日本血吸虫31／32kDa循环免疫复合物的检测及其在疾病 …

检测血吸虫循环免疫复合物（ＣＩＣ）有助于提高疾病的检出率。利用抗血吸虫单克隆抗体Ｈ２２６和碱性磷酸酶标记的羊抗人ＩｇＧ（Ｆｃ）作捕捉ＥＬＩＳＡ，检测Ｅ要血吸虫病患者血清ＣＩＣ，其阳性检出率为９２．２％，其中，ＥＰＧ〈１００的病人阳性检出主继９０．０％，ＥＰＧ〉１００的病人阳性检出率为９５．５％，两者无显著性差异。由于羊抗人ＩｇＧ Ｆｃ段能与ＣＩＣ中人ＩｇＧ的Ｆｃ段特异性结合，因此不驻省去了常规分离     

捕捉ABC—ELISA检测肺结核病人特异性CIC方法的建立

利用抗人IgG及抗人IgA捕捉完整的结核特异性IgG型及IgA型循环免疫复合物(CIC),进而用抗结核菌抗体检测的原理,建立起捕捉ABC-ELISA检测肺结核病人特异性CIC方法。对100例肺结核病人检测IgG型及IgA型CIC分别为35％及73％(P<0.001),总阳性率为85％;对40例正常人非特异反应IgG型及IgA型CIC分别为2.5％及7.5％(P>0.05),总特异性为90％。结果既证明结核病人血清中确含完整的结核特异性CIC,又为检测之提供了一套候选方法。     

应用单克隆抗体ELISA检测包虫病人循环免疫复合物的研究

应用抗包虫单克隆抗体F1建立了检测包虫病人循环免疫复合物和解离抗原的ELISA法。102例包虫病人中CIC阳性率为50％(51/102),其中手术前检出率为62.62％(33/53),手术后检出率为36.73％(18/49)。二者差异显著(P<0.005)。用PEG沉淀的免疫复合物经尿素解离后检测包虫抗原的阳性率为58.82％(60/102)。CIC与解离抗原二者累积阳性率达到86.27％(88/102)。表明在包虫病人中CIC的存在是普遍的。F1单克隆抗体ELISA法在检测包虫病人CIC和解离抗原上有极高的敏感性,因而证明F1单克隆抗体所识别的抗原表位是形成CIC的主要抗原成分。在IgG型CIC阳性的血清中解离抗原的检出率仅为48.1％(23/51)。可能是由于IgG抗体的高度亲和力,由其形成的CIC解离效率低。在IgG-CIC阴性血清中,检出解离抗原的阳性率高达72.5％(37/51),证明了在相当部分包虫病人血清中可能存在着IgE和IgM型CIC。解离抗原阳性率高表明这类CIC容易解离。在17例特异性IgG抗体阴性的包虫病人中CIC阳性者10例(58.82％),解离抗原阳性者9例(52.94％),二者累积阳性率为76.47％(13/17)。说明CIC与解离抗原的检测在IgG抗体阴性的包虫病人诊断上有实际意义。     

包虫病人特异性IgM型和IgE型循环免疫复合物的初步研究

应用ABC-捕获ELISA法研究了包虫病人血清中特异性IgM型和IgE型循环免疫复合物(IgM-CIC,IgE-CIC)。IgM-CIC的检出率为47.6％,IgE-CIC为64.3％。肺包虫病人中CIC的检出率略高于肝包虫病,但无显著性差异。特异性IgE抗体阴性的包虫病人中,IgE-CIC检出率达89.5％,而抗体阳性者中为57.7％。在5例特异性IgM抗体阴性的肺包虫病人中,有4例IgM-CIC阳性,而4例IgM抗体阳性者中仅1例CIC阳性。表明由于CIC的形成可造成特异性IgM和IgE游离抗体的“耗竭”。用8M尿素处理后,三种特异性CIC均可被解离。用单克隆抗体检测解离抗原的阳性率在IgG-CIC中为51.3％,在IgM-CIC中为75.0％,在IgE-CIC中为75.9％,证明解离效率与相应抗体的亲和力呈反比。作者认为,包虫病人特异性抗体的检出,不能反映抗体应答的真正水平,抗体阳性加上抗体阴性而CIC阳性者,才能完全反映抗体应答的频率:在IgG为95.2％,IgM为85.1％,IgE为95.6％。提示包虫病人IgM-CIC和IgE-CIC的检测可大大提高免疫诊断的价值,有实际意义。     

血吸虫循环抗原检测在血吸虫病普查中的应用

目的了解血吸虫循环抗原（ScAg）检测在普查中的应用效果，发挥ScAg检测在血吸虫病传播阻断中的作用。方法在现场单盲试验普查条件下测试血吸虫病血清库质控血清，应用ScAg在不同程度感染的血吸虫病流行区批量检测。对ScAg阳性者作9送9检粪孵追踪观察。结果ScAg检测EPG≤8的血吸虫病患者血清阳性率为95．45％，EPG≥16的血吸虫病患者血清阳性为100．00％，对正常人和华支睾吸虫病患者血清的特异性为100．00％。对高、中、低流行区普查ScAg的阳性率分别为7．27％、3．06％、2．70％。对ScAg阳性的病例粪孵9送9检累计阳性率为34．29％，粪检累计总阳性数中，第1次只检出16．67％，连续第3次的累计阳性率仅为50．00％。连续化疗地区ScAg检测阳性的粪孵阳性率为12．50％，间歇性选择化疗地区ScAg阳性者的粪孵阳性率占75．00％以上。结论9送9检粪孵累计阳性结果显示，检出率随粪检次数增加而升高，粪检漏检多、工作量大、不能正确反映当地疫情。ScAg检测是一种敏感性高，特异性好的简便快速方法，普查结果能反映不同感染度流行地区的实际状况，有利于制定防治策略，了解防治后成效。     

微量血清血吸虫抗体检测层析卡的研制与评价

目的 研制血清用量少的血吸虫抗体检测层析卡。方法 采用Sephacryl S?300HR柱层析分离血吸虫可溶性虫卵抗原（SEA）,保留具有特异性反应活性的抗原蛋白组分。并研究标记浓度及稀释剂配方、样品缓冲液成分,组成最佳反应体系,建立血清用量5 μl的血吸虫抗体检测层析卡。结果 血吸虫抗体检测层析卡对血吸虫虫卵阳性病人的敏感性为93.7%,EPG <24的低感染度检出率达91.3%,健康人群的特异性为97.1%,并殖吸虫病人的交叉反应率为5.6 %,Youden指数为0.91。与ELISA平行检测流动人口的总符合率为96.1％,Kappa值为0.81。结论 血吸虫抗体检测层析卡具有血清用量少、敏感性高、特异性强、 交叉反应低、应用范围广等特点,具有现场实用价值。     

改进改良加藤厚涂片法诊断血吸虫病的研究

目的将尼龙绢集卵法与改良加藤厚涂片法（简称加藤法）相结合加以改进,进行实验室预试验和现场研究。方法实验室设计制作EPG（每克粪虫卵数）分别为4、8、16、24、48、96六种阳性粪便标本,现场选定低、中、高三个不同感染率的流行村各粪检居民200人,对各EPG组和每人份粪便按30g、5g、41．7mg粪量分组,分别用改进法和加藤法作粪检,进行阳性检出率和EPG值的比较。结果改进法两组阳性检出率均高于加藤法,EPG在4时,加藤法未能检出,漏检率100％,EPG在24及其以上（新鲜血吸虫卵）两法检出率相等,检出EPG值相近。低度流行村,加藤法未能查出病人,改进法两组均可查出,且30g组优于5g组。中、高度流行村,加藤法检出阳性率渐增,但仍低于改进法;检出阳性病人EPG值,改进法的30g组与加藤法相近。结论EPG在24以下及低度流行区不宜用加藤法查病,采用改进法较好,EPG在24以上和中、高度流行区查病,两种方法均可采用。     

联合检测循环抗原和抗体在血吸虫病基本控制地区应用价值的研究

血吸虫病基本控制地区７岁以上的人群共８４７名为检测对象，应用抗日本血吸虫ＣＣＡ单克隆抗体ⅢＤ１０建立的Ｄｅｔ－ＥＬＩＳＡ检测血吸虫循环抗原（ＣＡｇ），ＥＬＩＳＡ和ＩＨＡ检测循环抗体（ＣＡｂ）。结果ＣＡｇ的阳性检出率为３．１９％，ＥＬＩＳＡ和ＩＨＡ检测ＣＡｂ的阳性检出率分别为６．２６％和９．４５％，两者的检出车间差异有显著性（Ｐ＜０．０１），ＣＡｇ与ＣＡｂ（ＥＬＩＳＡ和ＩＨＡ检测）的联合检出率分别为６．８５％和１０．５１％，两者联合检测能提高检出率。     

单克隆抗体鉴别日本血吸虫感染性钉螺的初步研究

在筛选McAb与受检钉螺样本的基础上，建立了McAb－ELISA，鉴别钉螺体内日本血吸虫抗原。每只受检钉螺．事先镜检作为对照，感染血吸虫钉螺36只，血吸虫抗原阳性检出率97.22％（35／36）。合并感染其他寄生生钉螺7只，其阳性检出率为100％（7／7）。感染其他寄生虫钉螺8只，无感染钉螺22只，血吸虫抗原阴性符合率均为100％。以上结果表明，McAb－ELISA与镜检法所得的结果是一致的，这为现场检测钉螺感染血吸虫与否将提供一种简便、有效的方法。     

金标法检测广州管圆线虫特异性IgG抗体的研究

目的探索简易、快速的金标免疫渗滤法检测广州管圆线虫特异性抗体的诊断价值。方法以广州管圆线虫成虫可溶性抗原点样于固相硝酸纤维素膜上，以胶体金-proteinA为标记物，采用自行设计的渗滤装置，检测病人血清中广州管圆线虫特异性IgG抗体。结果检测21人份广州管圆线虫病人血清，检出阳性19份，敏感性为90．5％（19／21）；100人份健康人群血清2份阳性，假阳性率为2％（2／100）；与旋毛虫病人、血吸虫病人血清交叉反应率分别为26．7％（8／30）、3．3％（1／30），未发现与肺吸虫、囊虫、华支睾吸虫、肺结核病人血清有交叉反应。结论金标免疫渗滤法快速检测血清中广州管圆线虫特异性IgG不仅操作简便、快速，结果判读容易，不需特殊仪器设备，而且敏感性高，有较好的辅助诊断价值，与旋毛虫病人血清存在交叉反应。     

A Flow Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothio-cyanate (FLTC)-labelled polyclonal goat anti-human IgG antibody or FITC-labelled non-immune goat IgG. Results were expressed as relative fluorescence intensity (RFI) defined as the ratio of specific fluorescence (mean fluorescence of platelets incubated with the FITC anti-IgG) over non-specific fluorescence (mean fluorescence of platelets incubated with FITC non-immune goat IgG). A normal range was formed by analysing platelets from 71 healthy subjects. Platelets from 16 patients with a firm clinical diagnosis of immune-mediated thrombocytopenia had a mean RFI significantly higher (p <0.001) than the controls, whereas platelets from 9 patients thought to have non-immune thrombocytopenia had an RFI not significantly different from the normal controls. From a prospectively studied group of 62 patients with no clinically obvious cause for their thrombocytopenia or impaired platelet function 35.5% had raised PSIgG. In order to express the results as number of IgG molecules per platelet, reference curves were created by using FC to nieasurc PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein IIb-IIIa complex. Normal platelets had an average 1,463 (SD = 927) molecules of PSIgG. In patients with immune-mediated thrombocytopenia the levels ranged from 690 to 32,328 (mean 11,535) molecules per platelet.
Flow-cytometric PSIgG estimation was sensitive, fast and easy to perform and therefore suitable for both research and clinical service purposes.     

A Flow Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothio-cyanate (FLTC)-labelled polyclonal goat anti-human IgG antibody or FITC-labelled non-immune goat IgG. Results were expressed as relative fluorescence intensity (RFI) defined as the ratio of specific fluorescence (mean fluorescence of platelets incubated with the FITC anti-IgG) over non-specific fluorescence (mean fluorescence of platelets incubated with FITC non-immune goat IgG). A normal range was formed by analysing platelets from 71 healthy subjects. Platelets from 16 patients with a firm clinical diagnosis of immune-mediated thrombocytopenia had a mean RFI significantly higher (p <0.001) than the controls, whereas platelets from 9 patients thought to have non-immune thrombocytopenia had an RFI not significantly different from the normal controls. From a prospectively studied group of 62 patients with no clinically obvious cause for their thrombocytopenia or impaired platelet function 35.5% had raised PSIgG. In order to express the results as number of IgG molecules per platelet, reference curves were created by using FC to nieasurc PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein IIb-IIIa complex. Normal platelets had an average 1,463 (SD = 927) molecules of PSIgG. In patients with immune-mediated thrombocytopenia the levels ranged from 690 to 32,328 (mean 11,535) molecules per platelet.
Flow-cytometric PSIgG estimation was sensitive, fast and easy to perform and therefore suitable for both research and clinical service purposes.     

建立和评价ELISA法检测血清和唾液中旋毛虫IgG抗体的研究

目的 建立检测血清和唾液中旋毛虫抗体的酶联免疫吸附试验(ELISA)方法.方法 应用方阵滴定法,筛选适宜的旋毛虫抗原(肌肉幼虫可溶性抗原、肌肉幼虫排泄分泌抗原、成虫可溶性抗原、成虫排泄分泌抗原)浓度、血清和唾液稀释度、辣根过氧化物酶标记的山羊抗兔、山羊抗人IgG抗体稀释度.共有20份旋毛虫感染兔、10份旋毛虫病患者血清和唾液用于这4种抗原的敏感性试验,20份健康兔与38份其他寄生虫感染兔和患者的血清和唾液用于这4种抗原的特异性试验.结果 这4种抗原适宜包被浓度依次为8.0 μg/ml、6.0 μg/ml、10.0 μg/ml、9.0 μg/ml.适宜的血清稀释度依次为1:100、1:200、1:50、1:200,唾液均用原液.适宜的山羊抗兔、山羊抗人IgG稀释度分别为1:2 500和l:2 000.这4种抗原检测旋毛虫感染兔血清和唾液的敏感性分别为100%和80%~100%,检测旋毛虫病患者血清和唾液的敏感性分别为100%和60%~80%;检测血清和唾液的特异性依次为81.03%、89.65%、77.59%、82.76%和93.10%、96.55%、89.65%、91.35%.结论 建立了检测兔和人血清及唾液中旋毛虫lgG抗体的ELISA方法.当采集血清标本有困难时,可将唾液替代血清用于检测旋毛虫病.     

应用快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体

目的　建立快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体 ,并对其敏感性和特异性进行评价。 方法　以胶体金标记纯化的基因工程重组抗原 ,在玻璃纤维上预包被金标记的纯化抗原 (Au -Ag)和鼠IgG ,在硝酸纤维素膜上检测线处包被羊抗人 μ链、羊抗人IgG或重组抗原 ;在对照线处包被羊抗鼠IgG ,组装成检测试纸。在试纸条的玻璃纤维上加待检血清 ,再滴加 2滴反应液。观察金标抗原释放后NC膜上检测线和对照线的反应情况 ,检测线和对照线同时出现红色条带判为阳性 ,仅对照线出现红色条带判为阴性 ,对照线未出现条带为无效试验。结果　间接免疫荧光法IFA检测IgG和金标免疫层析法检测IgM、IgG、总抗体的敏感性分别为 88 1% ,92 3% ,90 9%和 98 6 % ;特异性分别为 96 0 % ,94 7% ,94 7%和93 3%。结论　金标免疫层析法可替代传统方法用于检测恙虫病东方体特异性抗体。     

Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection.

OBJECTIVE: To understand the usefulness of detecting tuberculous IgG antibodies against mycobacterial excretory-secretory 31 kDa serine protease antigen (SEVA TB ES-31) and circulating free and circulating immune-complexed (CIC) serine protease in TB patients with and without HIV infection. DESIGN: Serum was collected from 144 individuals: patients with TB, with TB-HIV co-infection and HIV infection only, and ill and healthy controls. SEVA TB ES-31 antigen, a serine protease isolated from Mycobacterium tuberculosis H37Ra culture fluid, was used in indirect penicillinase ELISA to detect tuberculous antibodies. Similarly, affinity purified anti-ES-31 antibody was used in sandwich ELISA to detect circulating free and CIC serine protease. RESULTS: There was less sensitivity for tuberculous antibody in HIV-infected TB patients (46%) than in those with TB alone (87%) using mycobacterial serine protease. However, the sensitivity of detection of TB in the presence of HIV increased to 87% by concomitant detection of circulating free and CIC serine protease antigen. CONCLUSION: Detection of free and CIC tuberculous serine protease antigen along with antibody is more useful for detecting TB in the presence of HIV co-infection.     

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.     

直接法McAb-Dot-ELISA检测日本血吸虫感染耕牛血清循环抗原的研究

将抗日本血吸虫单克隆抗体标记辣根过氧化物酶,进行直接法单克隆抗体斑点酶联免疫吸附试验(McAb-Dot-ELISA),检测日本血吸虫感染耕牛的血清循环抗原。结果,该方法对实验感染耕牛的阳性检出率为100％(32/32);对安徽、江西和湖南3省血吸虫病流行区自然感染耕牛(粪孵阳性)的阳性检出率分别为93.93％(418/445),88.50％(100/113)和81.71％(143/175);对健康耕牛的阴性符合率为98.51％(66/77),对16份感染锥虫和8份实验感染肝片吸虫的耕牛血清均未见交叉反应。提示,该方法具有操作简便、快速等优点,可作为一种新的耕牛血吸虫病免疫诊断方法在疫区基层推广应用。     

日本血吸虫31／32kDa抗原纯化及诊断应用的研究

采用超凝胶柱层析结合超滤、透析、沉淀等方法分离纯化了日本血吸虫成虫３１／３２ｋＤａ抗原。经ＳＤＳ－ＰＡＧＥ、银染和免疫印迹分析，证明免疫及生化纯度。银染及ＰＡＳ证实此抗原是一种不含糖的多肽类组分。用于ＥＬＩＳＡ和ＩＨＡ诊断日本血吸虫病，与ＳＥＡ同样敏感，而特异性明显较优。     

棘球蚴病患者IgG抗体阴性反应血清再检测的研究

目的　探索棘球蚴病患者抗体应答假阴性反应原因 ,以改进棘球蚴病的免疫诊断方法。　方法　采用间接ELISA和双抗体夹心ELISA方法 ,检测 42例IgG抗体阴性反应棘球蚴病患者血清的IgG亚类 (IgG1、IgG2、IgG3和IgG4 )、IgA、IgM、IgE抗体及抗原和循环免疫复合物。　 结果　 42例阴性血清中 ,32例IgG亚类或IgA、IgM、IgE抗体阳性 ,1 0例血清抗体全部阴性。其中IgG1、IgG4及IgA、IgM、IgE的检出率明显高于正常人 ,分别为 42 .9%、1 1 .9%、2 8.6 %、2 6 .2 %和 2 1 .4 %。小儿的IgM高于成人。肝棘球蚴病患者的IgG亚类高于肺棘球蚴病患者。IgG1与其它抗体联合检测 ,以IgG1 +IgA +IgM检出率最高 ,为 64 .3 %。IgG阴性患者血清的CAg和CIC阳性率分别为 2 8.57%及30 .95 %。　结论　抗棘球蚴总IgG抗体表达水平低下 ,抗体表达种类不同及循环免疫复合物的形成 ,是造成棘球蚴病患者IgG抗体反应阴性的主要原因。IgG1 +IgA +IgM检测可提高棘球蚴病患者的诊断率     

Detection of specific circulating antigen, immune complexes and antibodies in human hydatidosis from Turkana (Kenya) and Great Britain, by enzyme-immunoassay

Circulating antigen, specific immune complexes (IgG and IgM) and specific antibodies (IgG, IgM, IgE and IgA) were detected by enzyme-linked immunosorbent assay (ELISA) in the sera of hydatid (Echinococcus granulosus) patients from Turkana (Kenya) and the UK. Specific IgG and IgM antibodies predominated in current UK hydatid infections, while all classes of specific antibodies were lower in the Turkana patients. Circulating antigen, detected in 3% polyethylene glycol (PEG) precipitated complexes, using peroxidase conjugated hyperimmune human hydatid IgG (Fab) was more specific in ELISA than either antibody or immune complex assays where peroxidase conjugated anti-human IgG was used. Anti-human immunoglobulin ('rheumatoid' factor) was not detected in hydatid sera. Serum antigen, specific IgM immune complexes and specific IgM antibodies were associated with UK cases of current hydatid infection in contrast to patients with previous histories of hydatidosis. In 3 hydatid patients (from UK) levels of circulating antigen and specific IgM immune complexes rapidly declined within 1-4 months after surgical cyst removal. The detection of specific IgG and antigen in PEG precipitated immune complexes from false-negative/low responder Turkana hydatid sera, suggests that antibody 'mopping' by specific antigen may be occurring. After SDS-PAGE/immunoblotting analysis, antigen of mol. wt 67 000, present in hydatid cyst fluid and protoscoleces, was identified as putative circulating antigen in 3% PEG precipitates of sera from albendazole treated hydatid patients.