食蟹猴疟原虫和恶性疟原虫两种抗原检测不同疟区人群中间接荧光抗体的实用性

[目的 ]比较食蟹猴疟原虫 (P c.)和恶性疟原虫 (P f.)两种抗原在不同疟区人群疟疾抗体检测的实用性。 [方法 ]1997年 5～ 10月在海南省间日疟和恶性疟混合流行区及河南省单纯间日疟区用P c 和P f 两种抗原测试人群疟疾抗体。 [结果 ]在海南省间日疟和恶性疟混合流行区P c 和P f 两种抗原检测人群疟疾间接荧光抗体阳性率分别为 37 4%和 31 3% ,其阳性符合率为 83 9% ;河南省单纯间日疟流行区的P f和P c两种抗原阳性率分别为 2 3 0 %和 9 7% ,阳性GMRT分别为 42 9%和 2 9 3%。 [结论 ]间日疟和恶性疟混合流行区P f和P c两种抗原均可用于人群疟疾抗体的检测 ,而单纯间日疟地区则以P c 抗原为优。     

间接荧光抗体试验在上海疟疾监测中的应用

以体外培养的恶性疟原虫 (Plasmodium falciparum,P.f )为抗原 ,代替以食蟹猴疟原虫 (Plasmodium cynomolgi,P.c)作为检测人体间日疟的替代抗原 ,用间接荧光抗体试验(Indirect luorescent antibody test,IFAT)在间日疟流行地区的人群中 ,进行疟疾血清学调查 ,其特异性与敏感性均较高 ,且节约人力、物力、财力 [1 ] 。 1987～ 1998年 ,作者以 IFAT对当地人群 (包括 15岁以下儿童 )和外来人口进行疟疾监测 ,以期说明应用 IFAT的效果 ,并以此推算人群中的疟疾年感染率。材料和方法1　抗原　以自制的人工培养 P.f制备抗原片 ,冷藏备用。…     

食蟹猴疟原虫实验感染恒河猴后原虫密度消长的观察

目的　探讨食蟹猴疟原虫感染恒河猴后原虫密度消长和抗体滴度变化及相互关系。　方法　食蟹猴疟原虫实验感染恒河猴后 ,在 7年多的时间内 ,定期进行病原学检测和疟疾荧光抗体检测 ,记录分析原虫密度消长和抗体滴度变化等数据。　结果　实验恒河猴接种食蟹猴疟原虫后 ,最早 3d可检出原虫 ,12～ 19d可达到原虫血症高峰期 ,2 483d(时间最长者 )时还可检出原虫 ;荧光抗体试验显示感染第 8d时可检出阳性 ( 1∶ 2 0 ) ,15 8～ 181d达到高峰滴度 ( 1∶ 6 40～ 1∶ 2 5 6 0 ) ,持续 6 0～ 85 d后抗体滴度逐渐下降 ,到实验结束时各猴抗体滴度还保持在 1∶ 40～ 1∶ 16 0。　结论　食蟹猴疟原虫是一种比较理想的实验虫种 ,用它在疟疾研究和抗疟工作中适时的替代间日疟原虫能获得较好的效果。     

应用猴抗食蟹猴疟原虫抗体检测间日疟抗原

检测疟原虫抗原是最近发展起来的一种免疫学技术,已被应用于恶性疟和间日疟的研究。我们也曾应用P.v.病人的混合抗体成功地进行P.v.抗原的检测,获得较满意的结果。为解决抗体来源的问题,本实验应用熊猴抗食蟹猴疟原虫抗体(简称抗P.c.抗体)取代病人混合抗体检测P.v.抗原,并     

间接荧光抗体试验应用于流动人口疟疾监测

为了巩固灭疟成果，控制输人性疟疾，我们于1993～1997年应用间接荧光抗体试验（IFAT）对流动人口进行疟疾监测。1材料和方法4．l监测对象为来自高疟区的外来人口并在本地已居住一周以上。于疟疾传播期末的10月下旬进行IFAT。并以本地常住居民作对照。1．2间接荧光抗体试验（IFAT）血标本采集均用滤纸干血滴法。食蟹猴疟原虫抗原片由中国预防医学科学院寄生虫病研究所提供，羊抗人IgG荧光抗体系上海生物制品研究所产品。按常规方法操作D’。抗体滴度＞1：20者为阳性，以阳性反应的最高稀释度作为终点滴度。IFAT统一由江苏省寄生虫病…     

猴疟感染过程特异性IgM抗体的变化

以食蟹猴疟原虫猕猴动物模型,用间接荧光抗体试验法(IFAT)观察疟疾感染过程中特异性IgM抗体的变化,共观察无复燃或复发的短原虫血症期(15天内,5只猴)、长原虫血症期(26～37天,2只猴)及出现复燃(5只猴)3种类型的疟疾感染。疟疾IgM抗体初次出现于子孢子感染后13～16天或阳性血感染后3～9天,抗体滴度高峰出现于子孢子感染后17～25天或阳性血感染后12～26天。尔后抗体逐渐降低,抗体消失时间在原虫血症短者为感染后2个月,在原虫血症长者约3个月,复燃出现在感染终止后约1个月。结果表明,疟疾特异性IgM抗体的出现与存留,是新近疟疾感染的一个佐证。     

生物信息学技术在疟原虫基因组研究中的应用

疟疾是一种流行广泛的热带寄生虫病 ,一直得到世界范围的广泛重视。恶性疟原虫 (Plasmodium falci-parum,P. f .)基因组研究从 1983年开始 ,1996年12月建立疟原虫数据库 ,主要内容包括以下方面 :核苷和蛋白信息、核苷和蛋白数据文件、疟原虫基因图谱数据和 Mal DB疟原虫基因组数据库[1] 。随着 PE公司快速测序仪的推出 ,疟原虫基因组数据将成倍增长。目前 ,P.f.2、 3和 9号染色体序列测定已经完成 ,预计在未来的 2～ 3年内 ,将有可能完成其基因组序列分析 ,疟原虫即将步入后基因组时代。大量的基因组数据必须借助生物信息学技术进行自动…     

应用酶联免疫吸附试验检测疟疾抗体

本文对采自我省不同疟区162份干血滴,包括恶性疟51例与间日疟111例,采用由当地分离的恶性疟原虫可溶性抗原进行ELISA试验,并用食蟹猴疟原虫(P.cynomolgi)替代抗原作对比,结果显示使用两种抗原的阳性符合率基本一致,但是用P.f抗原的几何平均滴度显著高于用P.c抗原者(371.76,324.68;P<0.05),用P.f抗原测恶性疟患者血清阳性几何平均滴度显著高于用P.c抗原(527.8,383.71:t=1.72 P<0.05)。在单纯间日疟区采集的49份血样使用P.c抗原的抗体阳性几何平均滴度显著高于P.f抗原,前者为388.84,后者为250.79(t=2.91 P<0.01。)     

重组表达的恶性疟原虫顶端膜抗原1片段诱导小鼠保护性抗体应答

目的分析恶性疟原虫顶端膜抗原(apicalmembraneantigen1,AMA1)主要结构域在诱导保护性抗体应答中的作用,为正确选择疫苗应用片段提供依据。方法PCR扩增编码P.fAMA1相应结构域的基因序列,构建pET原核表达载体,用纯化重组蛋白免疫BALB/c小鼠,ELISA测定抗体效价,用免疫荧光和免疫印迹分析抗体的特异性,用小鼠免疫血清进行疟原虫体外生长抑制实验。结果成功表达并纯化了代表AMA1不同结构域的重组抗原片段,包括完整的胞外域片段E、结构域Ⅰ+Ⅱ、Ⅰ、Ⅱ和Ⅲ,免疫小鼠后分别诱导出不同滴度的IgG抗体,免疫血清可特异地识别天然AMA1抗原,重组蛋白E和Ⅰ+Ⅱ免疫血清可明显抑制疟原虫的体外生长。结论AMA1胞外结构域的完整性是构成保护性抗体表位的重要基础,保护性抗体表位主要分布在结构域Ⅰ中。     

恶性疟原虫乳酸脱氢酶单克隆抗体的制备及其鉴定

[目的 ]制备抗恶性疟原虫乳酸脱氢酶 (LDHp)单克隆抗体 (McAb) ,并对其特异性进行鉴定。[方法 ]用纯化的LDHp重组抗原免疫BALB/c小鼠 ,采用杂交瘤技术制备McAb ,筛选分泌高滴度McAb的杂交瘤细胞株 ,测定其免疫球蛋白亚类及其效价 ,ELISA、Westernblot试验分析其特异性。 [结果 ]筛选出 2A5和1H10两株能稳定分泌抗LDHpMcAb的杂交瘤细胞株 ,两株单抗均为IgG2b,2A5和 1H10培养上清的ELISA效价分别为 1∶5 12和 1∶2 5 6 ,腹水效价分别为 1∶2 5 6 0 0和 1∶12 80 0 ,两株单抗与间日疟原虫、红细胞、弓形虫、日本血吸虫等抗原均不发生交叉反应 ,能识别恶性疟原虫 33kDa的虫源蛋白。 [结论 ]制备的抗LDHp杂交瘤细胞株能分泌高滴度和高特异性的单抗     

间日疟原虫和恶性疟原虫可溶性抗原免疫反应性比较

目的分析和比较间日疟原虫(Plasmodiumvivax,P.v)和恶性疟原虫(P.falciparum,P.f)可溶性抗原与间日疟感染者混合血清和单克隆抗体(McAb)M26-32免疫反应性的差别,以期寻找潜在的疟疾诊断抗原。方法取P.v感染者血样,用Plasmodipur滤器分离去除白细胞,经60%Percoll浓集其中的感染红细胞(i RBC)。分别制备P.v、P.f和正常红细胞(nRBC)可溶性抗原,应用P.v感染者、正常对照混合血清和M26-32单抗与相应的抗原进行免疫印迹分析。结果免疫印迹分析表明,P.v感染者能特异性识别26k、33、49、115kDaP.v抗原;100、102、110、150、175kDaP.f抗原;能够交叉识别的条带有:31、59、63、70、120kDa。M26-32单抗能识别P.f、P.v抗原中31kDa等抗原条带。结论P.v感染者能特异识别间日疟抗原组份,并能交叉识别P.f抗原,其中31kDa抗原组分具有较强的免疫原性,同时能被M26-32单抗识别,其作为P.v特异性诊断抗原的价值有待于进一步研究。     

The seroepidemiology of malaria in Middle America. II. Studies on the Pacific coast of Costa Rica.

Serologic studies for malaria using the indirect fluorescent antibody technique suggest that active transmission is either absent or very low in 6 villages on the Pacific side of Costa Rica. Positive titers (1:20 or higher) were seen in the under-15-year age group in three of the study localities, but only 5 such responses were encountered among 249 people examined in this age range. In the adults (15 years and over) from the same 3 villages there were 68 positive titers among 161 examined. There were 43 positive responses in 189 adults from the remaining 3 villages where none of 307 persons under 15 years of age showed a titer of 1:20 or higher to any of the 3 malaria antigens tested (Plasmodium falciparum, P. vivax and P. malariae). These data suggest that the positive responses in the latter villages are more likely to be associated with old or imported cases than with current local transmission. Serologic responses of 1:80 or higher to the P. falciparum antigen suggested the continued presence of this parasite in the population in spite of the paucity of positive blood smears with this species in recent years. Positive titers with the P. malariae antigen suggest that this parasite is probably still present in the area. Such serologic studies help to indicate areas where malaria transmission is active and provide information on parasite reservoirs in particular populations.     

Evaluation of four rapid diagnostic tests for the diagnosis of Plasmodium vivax in Korea

Objective To evaluate 4 rapid malaria diagnostic kits (RDTs) in Korea: OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria P.f/Pan antigen test and CareStart? Malaria Pf/Pv Combo test. Methods Hundred malaria patients with Plasmodium vivax (P. vivax) and 100 healthy volunteers were recruited. The results from earlier four RDTs were compared with the reference standard, the Giemsa‐stained traditional microscopic diagnosis. Results Compared with the reference standard, the sensitivity and specificity for Plasmodium vivax were 92.7 and 100% for SD BIOLINE Malaria Ag P.f/Pan; and 94.6% and 100% for OptiMAL; 95.5% and 100% for both Humasis Malaria P.f/Pan antigen test and CareStart? Malaria Pf/Pv Combo test. Conclusion The performances of all four malaria RDT kits were acceptable, although Humasis Malaria P.f/Pan antigen test and CareStartTM Malaria Pf/Pv Combo test gave superior performances with ROK isolates.     

Differential immunoglobulin E and cytokine responses in BALB/c and C57Bl/6 mice during repeated infections with blood-stage Plasmodium chabaudi malaria

Repeated blood-stage Plasmodium chabaudi chabaudi AS challenge infections in BALB/c and C57Bl/6 mice result in increased serum immunoglobulin (Ig) E levels and splenic cytokine production. The genetic background of the host influences both the cytokine response as well as the development of IgE antibodies. BALB/c mice showed high interleukin (IL)-4 secretion from splenocytes after in-vitro stimulation with malaria antigen after repeated P. chabaudi challenges and this was closely followed by higher levels of total IgE. Despite slightly elevated serum IgE levels, splenocytes from C57Bl/6 mice did not secrete any detectable IL-4 but produced interferon (IFN)-gamma in response to malaria antigen-stimulation in vitro. These data suggest that induction of IgE antibodies during murine malaria infection is genetically regulated.     

A longitudinal study of naturally acquired cellular and humoral immune responses to a merozoite surface protein (MSP1) of Plasmodium falciparum in an area of seasonal malaria transmission

A longitudinal study of cellular and serological responses to the major merozoite surface protein of Plasmodium falciparum (PfMSP1) has been conducted in a malaria immune population living in The Gambia, where malaria transmission is seasonally endemic. Recombinant or native proteins representing the sequence of PfMSP1 from the Wellcome strain of P. falciparum were used in in vitro lymphocyte proliferation, cytokine and antibody assays. Cellular responses of individual donors fluctuated over time, independent of seasonal changes in malaria transmission whereas anti-PfMSP1 antibody levels were remarkably stable. At a population level, IFNγ responses were both more prevalent and of greater magnitude at the end of the rainy (malaria transmission) season than during the dry season. Responses of individuals lving in a rural village were compared with those of individuals living in an urban area with much lower levels of malaria transmission. Malaria infections were more likely to be symptomatic in urban dwellers than in inhabitants of rural villages but no significant differences in the level or prevalence of cellular or serological responses were seen between the two groups. However, urban dwellers with current symptomatic malaria infections had somewhat lower anti-PfMSP1 antibody levels than their healthy, non-parasitaemic neighbours.     

The enzyme-linked immunosorbent assay (ELISA) for malaria. I. The use of in vitro-cultured Plasmodium falciparum as antigen.

Using the Panama II strain of Plasmodium falciparum obtained from continuous in vitro culture as antigen, the micro enzyme-linked immunosorbent assay (ELISA) was used to test serum samples from 50 persons from the southeastern United States and serum specimens collected weekly from four non-immune and nine semi-immune patients infected with P. falciparum. None of the 50 sera from the United States had ELISA antibody titers greater than 1:80. The nine semi-immune patients had rapid ELISA antibody responses (titers greater than 1:2560) following patent parasitemia. ELISA titers remained elevated despite disappearance of patent parasitemia, and declined gradually following curative antimalarial therapy. The ELISA responses observed in the four non-immune patients were more variable, though positive titers appeared rapidly with patent parasitemia. Maximum titers were lower than those observed in semi-immune patients. These results demonstrate that P. falciparum obtained from continuous in vitro culture is an excellent antigen for the micro-ELISA test for malaria. However, further assessments of the ELISA are needed to identify the conditions associated with positive responses.     

疟疾复合PCR检测系统的建立

目的：建立简易、快速的复合ＰＣＲ系统,用于检测间日疟、恶性疟及混合感染。方法：以疟原虫小亚单位核糖体核糖核酸基因为靶片段,设计疟原虫属特异性上游引物Ｓ１和间日疟原虫、恶性疟原虫种特异性下游引物Ｓ２和Ｓ３,建立双温度点复合ＰＣＲ扩增系统并用于临床血样的检测。结果：从间日疟原虫和恶性疟原虫感染血样中分别扩增出７０５ｂｐ和５７５ｂｐ特定扩增带,而食蟹猴疟原虫、诺氏疟原虫及健康人血样均未见扩增带。检测原虫水平达２－１０虫／μｌ全血。限制性内切酶酶切分析证实扩增产物为目的片段。检测１０４份镜检确诊疟疾患者血样,其中８１份与镜检结果相符,并查出镜检未发现的１７份混合感染和２份虫种鉴别失误的恶性疟。结论：本系统敏感性高,特异性强,操作简便,可在一次扩增中同时检出间日疟和恶性疟两种原虫。     

Malaria endemicity among Orang Asli (Malaysian aborigines) as determined by indirect fluorescent antibody tests.

Fluorescent antibodies were detected in 89% of 288 Orang Asli (Malaysian aborigines) with Plasmodium falciparum antigen and in 62% with P. brasilianum (for P. malariae) antigen. Blood films from 18 donors were positive for P. falciparum; 2 of them had mixed infection with P. vivax. Seven of the P. falciparum-positive blood films were from children in the 2- to 9-year age group. Of 17 sera from cord blood, 16 had significant levels of P. falciparum antibody and 14 of P. malariae antibody, the levels being the same as those of the mothers. None of these babies had congenital malaria. A higher percentage of male donors reacted to both antigens. There was an age dependent increase in the number positive and the maximum titers.     

A single gene copy merozoite surface antigen and immune evasion?

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood-stage antigens, exposed or non-exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant 'protective' antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N- or C-terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistingishable from controls despite significant anti-MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C-terminus recombinant polypeptide and a Triton X-100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co-immunization of E with the N-terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.