改良凝集试验（MAT）与间接血凝试验（IHAT）和酶联免疫吸附试验（ELISA）检测弓形虫抗体的比较分析

为了评价用于检测弓形虫抗体的改良凝集试验(MAT)、用MAT、间接血凝试验(IHAT)、酶联免疫吸附试验(ELISA)对动物和人的血清进行检测,比较阳性检出率与符合率,并对检测的结果进行统计分析.结果显示MAT和IHAT、MAT与ELISA检测动物和人血清的阳性检出率都无显著差异(P>0.05).MAT与IHAT检测动物血清的总符合率为78.7%(59/75),检测人血清的总符合率为81.6%(71/87);MAT与ELISA检测动物血清的总符合率为74.0%(54/73),检测人血清的总符合率为79.3%(69/87).应用配对资料卡方检验分析显示MAT与IHAT、MAT与ELISA检测的结果一致.因此,检测弓形虫抗体的MAT与IHAT和ELISA具有良好的符合性,这3种方法都能用于弓形虫抗体的常规普筛和血清流行病学研究.     

重组弓形虫P30抗原诊断弓形早病的初步探讨

为探讨重组弓形虫P30抗原（rP30)对弓形虫病的诊断价值。将rP30包被于酶标反应板微孔,用常规ELISA检测血清抗体。结果4例染色试验阳性血清,抗-rP30IgG全部阳性,阳性符合率为100%;27例虫体IgG/IgM抗体阳性血清中,22例rP30I-ELISA阳性,符合率为85.2%;100例门诊病人和50例献血员血清的阳性检出率分别为9.0%和8.0%。说明rP30检测弓形虫病患血清具有一定的敏感性和特异性,可望替代天然的弓形虫抗原用于弓形虫病的诊断。     

重组主要表面抗原1用于弓形体病IgG免疫诊断的研究

目的　用纯化的重组刚地弓形虫RH株主要表面抗原 1(SAG1)作为检测抗原 ,建立间接法rSAG1 ELISA ,检测弓形虫IgG抗体并与国外进口试剂盒进行比较 ,以观察其阳性和阴性符合率 ;并对rSAG1 ELISA检测的精确度、灵敏度和特异性进行评价。方法　接种重组菌至LB肉汤中 ,IPTG诱导表达后 ,用Ni2 螯合柱进行亲和纯化 ;分别用不同浓度的重组抗原rSAG1包被聚苯乙烯酶标条 ,检测不同稀释度的阳性和阴性血清 ,以辣根过氧化物酶标记的羊抗人IgG为二抗 ,采用正交试验确定rSAG1 ELISA的最佳检测条件 ;按rSAG1 ELISA法的最佳检测条件对混合弓形虫IgG阳性和阴性血清重复测 2 0次进行精确度的检测 ;采用抗体抑制试验检测其特异性 ;同时对用进口试剂盒筛得的 5 2份弓形虫IgG阳性血清和 4 0份弓形虫IgG阴性血清进行检测。结果　制备的rSAG1重组蛋白纯度在90 %以上 ;以该重组抗原建立的rSAG1 ELISA的最佳检测条件为 :重组抗原rSAG1的包被浓度为 5 μg/ml,血清稀释度 1∶10 0 ,酶标记的羊抗人IgG 1∶2 0 0 0稀释 ;用rSAG1 ELISA对混合弓形虫IgG阳性和阴性血清的重复检测表明 :IgG阳性血清的检测值的变异系数 (CV值 )为 10 .9% ,IgG阴性血清的CV值为 10 .7% ;灵敏度检测表明血清稀释度在 1∶5 0～ 1∶2 0 0均可检出阳性 ;特异性     

重症肌无力患者抗Ryanodine受体抗体检测及其意义

本实验应用Westernblot分析 4 4例伴不同胸腺病理类型重症肌无力 (MG )患者血清中Ryanodine受体 (RyR )抗体特异性 ,同时应用ELISA法检测 1 6 9例伴不同胸腺病理类型MG血清中RyR抗体水平。结果显示在Westernblot法分析中 ,2 4例伴胸腺瘤重症肌无力 (MGT )患者血清中有 1 9例可见到RyR抗体阳性条带 ,2 0例非胸腺瘤重症肌无力 (NTMG )患者血清中仅1例可见RyR抗体阳性条带 ,2 5例非MG个体 (NMG )均未见RyR抗体阳性条带。MGT患者血清中RyR抗体阳性条带检出率明显高于NTMG和NMG组 (P <0 0 1 )。在ELISA法检测中 ,MGT组患者血清中RyR抗体水平明显高于NTMG组、其他神经系统疾病 (OND )组、正常对照 (NC )组 (P <0 0 1 ) ;5 9例MGT患者血清中 4 6例RyR抗体阳性 ,2 83例NTMG、OND、NC组检测血清中 1 9例RyR抗体阳性 ,ELISA法检测MGT患者血清中RyR抗体敏感性为 78% ,特异性为 93 3%。结果表明RyR抗体检测是诊断MGT特异性较高的实验室指标 ,具有重要的临床意义     

应用双抗原夹心ELISA法筛查献血员梅毒螺旋体抗体

目的：优选一种适宜于献血员梅毒筛查的试验方法。方法：采用检测梅毒特异性抗体的双抗原夹心ELISA法对献血员进行抗体检测，并与RPR检测结果进行比较，对两种方法检测结果不一致的标本，再用TPHA法进行确证。结果：ELISA法阳性检出率0．36％(41／1271)、RPR法阳性检出率0．26％(29／11271)。ELISA法与TPHA法总符合率97．5％(40／41)、RPR法与TPHA总符合率63．41％(26／41)。结论：ELISA法优于RPR法，具有较高的灵敏度和特异性，适宜于献血员的筛查．有利于控制和减少梅毒的输血传播。     

快速胶体染料试纸条法检测弓形虫病IgG抗体的研究

目的研制一种检测弓形虫病IgG抗体的快速胶体染料试纸条法(DDIA),与国外进口试剂盒进行比较,以评价其临床应用价值。方法以本实验室筛选的胶体染料D1标记羊抗人IgG,以此标记物与弓形虫病人血清中的抗弓形虫IgG抗体结合,用点有弓形虫可溶性抗原的硝酸纤维膜通过层析捕获染料标记羊抗人IgG与弓形虫IgG抗体的复合物,采用正交试验确定DDIA的最佳检测条件;同时对弓形虫病流行病学筛查时的88份血清进行检测并与进口试剂盒的检测结果进行比较。结果DDIA的最佳检测条件为弓形虫可溶性抗原的点膜浓度为1mgml,人IgG点膜浓度为2mgml,血清用量为10μl和羊抗人IgG胶体染料检测液作1∶4稀释时的检测带清晰且背景较浅;DDIA与进口试剂盒的总符合率为97.7%,其中阳性和阴性符合率分别为100%(5252)和94.4%(3436)。结论DDIA与进口试剂盒有较高的符合率,表明该法具有较好的弓形虫病诊断价值,可进一步推广应用。     

甲状腺激素自身抗体(抗T3、T4)放射免疫分析

目的 :本实验主要建立内源性甲状腺激素自身抗体 (抗T3 、T4)放射免疫分析的检测方法。方法 :用12 5I标记T3 、T4衍生物作为示踪剂 ,血清样本与T3 、T4衍生物 (12 5IT3 、T4衍生物 )在室温 16h孵育。用抗人IgG二抗分离抗体抗原复合物 ,测定放射性活度。结果 :12 5I标记T3 、T4衍生物与人血清T3 、T4自身抗体特异性结合。健康人群中甲状腺激素自身抗体阳性检出率为 1 0 7% ;甲状腺功能异常组甲状腺激素自身抗体阳性检出率为 14 4 % ;甲状腺功能亢进组 13 5 % ;甲状腺功能减退组 15 2 %。甲状腺激素自身抗体阳性血清标本经加入非标记T3 或T4,血清中T3 或T4自身抗体可被中和。T3 或T4自身抗体滴度较原滴度明显下降 (P <0 0 5 ) ,用PEG沉淀法去除血清中免疫球蛋白 ,高水平的FT3 、FT4均明显下降 (P <0 0 5 )。结论 :本文结果提示 ,正常人群中甲状腺自身抗体阳性的发生率较低 ,甲状腺功能异常患者中甲状腺激素自身抗体阳性检出率达14 4 %。内源性甲状腺激素自身抗体对FT3 、FT4的测定存在干扰 ,结果常与临床表现不一致。经PEG将免疫球蛋白去除后 ,重新评估了血清中FT3 、FT4的含量 ,其结果与TSH趋于一致。     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

丙肝病毒单片段抗体检测分析

目的 探讨丙型肝炎病毒感染者抗.HCV各区段抗体的反应性及单片段抗体ELISA测定临床应用的可行性和应用价值.方法 留取经2种第三代丙肝病毒总抗体ELISA检测试剂A、B检测结果为阳性的血清36份、可疑血清2份、阴性血清40份,用丙肝单片段抗体检测试剂C对其进行检测,并以CHIRON公司第三代免疫印迹丙肝抗体确认试剂D检测结果作为对照.结果 78份血清中,抗HCV-C、NS3、NS4、NS5四区单片段抗体阳性检出率分别为44.87%、47.44%、30.77%、28.21%;单片段抗体检测试剂C的阳性率为43.59%,与第三代总抗体检测试剂A、B的阳性率(分别为48.72%和46.15%)无显著差异;试剂A、B检出的阳性血清36份中,根据试剂C的核心区、NS3、NS4及NS5区单片段抗体检测结果综合判断确定阳性结果34份,不确定结果2份,阳性符合率为94.44%;2份可疑血清根据单片段抗体检测结果综合判断均为不确定结果;40份阴性血清中,单片段抗体检测检出阴性结果40份,阴性符合率为100%.试剂C对所有78份血清检测结果为阳性34份,不确定4份,阴性40份;而确认试剂D检测结果为阳性34份,不确定3份,阴性41份.结论 NS3区和c区为抗-HCV ELISA检测中的主要血清学标志;单片段检测试剂C与第三代总抗体检测试剂A、B之间的检出率无显著差异,结果符合良好,且与国际上公认的CHIRON第三代免疫印迹丙肝抗体确认试剂检测结果高度一致.单片段抗体检测在临床判断病情、病毒复制情况及预后方面较总抗体检测能提供更多信息.     

抗人游离前列腺特异性抗原(f-PSA)单克隆抗体的制备及应用

目的建立产生抗人游离前列腺特异抗原单克隆抗体(f-PSA m Ab)的细胞株,获得抗体后建立抗人f-PSA化学发光免疫分析法(CLIA)并进行应用。方法采用杂交瘤技术获得2株稳定分泌抗f-PSA m Ab的细胞株,将细胞株用转瓶扩大培养并将上清液采用亲和纯化法获得抗体,ELISA测定抗体效价、特异性及表位,表面等离子共振法测抗体亲和力;建立双抗体夹心CLIA,采用标准曲线定量法对该体系进行分析性能评估,同时对426例临床血清样本进行检测[其中130例的总PSA水平在(4～10) ng/mL,为"诊断灰区"],评价该试剂与罗氏试剂的检测一致性。结果获得抗人f-PSA m Ab的杂交瘤细胞株(f-10-1、f-14-1)。用f-10-1和f-14-1建立的CLIA的检测范围为(0. 1～30) ng/mL,灵敏度为0. 05 ng/mL,检测结果的相对偏差均在±5%内,与癌胚抗原(CEA)、甲胎蛋白(AFP)及结合态PSA(c-PSA)无交叉反应,该试剂与罗氏试剂相关系数达到0. 99,灰区样本阳性符合率、阴性符合率、总符合率均高于90%。结论成功制备了抗人f-PSA m Ab,建立了特异性定量检测人f-PSA的双抗体夹心CLIA。     

Comparative study of three tests (dye test, indirect haemagglutination test, latex agglutination test) for the detection of antibodies to Toxoplasma gondii in human sera.

An evaluation has been made of a commercial latex agglutination test, Toxotest-MT (TMT) (Eiken, Japan), for the detection of antibodies to Toxoplasma gondii in human sera. In qualitative studies, 878 sera were examined in both the TMT and dye test (DT) and 96.6% agreement was found, In quantitative studies 339 sera were titrated in the TMT and DT, with 337 of these sera also titrated in the indirect haemagglutination test (IHAT). Agreement between the DT and TMT was best, 78% of the sera showing titres within +/- 1 dilution in the two tests. The IHAT and TMT gave 66% agreement, while the DT and IHAT showed least agreement, 40.9%. The results suggest that the pattern of antigenic determinants to which antibody levels are measured are different in the three test systems. The TMT is a better substitute for the DT than the IHAT.     

2005年湛江市、韶关市、汕头市人兽弓形虫感染状况调查

目的了解广东省湛江市、韶关市、汕头市的人兽弓形虫感染情况。方法收集3个地区人、猪、鼠血清，采用酶联免疫吸附试验（ELISA）间接法检测弓形虫IgG抗体。结果湛江市检测猪血清279份，阳性13份，阳性率为4．66％，检测鼠血清93份，阳性7份，阳性率7．53％；韶关市检测人血清42份，阳性0份，检测鼠血清95份，阳性3份，阳性率3．16％；汕头市检测人血清50份，阳性6份，阳性率为12．00％，检测鼠血清100份，阳性3份，阳性率为4．00％。结论汕头市人群存在弓形虫感染，湛江市、韶关市、汕头市兽类存在弓形虫感染，3个地区的鼠类弓形虫感染率无统计学意义，家鼠、野鼠的弓形虫感染率也无统计学意义。     

Comparison of six commercial enzyme linked immunosorbent assays for detecting IgM antibodies against Toxoplasma gondii.

To evaluate the usefulness of different commercial enzyme linked immunosorbent assays (ELISAs) for the detection of IgM antibodies against Toxoplasma gondii the results of six of these assays for a panel of 81 sera were compared. The following tests were selected: Toxoplasma gondii IgM ELISA (Clark Laboratories), Toxoplasma IgM EIA (Labsystems), Toxo-M EIA (Abbott), Toxonostika M (Organon), Toxo M Enzyme Immunoassay (Hybritech) and Platelia Toxo IgM (Diagnostics Pasteur). An antibody capture ELISA developed at our laboratory was used as the reference test. An IgM immunoblotting assay was also performed. Four (Toxoplasma IgM EIA, Tox-M EIA, Toxonostika M, and Platelia Toxo IgM) of the commercial IgM ELISAs gave a high sensitivity and a high specificity. Toxo-M EIA, Toxonostika M, Toxoplasma IgM EIA and the Toxo M Enzyme Immunoassay were too insensitive, and the Toxoplasma gondii IgM ELISA was both insensitive and unspecific. No remarkable differences were observed between the results of indirect or antibody capture ELISAs, and between the results of ELISAs performed with polyclonal or monoclonal antibodies.     

Use of MAG1 recombinant antigen for diagnosis of Toxoplasma gondii infection in humans

This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.     

A Standardized Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Antibodies to Toxoplasma Gondii

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed.

This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents.

In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficients of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.     

Immunochemical identification and detection of a 36-kDa Toxoplasma gondii circulating antigen in sera of infected women for laboratory diagnosis of toxoplasmosis

The detection of Toxoplasma gondii circulating antigens has been indicated to be a reliable diagnostic approach of active human toxoplasmosis. However, few reports have appeared in the literature regarding the diagnostic potential of T. gondii circulating antigens. Here, a specific antibody and western blot analyses were used to demonstrate the presence of a highly reactive antigen of 36-kDa, not only in the extract of T. gondii tachyzoites, but also in selected sera of women with confirmed laboratory and clinical signs of recent toxoplasmosis. The 36-kDa Toxoplasma antigen was purified from T. gondii tachyzoites and human serum using electroelution from preparative polyacrylamide gels. The purified polypeptides showed a single peak at 10.9 min when analyzed by capillary zone electrophoresis. Based on the above encouraging results, we have developed an ELISA format for the detection of target Toxoplasma antigen (TAg-ELISA) in human serum samples. The TAg-ELISA detected the target antigen in 88% sera of acutely infected women and showed high degree of specificity (91%) among sera from non-infected women. In conclusion, the detection of 36-kDa Toxoplasma circulating antigen in human sera appears to be a promising alternative approach for laboratory diagnosis of active T. gondii infection.     

Enzyme-linked immunosorbent assay for quantitation of toxoplasma antibodies in human sera.

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of toxoplasma antibodies using a single serum dilution (1:800) in conjunction with a standard curve Antigen was prepared from Toxoplasma gondii cultivated in human cell cultures. A nearly linear relationship was found between the logarithms of the absorbance values of 120 human sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was excellent; the coefficients of variation for within-day and day-to-day tests were less than 15%. A close correlation was found between the results obtained with ELISA, indirect immunofluorescence (IF), and complement fixation (CF). The titres in ELISA were 20 to 40 times higher than in IF and 200 to 1000 times higher than in CF.     

Characterization of Toxoplasma gondii SAG2 expressed in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.     

Antitoxoplasma antibody in clinically suspected cases of human toxoplasmosis

(1) Seventy sera from a variety of patients suffering from different diseases suspected to be caused by Toxoplasma gondii infection and forty from non-toxoplasmic hospital cases and laboratory and hospital staff were collected. (2) Antitoxoplasma antibody was detected in those sera by Indirect Haemagglutination test (IHA test) and Enzyme Linked Immunosorbent Assay (ELISA). (3) Twenty one (30%) sera out of 70 test samples and 2 (5%) out of 40 control samples were positive by ELISA test. With IHA test only 17 (24.3%) of test samples and same 2 (5%) of control samples were positive. (4) Sera collected from Paediatric Department showed the highest positivity (40%) followed by Opthalmological group (35.7%) and obstetrics and Gynaecological group (13.6%). (5) No significant co-relation was found between the seropositivity with sex, diet and history of cat contact of the patients.     

Reproducible enzyme-linked immunosorbent assay with a magnetic processing system for diagnosis of toxoplasmosis.

An enzyme-linked immunosorbent assay with polycarbonate-coated iron beads as the solid phase and with magnetic processing devices was evaluated for the quantitation of antibodies to Toxoplasma gondii in human serum samples. Under the parameters and other basic conditions determined in this study, the assay was highly reproducible: coefficients of variation for the absorbance values obtained with the positive serum were 2.42% in same-day tests and 3.75% in day-to-day tests. Significant correlations were observed between the present assay system and other conventional serological tests: correlation coefficients were 0.960 with the dye test and 0.929 with the latex agglutination test. Statistical analysis based on the frequency distribution of absorbance values for dye-test-positive and dye-test-negative serum samples gave feasible border lines for distinguishing between positive and doubtful samples (0.357) and between doubtful and negative samples (0.266). Under this diagnostic criterion, the results of our assay system agreed remarkably well with those obtained by the dye test and the latex agglutination test, with consistencies of 94.9 and 93.9%, respectively.