Dot—ELISA检测抗丝虫抗体的初步实验研究

本文报告以马来丝虫成虫可溶性抗原作Dot-ELISA检测丝虫病人抗丝虫抗体的初步结果。62例马来丝虫微丝蚴血症者的阳性符合率为96.8％,49例班氏丝虫微丝蚴血症者的阳性符合率为85.7％,50例和39例非流行区正常人的假阳性率分别为4.0％和5.1％,与钩虫和蛔虫混合感染者的血清有一定的交叉反应。实验结果显示,Dot—ELISA检测微丝蚴血症者抗丝虫抗体具有敏感性高,且方法简便,可望用于丝虫病监测。     

马来丝虫不同发育期虫体抗原检测班氏丝虫病血清抗体

以马来丝虫不同发育期虫体为抗原,用间接荧光抗体试验检测班氏丝虫微丝蚴血症者的血清抗体.结果表明,木瓜蛋白酶处理的微丝蚴整体抗原和成虫冷冻切片抗原的敏感性和特异性无显著差异,检出率分别为95.3％和93.7％。假阳性率分别为3.9％和0.感染期幼虫抗原的敏感性和特异性较差,检出率为76.2％,非流行区对照组假阳性率和交叉反应率分别为11.5％和18.8％。     

应用单克隆抗体酶联免疫吸附试验双抗体法检测人体丝虫病循环抗原

应用兔抗微丝蚴抗体及抗班氏丝虫微丝蚴代谢抗原(ES34株)或抗马来丝虫3期幼虫抗原(HC11株)单克隆抗体(McAb)酶联免疫吸附试验(ELISA)双抗体法检测人体丝虫病循环抗原时,微丝蚴血症者阳性率分别为94.5％(103/109)及89.0％(97/109),且ES34株单克隆抗体ELISA检测结果显示微丝蚴密度与抗原滴度呈正相关;部分微丝蚴血症者尿液中亦可测得丝虫抗原;晚期丝虫病人阳性率分别为57.4％(31/54)及61.1％(33/54);获自美国及中国贵阳非丝虫病流行区正常人血清阳性率分别为0～4.1％及2.8～4.1％;30份肠道蠕虫感染者血清全部为阴性。血清中丝虫循环抗原的存在似与活动性感染有关。     

单克隆抗体ELISA检测班氏丝虫病人血清循环抗原

应用抗马来微丝蚴抗原4B_1株单克隆抗体(McAb)和抗马来成虫代谢抗原4B_7株McAb酶联免疫吸附试验(ELISA)检测班氏微丝蚴血症者血清中循环抗原,阳性率分别为78.46％(102/130)和87.13％(88/101);检测51例晚期丝虫病人血清,阳性率分别为3.92％和1.96％;36例经治疗后微丝蚴血症阴转者血清,阳性率分别为13.89％和8.33％。丝虫病非流行区99例肠道蠕虫感染者阳性率分别为16.16％和5.05％。结果表明抗马来成虫代谢抗原4B_7株McAb应用于诊断班氏丝虫感染的效果优于抗马来微丝螺4B_1株McAb。     

班氏丝虫病人治疗前后血清抗体水平及免疫学诊断方法的研究

应用马来丝虫成虫冰冻切片抗原免疫酶染色技术(IEST),检测班氏丝虫微丝蚴阳性病人107例及治疗后一年微丝蚴阴转者35例和治后三年者112例血清特异IgG、IgE和IgM抗体水平,结果表明:治前病人血清IgG抗体滴度≥1:40者阳性率为96.3％,阳性GMRT为116.6;IgE抗体滴度≥1:20者阳性率为86.9％,阳性GMRT为37.1;IgM抗体滴度≥1:20者阳性率为88.8％,阳性GMRT为31.0,该三种抗体从阳性滴度     

异种丝虫成虫抗原间接荧光抗体试验检测班氏丝虫病人血清抗体的比较

用马来丝虫成虫和牛丝虫成虫切制成4μm厚的冰冻切片作抗原,以间接荧光抗体试验检测班氏丝虫病人血清中抗体并进行比较,结果在班氏丝虫微丝蚴血症阳性101例中,马来丝虫成虫抗原片呈阳性反应92例,阳性率为91.1％;牛丝虫成虫抗原片呈阳性反应86例,阳性率为85.1％。经统计学处理无显著差异(x~2=1.18,P>0.25),作者认为两者对诊断班氏丝虫病都有一定参考价值。而牛丝虫来源容易,阳性率大于85％,在马来丝虫成虫短缺的情况下,可作为异种抗原使用。     

IEST和IFAT检测丝虫病人血清抗体的比较研究

应用马来丝虫成虫冰冻切片抗原作免疫酶染色试验(IEST)和间接荧光抗体试验(IFAT),检测班氏微丝蚴血症者79例和马来微丝蚴血症者35例,结果IEST的阳性率分别为93.7％和94.3％;IFAT的阳性率分别为89.9％和97.1％;用IEST与IFAT检测健康人98名的假阳性率分别为2.0％和5.1％。IEST与肠道线虫感染者和脑囊虫病人均无交叉反应;IFAT与肠道线虫感染者血清有6.8％的交叉反应,与脑囊虫病人无交叉反应。表明两法检测丝虫抗体均具有较高的敏感性和特异性,但IEST简便、快速,不需要特殊仪器,适合于现场应用。     

单克隆抗体与马来丝虫、班氏丝虫表皮抗原的相互作用

为了寻找可产生抗感染作用的丝虫功能性抗原,制备了抗班氏丝虫微丝蚴分泌代谢抗原的McAb(F_((3)2))和抗马来丝虫第Ⅲ期幼虫(L_(3))的McAb(F_(46))。免疫扩散试验证实两种McAb均为IgM_(0)用ELISA方法检测丝虫抗原结果表明,F_(46)对马来、班氏及彭亨丝虫L_(3)抗原具有高度反应性,而对这些丝虫的微丝蚴(mf)抗原反应性低得多,与钩虫、     

圈形盘尾丝虫成虫抗原间接红细胞凝集试验诊断丝虫病的观察

应用圈形盘尾丝虫成虫和马来丝虫成虫抗原致敏绵羊红细胞作间接红细胞凝集试验,以诊断丝虫病。对84例马来微丝蚴阳性血清标本检测,结果圈形盘尾丝虫成虫抗原阳性率为90.5％(76/84);马来丝虫成虫抗原阳性率为93.8％(78/84),经统计学处理,两者无显著性差异(x~2=0.312,P>0.05)。两种抗原对52例正常人血清检测的假阳性率均为7.7％(4/52),结果说明圈形盘尾丝虫成虫可作为异种抗原用于丝虫病诊断。     

间接荧光抗体试验用于基本消灭丝虫病地区人群抗体水平的监测

本文报告了用马来丝虫成虫冰冻切片抗原作 IFAT 检测班氏丝虫微丝蚴血症者抗体阳性率为88.89％;非流行区健康居民抗体阳性率为3.92％。基本消灭丝虫病后8年的地区原微丝蚴血症者和阴性居民抗体阳性率分别为13.54％和10.84％;基本消灭后15年原微丝蚴血症者和阴性居民抗体阳性率分别为6.45％和5.16％;基本消灭后24年原微丝蚴血症者和阴性居民抗体阳性率分别为3.67％和3.96％。基本消灭丝虫病后15年的地区人群抗体阳性率已降到非流行区健康人群水平(X~2=0.48 P>0.05)。因此认为 IFAT 可作为我省丝虫病防治后期和基本消灭丝虫病后流行病学监测的主要方法之一。此外,观察到微丝蚴血症者血清抗体阳性率和阳性 GMRT 与微丝蚴密度无相关性。     

Dot-IGSS检测日本血吸虫病人血清抗体的研究

运用斑点免疫金银染色法(Dot-IGSS)检测了日本血吸虫病人血清抗体,并与斑点酶联免疫试验(Dot-ELISA)作了比较。检测了急性血吸虫病人血清38份,两法结果全部阳性;慢性血吸虫病人血清70份,Dot-IGSS阳性69份(98.6％),Dot-ELISA阳性65份(92.9％);治疗后血吸虫病人血清40份,Dot-IGSS阳性15份(37.5％),Dot-ELlSA阳性11份(27.5％)。用两法检测华枝睾吸虫病人血清20份,均有1份为阳性;检测正常人血清40份,均未出现阳性反应。将8份阳性血清作倍比稀释(1:20～1:61440)后分别用该两法检测,Dot-IGSS的敏感性明显高于Dot-ELISA(P<0.05)。对120例过去无血吸虫病史、接触疫水且皮试阳性者血清检测结果表明,Dot-IGSS的阳性检出率明显高于Dot-ELISA、COPT及循环抗原测定法(P<0.01)。     

Parasite antigens in sera and urine of patients with bancroftian and brugian filariasis detected by sandwich ELISA with monoclonal antibodies

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigens in human lymphatic filariasis. The assay utilizes a polyclonal rabbit antifilarial antiserum to capture, and a monoclonal antibody to identify, circulating parasite antigen. Using this assay, we found that greater than 95% of sera from microfilaremic donors with bancroftian or brugian filariasis, approximately 60% of sera from microfilaremic patients with hydroceles, chyluria, or elephantiasis, and 15%-20% of sera from asymptomatic residents of filariasis-endemic areas evidently contain filarial antigens. Antigen was also detected in the urine of some microfilaremic patients. Serum levels of antigen detected with one monoclonal antibody, ES34, correlated well with microfilarial density in night blood. In contrast, less than 5% of sera from residents of areas where lymphatic filariasis is not endemic reacted in the assay, even though approximately one-third of the donors whose sera were tested were known to be infected with intestinal nematodes. The assay was designed to be flexible enough to allow the parallel use of multiple monoclonal antibodies with different specificities and simple enough to be applicable in most areas where lymphatic filariasis is endemic.     

Comparison of Dot-ELISA with Sandwich-ELISA for the detection of circulating antigens in patients with bancroftian filariasis

We compared the performance of a newly developed Dot-ELISA with that of a previously described Sandwich-ELISA to detect parasite antigens in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same sera were used in both tests. In the Dot-ELISA, 67 of 70 sera from microfilaremic donors were deemed to contain filarial antigens when screened at a dilution of 1:50. End titers were 1:80-1:1280. With the Sandwich-ELISA, 64 of the same sera were positive at a dilution of 1:10 and 42 were positive at a dilution of 1:50. End titers were 1:10-1:320. The specificity of both assays was greater than 95%, but their sensitivity was remarkably different. The Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human sera, whereas the lower limit with the Sandwich-ELISA was 10 ng/ml parasite antigen. Additionally, the Dot-ELISA does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.     

检测血吸虫循环抗原的多价单克隆抗体诊断试剂盒的研制和应用

用杂交瘤技术生产抗血吸虫肠相关循环阳极抗原（ＣＡＡ）单克隆抗体与血吸虫卵糖蛋白单克隆抗体经混合后标记辣根过氧化物酶（ＨＲＰ），制成Ｄｏｔ－ＥＬＩＳＡ诊断试剂盒，检测轻、中、重不同疫区粪检阳性的血吸虫病人血清中循环抗原，阳性率分别为８４．３％、８７．２％和９１．５％。累计阳性率为８９．２％（６１９／６９４）。对健康人、肝吸虫、肺吸虫病人血清均未出现阳性反应，显示有较高的敏感性和特异性。ＥＰＧ＞８０的病人循环抗原检出率高干ＥＰＧ／８０的病人。说明循环抗原检出率与感染度有关。血吸虫病人经吡喹酮治疗后半年和１年循环抗原转阴率分别为７１．４％和８８．６％。结果表明．该试剂盒可用于确诊病人和考核疗效。试剂盒质量稳定，操作简便快速，整个试验可在１小时内完成，且不需特殊仪器，适于现场大规模查病应用。     

Antibodies to somatic L3 antigen not protective against Brugia malayi infection

Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.     

用虫体抗原进行斑点酶联免疫吸附试验检测弓形虫抗体的研究

应用完整弓形虫速殖子为抗原,以HRP-SPA代替酶标第二抗体进行Dot ELISA检测58例兔血清中弓形虫抗体,同时用速殖子可溶性抗原检测比较。两种抗原检测结果一致。阳性反应最高稀释度为1:25600。本法检测阿米巴阳性兔血清、感染球虫的兔血清,血吸虫阳性兔血清无交叉反应。阻抑试验显示高抗体滴度的阳性兔血清经抗原吸收后,反应明显抑制。实验表明本法特异性强、敏感性高、重复性好抗原制备简单不需特殊设备。为人畜弓形虫病临床诊断、流行病学现场调查、单克隆抗体杂交瘤细胞的筛选提供了一种较理想的简易可行的方法。     

Detection of circulating antigen in bancroftian filariasis by using a monoclonal antibody

A monoclonal antibody designated Gib 13-5-2 (Gib 13) and directed against the cattle parasite Onchocerca gibsoni was used in a two-site immunoradiometric assay (IRMA) for detection of circulating antigen in the sera of Wuchereria bancrofti-infected individuals from Sri Lanka and Papua New Guinea. The microfilaremic patients were, in general, serum antigen positive by the Gib 13 IRMA. Among the amicrofilaremic patients, 47% of those with lymphedema, lymphangitis, hydrocele, etc., and 25% of those with elephantiasis had circulating antigen. Correlation of the presence of serum antigen with clinical status indicated that the Gib 13 target antigen in serum is probably an indicator of either active or early infection, or of both. The antigen was also detected in the urine of some patients. By sodium dodecyl sulphate polyacrylamide gel electrophoresis immunoblotting, Gib 13 target antigens of molecular weights 67,000 and 52,000 were identified.     

血吸虫循环抗原检测的现场试验Ⅰ

将血吸虫肠相关趋阴极抗原的单克隆抗体用于Dot—ELISA,共检测2175份血清,其中急性血吸虫病血清210份,阳性率为91.9％;慢性血吸虫病血清798份,阳性率为86.7％;正常人血清752份,假阳性率为1.1％;除肺吸虫病外,对其他寄生虫病或非寄生虫感染疾病血清无明显交叉反应。用单盲法Dot—ELISA检测3批血清,结果敏感性为86.5％～95.1％;特异性为96.9％～100.0％。检测流行区居民547份血清,Dot—ELISA阳性反应130份;粪检阳性72例。两者均为阳性者55例,相符率为76.4％。上述结果表明,Dot—ELISA检测循环抗原确实具有较好的敏感性与特异性,适于大规模应用及流行病学调查。     

抗日本血吸虫单克隆抗体的制备及其在诊断中的应用

The present paper reported on an anti-CCA monoclonal antibody, McAb-IIID 10, which could be used in determinations of both parasite-oriented circulating antigens and specific anti-CCA antibodies. The established competitive ELISA (C-ELISA) using McAb-IIID 10 to detect schistosome-antibodies showed high sensitivity and specificity in the diagnosis of schistosomiasis with few cross-reactions. In field trials, coincident rates in 3 separate batches of serum samples when subjected to double-blind detections were obtained. A total of 1,915 serum samples had been determined by C-ELISA, among them 113 acute cases achieved a 100% positive rate, 765 chronic and 25 late cases showed 96.3% and 72% positive respectively. 70% of the 66 cured schistosomiasis cases turned to be negative. None of the 750 normal individuals showed positive reactions. No cross reaction was found in 27 sera from hydatidosis, whereas 1 and 2 positive reactions were found in 43 paragonimiasis sera and 126 clonorchiasis sera respectively. The established McAb-IIID 10 involved Dot-ELISA was found of value in the assessment of effective chemotherapy and showed a high negative conversion rate of 97.9% in 48 cured schistosomiasis patients. In 16 experimentally infected rabbits, 12 became negative in Dot-ELISA determinations at the 8th week post treatment, and the remaining 4 treated ones, the titer as well as the reaction intensity were also found reduced. A good coincidence rate was also found between C-ELISA and Dot-ELISA, their detection results may be complementary each other.     

Differential recognition of Loa loa antigens by sera of human subjects from a loiasis endemic zone

Somatic antigens of Loa loa adult worms with molecular weights of 15-180 kDa were identified by Western blot analysis using sera from 3 categories of parasitologically and clinically defined subjects from a loiasis endemic zone. Sera of occult, amicrofilaremic (OL), and 'resistant' individuals with no clinical signs of infection (R) reacted with an antigen of 160 kDa; sera of highly microfilaremic individuals (ML) did not. ML sera strongly reacted with an antigen of 18 kDa which was recognized only weakly or not at all by OL and R sera. At higher dilutions, OL sera only reacted with antigens at 23 and 160 kDa and ML sera reacted with antigens at 18 and 23 kDa, whereas R sera reacted with antigens at 23, 42, 54, 70, 100, and 160 kDa. These data suggested that R sera contained a higher concentration of antibodies which reacted with denatured, nitrocellulose-bound antigens. The IgG4 isotype predominated for all groups of sera, while IgG3 antibody responses were observed only with R sera. IgG1 antibodies were seen in all groups but reacted with fewer antigens than IgG4 antibodies, and no IgG2 antibody responses were detected. Sera against Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis cross-reacted with somatic antigens greater than 70 kDa, whereas none reacted with Loa loa antigens less than 23 kDa.