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。     

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。     

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。     

间接荧光抗体试验应用于丝虫病诊断及流行病学调查的研究

1980～1985年我们应用马来丝虫成虫冰冻切片抗原作IPAT检测马来丝虫微丝蚴血症阳性228例、班氏丝虫微丝蚴血症444例与晚期病人32例,其IFAT的阳性率分别为99.1％(226/228)、92.8％(412/444)和96.9％(31/32),健康对照阴性者占99.3％(149/150)。本方法敏感性和特异性较强,可节省抗原材料,不需要夜间采血,简便易行,是丝虫病较为理想的辅助诊断方法。用于基本消灭丝虫病地区人群的血清流行病学调查可反映防治的成效。     

单克隆抗体免疫放射测定法检测丝虫抗原

应用抗马来丝虫成虫单克隆抗体结合免疫放射测定法，对马来丝虫成早抗原，马来丝虫成虫排泄－分泌抗原，马来微丝蚴抗原及班氏丝虫病病人血清中循环抗原进行了检测。马来丝虫成虫抗原检出的最低含量为０．４８ｐｇ，ＥＳ抗原，马来微丝蚴抗原可测得抗原结合量分别为０．３５μｇ和０．０６５μｇ，班氏丝虫病人血清中循环抗原的检测结果阳性率为８０％，线虫病流行区５０份微丝蚴阴性血清及丝虫病非流行区５０份肠道蠕虫感染者血清全     

晚期丝虫病患者血清特异性抗体及抗体亚型的分析

目的评价晚期丝虫病患者血清抗丝虫抗体及抗体亚型的免疫学特性.方法用马来丝虫成虫和微丝蚴抗原,以ELISA法检测120份血清标本抗丝虫特异性抗体和抗体亚型,FPT方法进行丝虫皮内试验.结果病原学检查120份受试者均为非微丝蚴血症.80例晚期丝虫病患者FPT试验阳性率为95%(76/80),晚期丝虫病患者血清抗微丝蚴和成虫特异性抗体阳性率分别为82.5%(66/80)和80%(64/80).流行区对照抗体阳性率为10%(2/20),非流行区正常人则均为阴性.晚期丝虫病患者血清抗体亚型以IgG2为主,阳性率92.5%,其它亚型分别为IgG11.25%、IgG322.5%、IgG46.25%.结论晚期丝虫病患者血清中存在抗丝虫特异性抗体,抗体亚型主要是IgG2,它也可能是晚期丝虫病患者特异的抗体亚型.     

晚期丝虫病患者血清特异性抗体及抗体亚型的分析

目的 评价晚期丝虫病患者血清抗丝虫抗体及抗体亚型的免疫学特性。方法 用马来丝虫成虫和微丝蚴抗原,以ELISA法检测120份血清标本抗 丝虫特异性抗体和抗体亚型,FPT方法进行丝虫皮内试验。结果 病原学检查120份受试者均为非微丝蚴血症。80例晚期丝虫病患者FPT试验阳性率为95％（76／80）,晚期丝虫病患者血清抗微丝蚴和成虫特异性抗体阳性率分别为82．5％（66／80）和80％（64／80）。流行区对照抗体阳性率为10％（2／20）、非流行区正常人则均为阴性。晚期丝虫病患者血清抗体亚型以IgG2为主,阳性率92．5％,其它亚型分别为IgG1 1.25%,IgG322.5%,IgG46.25%。结论 晚期丝虫病患者血清中存在抗丝虫特异性抗体,抗体亚型主要是IgG2,它也可能是晚期丝虫病患者特异的抗体亚型。     

单克隆抗体免疫放射测定法检测丝虫抗原

应用抗马来丝虫成虫单克隆抗体（ＭｃＡｂ）结合免疫放射测定法（ＩＲＭＡ），对马来丝虫成虫抗原、马来丝虫成虫排泄－分泌抗原（ＥＳ抗原）、马来微丝蚴抗原及班氏丝虫病病人血清中循环抗原进行了检测。马来丝虫成虫抗原检出的最低含量为０．４８ｐｇ，ＥＳ抗原、马来微丝蚴抗原可测得抗原结合量分别为０．３５μｇ和０．０６５μｇ，班氏丝虫病人血清中循环抗原的检测结果阳性率为８０％（４０／５０），丝虫病流行区５０份微丝蚴阴性血清及丝虫病非流行区５０份肠道蠕虫感染者血清全部为阴性。     

间接荧光抗体试验用于丝虫病诊断及监测的研究

本文报告用马来丝虫成虫冰冻切片抗原作IFA。125例班氏微丝蚴血症阳性者的血清和干滴血阳性率分别为99.2％和96.8％;311例非丝虫病流行区健康者均为阴性。289例其它疾病患者,有0～7.1％的交叉反应率;用IFA与常规血检方法检查班氏丝虫病流行区居民共801人,阳性符合率为97.0％。62例班氏丝虫微丝蚴血症阳性者经海群生治疗后3个月,其抗体水平已显著下降,6个月后下降非常显著;原微丝蚴血症者治后9年(马来丝虫病流行区)和10年(班氏丝虫病流行区)的阳性率和抗体滴度已降至接近流行区的健康人群水平。以上表明,IFA具有较高的敏感性和特异性,可作为诊断和监测的方法。     

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、     

Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis.

The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. malayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.     

Studies on human filariasis in Malaysia: immunodiagnosis using indirect immunofluorescence.

The indirect immunofluorescence test using sonicated microfilariae of Brugia malayi has been evaluated on 173 sera from patients and persons exposed to Wuchereria bancrofti and B. malayi in endemic areas of Peninsular Malaysia. In the microfilaria-negative group, without signs and symptoms of filariasis 55/62 sera (89%) had titers of 1:16 and less. In the microfilaremic groups and in the amicrofilaremic cases with clinical filariasis, all the sera tested were positive, with the antibody titers ranging generally from 1:16 - 1:256. Cross-reaction tests were done on 16 samples of onchocerciasis sera from West Africa using sonicated antigen as well as antigen-coated CNB1-activated sepharose. Antibody titers were detected in all the sera. The usefulness of the sonicated microfilarial antigen in serodiagnosis of filariasis is discussed.     

Sensitivity and specificity of skin reactivity to Brugia malayi and Dirofilaria immitis antigens in Bancroftian and Malayan filariasis in the Philippines.

Saline antigen extracts of microfilariae, adult worms and third-stage larvae of subperiodic Brugia malayi maintained in gerbils were prepared for use as skin test reagents. Patients were studied on three different islands in the Philippines, one endemic for Bancroftian filariasis (Sorsogon, Luzon), another endemic for Malayan filariasis (Palawan) and the third without endemic filariasis (Cebu). A dose-response curve was established initially in patients with Bancroftian filariasis: thereafter 1.0 microng of the B. malayi antigens and 0.05 microng of Dirofilaria immitis FST antigen (obtained from Dr. T. Sawada) were used. Sizes of reactions were measured by recording the diameters of wheals at 20 minutes, 24 and 48 hours. There was a very high correlation in immediate hypersensitivity reactions among the three B. malayi antigens. Reaction sizes followed a normal distribution. When an area of an antigen-induced wheal 3 X that of the saline control was considered a positive reaction, 99% of 150 patients with Bancroftian filariasis and 96% of 45 subjects with Malayan filariasis reacted to B. malayi larval antigen. Only 68% of patients with Bancroftian filariasis but 90% of those with Malayan filariasis reacted to D. immitis FST antigen. There was no relationship between skin reactivity and age, sex, microfilaremia or severity of clinical disease. Approximately half of 50 patients who lived in an endemic area for W. bancrofti but had neither patent infection nor clinical disease reacted to B. malayi antigens. A maximum of 7% of 120 age- and sex-matched controls from Cebu gave false positive reactions with any of the antigens. Only a small proportion of patients gave 24- and 48-hour reactions. It is concluded that the use of antigens prepared from a human parasite, subperiodic B. malayi, which is easily maintained in a laboratory animal host, improves the ability to diagnose filarial infections by immunological means.     

Current status of filariasis in Chavakad taluk, Trichur district, Kerala

A sample survey using parasitological, clinical and entomological indicators was carried out in all the 18 administrative units of Chavakad taluk, Trichur district, Kerala, India to assess the current filariasis situation. Cluster sampling procedure was followed to screen individuals. Both Wuchereria bancrofti and Brugia malayi species were found to be prevalent in this taluk. Microfilaria (mf) carriers with W. bancrofti were detected in eight areas while B. malayi was recorded only from one area. The two species were found to co-exist in another area. The highest infection rate registered for W. bancrofti was 1.51 while it was 0.3 for B. malayi. Infection due to W. bancrofti constituted 87.88% of the total 33 microfilaria cases. Prevalence of B. malayi was very low. Cases with clinical manifestation of filariasis were recorded in all the four areas surveyed. The present trend in the prevalence of infection (mf) and disease showed a decline in both the species when compared to earlier surveys of 1960s. At least 11 areas are still endemic for filariasis in this taluk. Although prevalence of mf was recorded for the first time in one of the areas viz., Elavalli, the rate was only 0.16%. Entomological surveys revealed the presence of 14 mosquito species, of which Culex quinquefasciatus contributed 84.85% and Mansonia 0.77%. While C. quinquefasciatus was recorded in all the 18 areas, Mansonia spp were found only in 8 areas. Only C. quinquefasciatus was found to harbour different developmental stages of W. bancrofti, with overall infection and infectivity rates of 1.94 and 0.97 respectively. The possible reason for the decline in vector density and infection in man are postulated.     

抗独特型抗体对马来丝虫感染沙鼠的保护性免疫效果观察

目的 :研究抗丝虫抗独特型抗体 (抗 fil-抗 Id- Ab)对丝虫感染沙鼠的保护性免疫作用。方法 :从班氏丝虫病乳糜尿和鞘膜积液患者血清中分离含有高滴度的 Ig G,免疫家兔 ,获得兔抗 fil-抗 Id- Ig G,免疫沙鼠 ,再用马来丝虫感染期幼虫攻击 ,观察其免疫效果。结果 :抗 fil-抗 Id- Ig G一次脾内注射或多次皮下及腹腔内免疫后 ,50 %和 80 %沙鼠产生保护性免疫效果 ,沙鼠体内查不到微丝蚴和成虫 ,而从马来丝虫成虫可溶性抗原或兔抗正常人 Ig G为对照组的沙鼠中 ,80 %沙鼠感染丝虫 ,与正常感染组相近似。结论 :用抗 fil-抗 Id- Ab对马来丝虫感染沙鼠 ,证实有保护性免疫作用。     

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.     

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.     

Combined detection of Brugia malayi and Wuchereria bancrofti using single PCR

A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.     

抗恶性疟原虫有性期单克隆抗体的制备和鉴定

以培养的恶性疟原虫ＮＦ５４（３Ｄ７）株配子体蛋白抽提液及我国云南现场采集的恶性疟原虫细胞骨架分别免疫ＢＡＬＢ／ｃ小鼠。取免疫小鼠脾细胞与ＳＰ２／０骨髓瘤细胞融合，以ＩＦＡ法筛选出８株抗恶性疟原虫有性期ＭｃＡｂ杂交瘤细胞株。经免疫球蛋白类别鉴定，６株为ＩｇＧ１（Ｍ２Ａ１０Ｃ９、Ｍ２Ｃ１Ｂ８、Ｍ４Ｃ７Ｂ１０、Ｍ４Ｇ１２Ｃ１、Ｍ５Ｂ７Ｅ６和Ｍ６Ｅ１Ｇ１１），２株为ＩｇＭ（Ｍ４Ｄ７Ｆ７和Ｍ６Ｆ４Ｄ６）。其中３株ＭｃＡｂｓ（Ｍ４Ｃ７Ｂ１０、Ｍ４Ｄ７Ｆ７和Ｍ６Ｅ１Ｇ１１）的靶抗原定位于配子体以及大滋养体和裂殖体期无性体原虫；其余５株仅定位于配子体。经Ｗｅｓｔｅｒｎ印迹试验，ＭｃＡｂ所识别的蛋白区带各异（１６－１２０ｋＤ），与已发现的有性期特异性抗原相比较，３２ｋＤ抗原国内外尚未报道。各株ＭｃＡｂ与猴疟（Ｐ．ｃｙｎｏｍｏｌｇｉ）红内期、鸡疟（Ｐ．ｇａｌｉｎａｃｅｕｍ）子孢子和杜氏利什曼原虫前鞭毛体均无交叉反应。     

Schistosoma mansoni: characterization of two protective monoclonal antibodies

Two monoclonal antibodies against the surface of S. mansoni schistosomula were found to confer significant passive protection to mice (M7B3A, range 28-70%; M22H12C, range 14-58%). No additive effect was observed when both were transferred together. Neither McAb bound to the cercarial surface but both bound to the surface of in vitro derived schistosomula and schistosomula recovered from mouse skin up to 3 days after infection. The McAbs were species specific, but not S. mansoni strain specific. M22H12C immunoprecipitated an 125I-labelled surface antigen of relative molecular weight (mol. wt) 32 000. In Western blotting of an NP40 schistosomular extract, M7B3A recognized an antigen smear of 13 000-18 000 with a dominant band at 16 000. This 16 000 antigen was recognized by serum from demonstrably immune mice and rats vaccinated with highly irradiated carcariae but not by sera from mice with chronic single sex or bisexual infections.