,通过膜蛋白表面活性剂浸泡、再经液氮反复冻融和硫酸铵沉淀提取成虫可溶性抗原。用制备的成虫可溶性抗原作为包被抗原,以胶体金标记G蛋白为检测探针,制备检测钩虫特异抗体的免疫层析试条。用该试条检测美洲钩虫、蛔虫、鞭虫、日本血吸虫和刚地弓形虫等寄生虫感染者的血清以及健康人血清,评价该试条检测的敏感性和特异性,同时用ELISA进行平行检测,以评价该试条的检测效能。结果制备了检测钩虫特异抗体的胶体金免疫层析试条。用该试条和ELISA法分别检测美洲钩虫、蛔虫、鞭虫、血吸虫、刚地弓形虫感染者血清95、10、11、10和10份,健康人血清74份。试条和ELISA检测美洲钩虫感染者血清的敏感性分别为88.4%(84/95)和90.5%(86/95),检测74份健康人血清假阳性率分别为4.1%(3/74)和6.8%(5/74);与蛔虫感染者血清的交叉反应分别为2/10和4/10,与鞭虫感染者血清的交叉反应分别为1/11和4/11,试条法与血吸虫和弓形虫感染者血清无交叉反应,ELISA法与血吸虫感染者的交叉反应为1/10,与弓形虫感染者血清无交叉反应。试条法与ELISA法的特异性分别为94.9%(109/115)和88.7%(102/115),检测效能分别为91.9%和89.5%。试条法与ELISA法敏感性、特异性的差异均无统计学意义(P0.05)。结论以美洲钩虫成虫可溶性抗原制备的快速检测美洲钩虫特异抗体胶体金免疫层析试条检测美洲钩虫感染者血清的敏感性和特异性均较高。     

血吸虫病快速免疫诊断—胶体染料免疫渗滤法的初步研究

目的开发一种适合现场应用的血吸虫病快速免疫诊断方法。方法将纯化的日本血吸虫虫卵可溶性抗原(SEA)点于硝酸纤维素膜上,以捕获血清标本中血吸虫特异性抗体,通过胶体染料R3标记的SEA来直接显色,阳性者出现红色斑点。结果胶体染料免疫渗滤法检测急性血吸虫病人血清35份、慢性血吸虫病人血清107份、晚期血吸虫病人血清35份,其敏感性分别为100％、97.2％和74.3％。检测健康人血清87份,特异性为100％。检测肝吸虫病人血清38份、姜片虫病人血清11份、钩虫病人血清20份,其交叉反应率分别为2.6％、0.0％和0.0％,整过试验过程仅需2～3分钟,操作简单。染料标记SEA在室温条件下保存6个月、37℃恒温中保存62天,其活性均未发生明显变化。结论胶体染料免疫渗滤法检测各期血吸虫病结果准确,操作简便、快速、廉价、肉眼观察结果不需特殊仪器,特别适用于基层现场查病。     

成虫三氯醋酸抽提抗原金标免疫渗滤法诊断日本血吸虫病及考核疗效的研究

目的探讨成虫三氯醋酸抽提抗原(AWA-TCA)金标免疫渗滤法在日本血吸虫病诊断和疗效考核中的潜在应用价值.方法用三氯醋酸提取AWA-TCA,以此抗原为探针,建立金标免疫渗滤法(AWA-TCA-DIGFA),检测血吸虫病人血清中特异性抗体,并与可溶性虫卵抗原金标免疫渗滤法(SEA-DIGFA)作平行对照.结果AWA-TCA-DIGFA和SEA-DIGFA平行检测146人份慢性血吸虫病人血清,敏感性分别为96.6%和97.3%,两者差异无显著性(P＞0.05);检测100人份健康人血清,仅1份两法检测均呈弱阳性,特异性为99.0%;43人份治后6个月病人血清中,AWA-TCA-DIGFA检出33份阳性,抗体阴转率为23.3%(10/43),SEA-DIGFA检出35份阳性,阴转率为18.6%,两者差异无显著性(P＞0.05);两法检测36份治后12个月病人血清,抗体阴转率分别为80.6%(29/36)和58.3%(21/36),两者差异有显著性(P＜0.05).检测其他蠕虫病人血清60人份,除与2例并殖吸虫病人血清有交叉反应外,其余均为阴性.结论AWA-TCA-DIGFA用于诊断血吸虫病,其敏感性和特异性与SEA-DIGFA相近,且有更好的疗效考核价值.     

血吸虫病快速免疫诊断——胶体染料试纸条法的研究

目的 开发一种血吸虫病快速免疫诊断方法。方法 用国产胶体染料Ｄ－１标记日本血吸早虫可溶性虫卵抗原（ＳＥＡ），以此标记物与血吸虫病人血清中的抗血吸虫抗体反应，再用点有羊抗人ＩｇＧ（第二抗体）的硝酸纤维层析膜以年捕获标记抗原与抗体的复合物。结果 用 纸条法检测急性血吸虫病人血清３０份，慢性血吸虫病人血清８４份，其敏感性分别为９６．７％和９４．０％，检测健康人血清６０份，其特异性为９６．７％，检测肝吸虫     

PCR technique for detecting Toxoplasma gondii in animal amniotic fluid

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.     

The ELISA-U: an enzyme-linked immunosorbent assay using urease as the enzyme marker for rapid detection of Plasmodium falciparum antibody in human serum

A visual, enzyme-linked immunosorbent assay using urease (ELISA-U) as the enzyme marker was adapted for rapid detection of antibody against Plasmodium falciparum. Flat-bottom, 96-well microtiter plates were coated with P. falciparum soluble antigen obtained by saponin and NP-40 treatment of parasite cultures. Antibody was detected by successive incubations with test sera, urease-conjugated rabbit-human antibody, and urease substrate. Reactive sera developed a definite and easily visualized purple color. Sera from patients with single infections of P. vivax or P. ovale were unreactive. No cross-reactivity was noted with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The procedure can be performed at room temperature and completed within 1 hr. The sensitivity of the assay is comparable to that of the indirect fluorescent antibody test at all but the lowest dilutions tested.     

Acute toxoplasmosis: evaluation of a thin-layer immunoassay technic for detection of IgM antibodies, anti-Toxoplasma gondii

A solid phase method, thin-layer immunoassay (IgM-TIA) was standardized and evaluated for the immunodiagnosis of acute toxoplasmosis, through the detection of IgM antibodies to Toxoplasma gondii. A total of 300 serum samples from serologically defined acute toxoplasmosis and, from non-related infections, was investigated by IgM-TIA. Statistical analysis were carried out in comparison with conventional tests, the immunofluorescence test for the detection of IgM antibodies (IgM-IFI) and hemagglutination test which uses 2-mercaptoethanol serum treatment (2ME-HA). Also the correlation coefficients were calculated for various Toxoplasma gondii antigen concentrations, as well as, the influence of the antigenic concentration on the relative indices of sensitivity and specificity were verified. The intra and inter test reproducibilities were demonstrated statistically, as well as, the reutilization of T. gondii antigen was proven to be possible for at least 10 times. The data indicated that antigenic concentrations, from 70 to 100 Cmg/ml, were able to provide maximum sensitivity and specificity. IgM-TIA displayed similar diagnostic efficiency to those two conventional tests here utilized, and may be employed to make diagnosis of acute toxoplasmosis, mainly if laboratory animals are available.     

Composition of circulating immune complexes in acute toxoplasmosis

The circulating immune complexes (CIC) that form in the hot just in early Toxoplasma gondii invasion can be present in the blood bed for a while. At the same time, the data on the antigenic composition of CIC in toxoplasmosis are fragmentary and rather contradictory. The investigation used enzyme immunoassay (EIA) to detect specific CIC that contain antigens to T. gondii tachyzoites in the sera of different populations and studied their antigenic composition by immunoblotting after 2-6% polyethylene glycol 6000-induced deposition. Examining 464 sera from groups of individuals with varying-stage T. gondii invasion indicated CIC in each, but showing their different frequency. CIC were virtually present in all sera from children who had been diagnosed as having congenital toxoplasmosis. In groups of seropositive pregnant women, CIC detection rates were noticeably higher in the samples showing both IgG and IgM antibodies (40.2 and 66.4%, respectively). CIC were also revealed in 9.8% of the seronegative blood donors; however, immunoblotting failed to confirm that they had no components that specifically reacted with T. gondii tachyzoite antigen antibodies. There were some differences in the composition of CIC in the serum yielding positive results in both EIA and immunoblotting. The serum CIC from pregnant women that exhibited only IgG antibodies contained mainly T. gondii antigens having molecular weights of 67 and 30 kD. The serum CIC from children with congenital toxoplasmosis and from pregnant women with serologically detected IgM antibodies to Toxoplasma antigens were found to contain 55-58-, 48-, 44-, 38-, 30-, and 26-kD components. The same molecular weight proteins were detected by electrophoretic studies of Toxoplasma excretory-secretory antigen (ESA). Comparing the findings suggests that in acute toxoplasmosis, the circulating complexes mainly contain ESA of the tachyzoites which appear in human blood just at the onset of invasion. Thus, this study demonstrates that specifically CIC are detectable in the sera of individuals infected with T. gondii and their antigenic composition varies with the stage of disease. In the authors' opinion, the detection of specific CIC and the determination of their antigenic composition may be serve an additional test in diagnosing acute toxoplasmosis.     

直接凝集试验（DAT）检测弓形虫IgG抗体的研究

本文报道了国内首次建立的检测弓形虫IgG抗体的微孔板法直接凝集试验(DAT)。采用的抗原系经胰蛋白酶处理和福尔马林固定后的弓形虫速殖子全虫抗原,被检血清用α-巯基乙醇排除IgM引起的非特异性反应。对包括实验感染动物在内的不同来源的动物血清和人血清进行了检测。结果表明:该试验具有较高的敏感性、特异性和重现性,并能测出感染较早期的IgG抗体,在4～6℃低温下保存达6～9个月的抗原仍保持其原有的抗原性。     

肺吸虫特异性IgG4快速检测方法的建立与应用

目的创建简易、快速、准确的肺吸虫病免疫诊断方法。方法用卫氏并殖吸虫成虫粗提液为抗原,以胶体金标记抗人IgG4单抗为检测探针,采用自行设计的垂直流渗滤装置,创建快速检测人血清肺吸虫特异性IgG4方法(P-IgG4-DIGFA),平行检测IgG作比较分析;以经典ELISA为对照,评价其诊断价值,应用P-IgG4-DIGFA检测有效治疗后病人血清,评价其疗效考核价值。结果P-IgG4-DIGFA检测人血清中肺吸虫特异性IgG4的敏感性和特异性分别为95.2%(40/42)、100%(50/50),与血吸虫病患者血清的交叉反应率仅为2%(1/50),未发现与华支睾吸虫病有交叉反应。P-IgG4-DIGFA的敏感性和特异性略高于经典ELISA,差异无统计学意义(χ2=0.25,P>0.05);应用P-IgG4-DIGFA检测有效治疗后3个月、6个月和12个月病人血清特异性IgG4,阴转率分别为0%、16.7%(1/6)和100%。结论金标渗滤法快速检测肺吸虫特异性IgG4,不仅操作简便、快速,而且敏感性高、特异性强,适用于临床检验和现场查病,对治疗后1年病人具有疗效考核价值,有待进一步开发应用。     

混合血清法检测献血员血清抗弓形虫抗体

将徐州市区义务献血员６人一组混合，用斑点免疫金银染色法（Ｄｏｔ－ＩＧＳＳ）进行检测，同时对所有血清用同样方法逐份进行检测，以研究混合血清法筛检抗弓形虫抗体的可能性。检测结果显示，混合血清法的敏感性为９２．３１％。特异性为９９．９６％，混合血清法与逐份血清法的一致率为９９．７３％，Ｋａｐｐａ值为０．９４７（Ｐ〈０．０１）。以全国弓形虫平均感染率（４．８６％）进行定量效益分析，用混合血清法筛检献血员，     

应用磁分离酶联免疫技术检测抗日本血吸虫虫卵抗体

目的 建立检测日本血吸虫病患者血清中特异性虫卵抗体的磁微粒分离酶联免疫法（MPAIA）。方法 采用异硫氰酸荧光素（FITC）标记日本血吸虫可溶性虫卵抗原（Sj-SEA）为检测抗原, 标记羊抗FITC抗体的磁微粒为固相载体, 碱性磷酸酶（ALP）标记羊抗人免疫球蛋白G（IgG）作为酶标二抗, 以单磷酸酚酞溶液为底物, 检测日本血吸虫病患者血清中虫卵特异性抗体。 结果 用磁微粒分离酶联免疫法检测日本血吸虫虫卵抗体, 阳性检出率为96.7%（116/120）, 与旋毛虫、并殖吸虫、嚢尾蚴等其他寄生蠕虫抗体无交叉反应现象, 检测试剂4 ℃可保存12个月。灵敏度参考品的灵敏度为1 ∶ 1 600, 精密度参考品的精密度（CV）＜10%。 结论 磁微粒分离酶联免疫法具有灵敏度高、特异性强、技术先进, 试剂保存时间长等特点。