间日疟原虫与异种疟原虫之间的红内期交叉反应原分析

本文用１％ＮＰ－４０分别提取间日疟原虫、恶性疟原虫、食蟹猴疟原虫、伯氏疟原虫及约氏疟原虫中溶性抗原，用间日疟病血清及两株抗间疟红细胞内原虫单克隆抗体６Ｈ７和２Ｃ３对上述抗原进行免疫印迹分析。结果表明，间日疟原虫与异种疟原虫之间有一系列交叉反应原，其中食触猴疟原虫有１４条蛋白带可被间日疟病人血清识别，多于另外三种疟原虫。五的虫共有的７９ｋＤ蛋白均可被间日疟病人血清识别。此外，间日疟病人血清还识别正常     

间日疟原虫与异种疟原虫之间的红内期交叉反应抗原分析

本文用1％NP－40分别提取间日疟原虫、恶性疟原虫、食蟹猴疟原虫、伯氏疟原虫及约氏疟原虫红内期可溶性抗原，用间日疟病人血清及两株抗间日疟红内期原虫单克隆抗体6H7和2C3对上述抗原进行免疫印迹分析。结果表明，间日疟原虫与异种疟原虫之间有一系列交叉反应抗原，其中食蟹猴疟原虫有14条蛋白带可被间日疟病人血清识别，多于另外三种疟原虫。五种疟原虫共有的79kD蛋白均可被间日疫病人血清识别。此外，间日疟病人血清还识别正常人红细胞膜抗原中的160kD、67kD蛋白带。McAb6H7识别不同疟原虫中相应的交叉反应抗原区带分别为：恶性疟原虫186kD、175kD；食蟹猴疟原虫133kD；伯氏疟原虫186kD、175kD及约氏疟原虫164kD，另外，McAb6H7还识别正常人红细胞膜中的184kD、170kD蛋白。McAb2C3识别四种疟原虫共有的60kD蛋白，该蛋白可能为疟原虫的种间共同抗原。     

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。     

抗疟原虫单抗 M26-32IgM 的单体化及其免疫学特性研究

目的改造IgM类抗疟原虫单抗M26-32为单体IgM亚单位，胶体金标记天然和改造后两种M26—32用于检测疟原虫抗原。方法复苏培养M26—32杂交瘤细胞，接种BALB／c小鼠，收集含有单抗的腹水，用商品HiTrap IgM HP柱纯化腹水中的IgM抗体，并用L-半胱氨酸裂解IgM类M26—32单抗成单体IgM亚单位。经间接免疫荧光检定两种抗体的效价，并用胶体金标记，分别检测恶性疟原虫和间日疟原虫可溶性抗原。结果接种20只小鼠，共采集约80mL单抗腹水，经HiTrap IgM HP柱纯化，获得了纯度较高的M26—32单抗。经L一半胱氨酸裂解后，Native电泳显示天然IgM被裂解为单体亚单位，IFA结果显示，亚单位M26-32与天然五聚体IgM滴度无差别。经胶体金标记后，两种金标抗体均分别能检测1：100稀释的恶性疟原虫和1：10稀释的间日疟原虫可溶性抗原。结论建立了L-半胱氨酸裂解IgM类M26—32单抗为单体IgM亚单位及其胶体金标记的方法，为利用M26—32进一步制备相应快速诊断工具奠定基础。     

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     

恶性疟原虫红内期145／102kDa抗原的超微结构定位

用LR White低温包埋法及胶体金标记免疫电镜细胞化学技术对保护性单克隆抗体M26-32识别的体外培养的恶性疟原虫FCC1/HN株红内期145/102 kDa抗原进行超微结构定位研究。发现金颗粒主要定位在该株原虫红内期环状体、滋养体、裂殖体及裂殖子的细胞质中;一些金颗粒则定位于原虫表面的复合膜或散布在感染红细胞的细胞质中。表明145/102kDa抗原是恶性疟原虫FCC1/HN株红内期各无性发育阶段的共同细胞质抗原,其一部分可途经原虫表面的复合膜而被转运到感染红细胞的细胞质。     

用抗间日疟原虫单克隆抗体鉴定食蟹猴疟原虫复合体中具交叉反应性的红内期抗原

鉴于间日疟原虫(P.v.)和食蟹猴疟原虫(P.c.)在进化上较接近,且后者可以体外培养,作者用抗间日疟原虫 Belem 株红内期原虫的 McAb 鉴定间日疟和食蟹猴疟的靶抗原,以确定这些抗原在功能上的作用。     

DNA探针检测间日疟原虫的初步研究

由于间日疟原虫目前尚不能体外培养,抗原制备困难,使间日疟的流行病学调查及诊断受到限制。本文报道以间日疟原虫红内期基因组DNA库0.24kb DNA片段为探针,用~(32)P标记后进行点杂交试验检测间日疟原虫的初步结果。 材料与方法 含间日疟原虫红内期基因组DNA库亚克隆VPL101/5 0.24kb DNA片段的重组质粒pUC19由澳大利亚昆士兰医学研究所Kidson赠送。按文献方法进行感受态细胞JM 109的制备、重组质     

单克隆抗体M26-32靶抗原基因片断融合蛋白的表达和特性分析

用温度诱导方法获得了 M2 6 - 32靶抗原基因片断表达的融合蛋白。 Western- blot分析显示 ,该融合蛋白能与单克隆抗体 M2 6 - 32反应 ,证实了获得的基因序列是单克隆抗体 M2 6 - 32的靶抗原基因片断。该融合蛋白能与抗间日疟原虫和抗恶性疟原虫血清反应 ,且抗该融合蛋白抗体和纯化融合蛋白的抗血清还能与间日疟原虫和恶性疟原虫的胞浆抗原反应 ,表明该靶抗原基因片断内含有间日疟原虫和恶性疟原虫的共同基因序列 ,且这一共同基因表达的蛋白存在于间日疟原虫和恶性疟原虫抗原之中     

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     

Purification of a specific immunodiagnostic Parastrongylus cantonensis antigen by electroelution from SDS-polyacrylamide gels

A 31-kDa glycoprotein antigen was purified by electrophoresing the crude extract of Parastrongylus cantonensis adult worms in a 12% SDS-polyacrylamide gel, identifying the 31-kDa component with prestained molecular weight standards, cutting the desired gel strip, and then isolating it by electroelution. Antigen fraction of 31 kDa was re-electrophoresed, transferred to a nitrocellulose membrane and found to be reactive with only the sera from patients with parastrongyliasis. No reactive band was observed with the sera from other related parasitic infections, eg, gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria, and the normal healthy control sera. This antigen fraction isolated showed 100% sensitivity and 100% specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of 31-kDa specific antibody in the sera from patients with parastrongyliasis. The P. cantonensis antigen of 31 kDa has been obtained by this means with a high degree of purity and applied successfully in conventional ELISA for the specific immunodiagnosis of human parastrongyliasis.     

Antinuclear antibody in juvenile rheumatoid arthritis sera reacts with 50-40 kDa antigen(s) found in HeLa nuclear extracts

To identify whether sera containing antinuclear antibody (ANA) from patients with juvenile rheumatoid arthritis (JRA) recognize a common antigen, extracts of HeLa nuclei were prepared by incubating the nuclei with buffers of sequentially increasing salt concentrations (0.1 M, 0.3 M, and 0.5 M NaCl). Western blot analysis using sera from 82 patients with JRA revealed that 63% of ANA positive JRA sera reacted with 2 bands (50-40 kDa) in the 0.3 M extract whereas only 14% of ANA negative JRA sera had antibody to these bands. Antibody to the 50-40 kDa antigen(s) strongly correlated with the presence of ANA by immunofluorescence and was found in 70% of patients with iritis; however, it did not correlate with disease subgroup or disease activity.     

Leishmania donovani: isolation of a concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis.

A glycoconjugate antigen of 27-39 kDa was isolated from a cell-free extract of Leishmania donovani by affinity chromatography using a Concanavalin-A sepharose-4B column and eluted with 0.5 M alpha-methylmannoside. The antigen was recognized specifically by sera from kala-azar (visceral leishmaniasis) patients and did not react with sera from tuberculosis, leprosy or malaria patients. The antigen may therefore be useful in developing a serodiagnostic assay for visceral leishmaniasis.     

Detection of an early activation-dependent cell surface antigen on human T-lymphocytes

The binding properties of a monoclonal antibody to an early activation marker of human lymphocytes are described. The monoclonal antibody BL-Ac/p26 binds to an antigen which is expressed on activated lymphocytes. This antigen is not detectable on resting lymphocytes or other blood cells. The surface radioiodinated antigen of activated T cells isolated by the monoclonal antibody BL-Ac/p26 is separated by SDS-PAGE in a major 26k Da component and a minor band (32 kDa) under reducing conditions. These polypeptide chains form disulphide linked dimers on the cell surface (Mr 55-77 kDa). This 26 kDa antigen is a very early activation marker of T lymphocytes. Human T lymphocytes stimulated by mitogens (PHA, ConA or PWM), anti-TCR/CD3 monoclonal antibodies or allogeneic leucocytes express this 26 kDa antigen after 2-4 hours.     

Characterization of three types of Schistosoma mansoni soluble egg antigen and determination of their immunodiagnostic potential by western blot immunoassay

On the search of highly sensitive and specific antigenic components for use in serological tests, the serologic activities of the various protein fractions of three types of Schistosoma mansoni soluble egg antigen (SEA) were compared in an immunoblot analysis for their ability to detect schistosomiasis mansoni infections . Three types of soluble egg antigen (SEA) were prepared from three suspensions of Schistosoma mansoni eggs; namely living SEA (L-SEA), dead SEA (D-SEA) and mixed SEA (M-SEA). The three antigens were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A total of 80 Egyptian individuals were enrolled in the present study. After being screened by clinical examination, urine and stool analysis, sigmoidoscopy rectal snip examination, abdominal ultrasonography and indirect haemagglutination test (IHAT), the study population were grouped into an active intestinal schistosomiasis group (group I, n=20), a schistosomiasis seropositive group by IHA test (group II, n=20), a parasite control group including 10 patients with hydatidosis & 10 patients with fascioliasis (group III, n = 20) and a normal control group (group IV, n=20). Sera of all subjects were studied by immunoblotting for the presence of IgG antibodies against the various protein fractions of the three prepared types of SEA. Several protein bands from the 3 types of SEA reacted with the schistosomiasis patients' sera in a heterogenous manner. However, a 31-32 kilo daltons (kDa) protein fraction of L-SEA reacted with 80% (16/20) of group I sera, 40% (8/20) of group II sera, one hydatidosis serum, but no reaction occurred with normal sera. Also, in the active intestinal schistosomiasis group, the 31-32 kDa fraction of L-SEA was more recognized by patients with early active intestinal schistosomiasis without organomegaly (100%, 12/12) than in those with organomegaly (50%, 4/8) (P < 0.05). On the other hand, a 80-82 kDa band of M-SEA was recognized by 70% (14/20) of group 1, 30% (6/20) of group II & sera from 3 hydatidosis and 2 fascioliasis cases, but not by normal human sera. So, it can be concluded that the 31-32 kDa protein fraction of L-SEA is highly immunogenic, with the least cross reaction with other parasitic infections, and may be a useful serologic marker for diagnosing and differentiating between early and chronic schistosomiasis mansoni infection.     

日本血吸虫24—26kDa抗原和重组Sj26抗原的...

This paper reports on the comparison of the recombinant Sj26 (rSj26) antigen originated from the Philippine strain and 24-26 kDa antigen isolated and purified from Chinese mainland strain of S. japonicum for their antigenicity and immunogenicity. The results showed that there were obvious cross reactions between rSj26 and 24-26 kDa antigen when rSj26 antigen was tested against specific antibodies in sera from mice infected with the mainland strain of S. japonicum or 24-26 kDa antigen was tested against specific anti-rSj26 antibodies by ELISA, IFA and Western blotting etc. Both of 24-26 kDa and rSj26 antigen had weak cross reaction with SEA antigen. The worm reduction rate after challenging with mainland strain cercariae in mice immunized with rSj26 was 26-32%, similar to that in mice immunized with 24-26 kDa antigen. It is suggested that rSj26 antigen can induce certain level of specific protective immunity to protect the host against infection by Chinese strain of S. japonicum cercaciae.     

Characterization of an S antigen synthesized by several isolates of Plasmodium falciparum.

The S antigen of a Papua New Guinean isolate of Plasmodium falciparum was identified by immunoblotting as the dominant antigen in culture supernatants. An antigen identical in molecular weight (Mr 220,000), isoelectric point (pI 4.2), and immunoreactivity with sera from individuals exposed to malaria was expressed by four Papua New Guinean isolates and one isolate of unknown origin. The Mr 220,000 antigen was not detected in culture supernatants derived from two isolates from Thailand and one from Ghana. The Mr 220,000, pI 4.2 S antigen may characterize a subpopulation of parasites common to many isolates of P. falciparum, which is selected for by continuous culture in vitro. A variant S antigen, 30 kilodaltons larger but with similar immunoreactivity, was expressed by 1 of 26 clonal populations derived by limit-dilution culture from one of the Papua New Guinean isolates of P. falciparum. The characteristics of the S antigen, defined by immunoblotting, allowed it to be identified in two-dimensional separations of [35S]methionine-labeled parasite proteins, thus confirming the parasite origin of the antigen.     

Two-site pan-species monoclonal antibody ELISA for detection of blood stage malaria antigen.

A two-site pan-species monoclonal antibody sandwich ELISA (MAb-MAb ELISA) was developed to detect both Plasmodium vivax and P. falciparum antigens in whole blood impregnated on filter paper. In this assay, the plates were coated with pan-species MAb 3F9 and another pan-species MAb M26-32 conjugated with alkaline phosphatase was used for detection of bound antigen. The sensitivity of this assay was 5, 10 and 10 parasites per 10(6) erythrocytes for cultured P. falciparum, patient-derived P. vivax and P. falciparum, respectively. The coincidence rates for this assay were 93% (92/99) with healthy individuals and 93% (42/45) with microscopically confirmed vivax malaria cases. After two weeks treatment, 77.7% (14/18) of vivax malaria were still positive by this assay but with diminished level of reactivities [corrected].     

两种疟原虫抗原片不同保存温度及时间稳定性比较

目的 观察比较食蟹猴疟原虫 （Plasmodium cynomolgi, P. c） 抗原片和人工培养恶性疟原虫 （Plasmodium falci? parum,P. f） 抗原片在不同保存温度与时间下检测疟疾抗体滴度的效果。方法 显微镜下计数2种抗原片原虫密度, 计算每个视野的平均原虫数。以间日疟和恶性疟病人的混合血清作为已知疟疾抗体血清, 稀释浓度为1∶5~1∶1 280。将2种抗原片置于4～6、 25～27、 33～35 ℃ 3组温度下, 于放置第3、 5、 7、 10天各取出2张, 用间接荧光抗体试验 （IFAT） 法检测抗体终点滴度, 并比较-20 ℃下贮藏1年和2年的2种抗原片, 在３个温度组保存3 d时抗体滴度的变化。结果 2种抗原片红细胞疟原虫密度分别为2.00×105 /μl和1.89×105 /μl, 每个视野平均疟原虫数分别为157±13和142±9。3个温度组P. c和P. f 抗原其抗体终点滴度均在5 d后呈下降趋势。2种抗原片在4～6 ℃组及以上组的平均抗体滴度分别为1∶440和1∶80, 差异有统计学意义 （t = 1.940, P＜0.05）。在4～6 ℃放置3 d, -20 ℃贮藏1年的P. c和P. f抗原片检测疟疾抗体的终点滴度均为1∶640; 贮藏2年的2种抗原片终点滴度分别为1∶320与1∶160, 差异均有统计学意义 （tP. c=11.362,PP. c＜0.01; tP. f = 38.845,PP. f＜0.001）。结论 P. c和P. f抗原片检测疟疾抗体终点滴度随存放温度的上升和时间的延长逐渐下降。