抗SEA鸡卵黄抗体用于血吸虫循环抗原检测的初步研究

目的:探讨抗可溶性虫卵抗原(SEA)鸡卵黄抗体(Immunoglobulin Y,IgY)用于血吸虫循环抗原检测的可行性。方法:以纯化的鸡抗SEA卵黄抗体为捕捉抗体,以酶标抗SEA单克隆抗体NP28-5B进行双抗体夹心酶联免疫吸附试验法(S-ELISA)检测急、慢性血吸虫病病人和健康人血清,并与常规检测抗体的酶联免疫吸附试验(SEA-ELISA)法做比较。结果:S-ELISA法测得SEA浓度(y)与A450值(x)呈明显的正相关(r=0.9481,y=459.22x-108.14)。S-ELISA法检测急性血吸虫病人循环抗原的阳性率为100%,慢性血吸虫病人循环抗原的阳性率为84.40%,健康人的特异性为96%。两种方法对血吸虫病的检出率差异无统计学意义(P>0.05)。结论:S-ELISA可用于血吸虫病的免疫诊断。     

日本血吸虫卵尿素溶解性抗原的简易提取及其化学和免疫学性质

血吸虫卵可溶性抗原(SEA)已广泛用于血吸虫病的免疫诊断。WHO(1982)对各种有效抗原在免疫诊断的应用进行了全面、系统的比较研究,结果以SEA的敏感性和特异性为高。Tsang等(1981)、陶义训等(1983)分别从曼氏血吸虫卵和日本血吸虫卵提取了可溶性的SEA,然后用尿素溶液从沉渣中提取另一类抗原,称为尿素溶解性虫卵抗原(简称JEU)。用动力学酶标固相免疫测定(K—ELISA),JEU的敏感性和特异性均优于SEA,抗原总活力相当于SEA的2.83,并可多获2.48倍的抗原量。原法提取JEU需用48,000—100,000g的高速及超速离心、超声处理及Bio—gelA50m…     

日本血吸虫成虫培养液及虫卵对家兎机体的影响

本文就家兔感染日本血吸虫病后所引起的机体反应及组织病理变化的主要发病因子进行了探讨。结果发现家兔在注射40对/8毫升成虫培养液后,除血沉有加速趋势外,血清蛋白组成部分的变化不明显。组织病理变化与家兔感染150条尾蚴后血吸虫产卵前的相似。家兔在注射40—60万虫卵(其中成熟虫卵约占40%)后出现血沉加速,血清白蛋白减少和α、β及γ球蛋白增加。组织病理变化与家兔感染150条尾蚴后5—6周、即虫卵成熟后所引起的酷似。结论认为虫卵、特别是成熟虫卵是血吸虫病发病学中的主要因子,但成虫代谢产物对机体的影响也不容忽视。对血沉与血清蛋白组成变化间及其与组织病理变化间的相互关系及发生机制进行了讨论。     

单克隆抗体抑制性酶联免疫吸附试验：（I—ELISA）检测日本血吸虫虫卵抗原

检测血吸虫抗原较之检测虫体的明显优点是可准确地估计虫荷以及判断是否活动性感染,有助于了解化疗效果。近年来国内已有用双抗体夹心酶联法检测血吸虫循环抗原的报道。使用高效特异的单克隆抗体可望进一步提高检测的敏感性。本文对单克隆抗体(McAb)I-ELISA检测日本血吸虫虫卵抗原的敏感性和特异性作了初步探讨。有关本法检测感染日本血吸虫的动物和病人血中血吸虫虫卵抗原的情况将另文报道。材料与方法McAb制备:用日本血吸虫虫卵抗原(SEA)免疫BALB/C小鼠,取其脾细胞与SP2/O小鼠骨髓瘤细胞融合,经克隆化后获得3株分泌抗日本血吸虫…     

中华硬蜱和二棘血蜱的交叉免疫反应

本文首次比较了经中华硬蜱(Ixodes sinensis)叮咬三次后再经二棘血蜱(Haemaphysalisbispinosa)叮咬的家兔与仅经二棘血蜱叮咬的家兔的交叉免疫抗性。二棘血蜱叮咬被中华硬蜱致敏的家兔时,吸血增重为:143.12±32.67mg,但二棘血蜱在正常家兔体上寄生,初次吸血增重为:181.30±44.35mg,两者之间有显著性差异(P<0.01)。中华硬蜱和二棘血蜱唾液腺提取物(SGE)经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),显示两者分别有24条和22条电泳带,中华硬蜱主带有6条,分子量分别为142、105、94、66/65、64和56kD,而二棘血蜱主带有5条,分子量分别为:215、114、105、66/65和58kD,经中华硬蜱叮咬致敏的家兔血清和经二棘血蜱叮咬致敏的家兔血清作免疫印渍,均显示出105kD这一电泳带。该实验表明中华硬蜱和二棘血蜱叮咬家兔两者之间存在着交叉免疫反应,提示105kD蛋白质抗原可能是两者的共同抗原。     

华硬蜱和二棘血蜱的交叉免疫反应

本文首次比较了经中华硬蜱(Ixodes sinensis)叮咬三次后再经二棘血蜱(Haimaphysalis bispinosa)叮咬的家兔与仅经二棘血蜱叮咬的家兔的交叉免疫抗性。二棘血蜱叮咬被中华硬蜱致敏的家兔时,吸血增重为：143.12±32.67mg,但二棘血蜱在正常家兔体上寄生,初次吸血增重为：181.30±44.35mg,两者之间有显著性差异(P<0.01)。中华硬蜱和二棘血蜱唾液腺提取物(SGE)经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),显示两者分别有24条和22条电泳带,中华硬蜱主带有6条,分子量分别为142、105、94、66/65、64和56kD,而二棘血蜱主带有5条,分子量分别为：215、114、105、66/65和58kn,经中华硬蜱叮咬致敏的家兔血清和经二棘血蜱叫‘咬致敏的家兔血清作免疫印渍,均显示出105kD这一电泳带。该实验表明中华硬蜱和二棘血蜱叮咬家兔两者之间存在着交叉免疫反应,提示105kD蛋白质抗原可能是两者的共同抗原。     

镰孢体外抗原的电泳及免疫印渍分析

用SDS-PAGE及免疫印渍法分析了三种镰孢的体外抗原(exoantigen)和菌丝体可溶性蛋白质的部分特性,并研究了培养基对体外蛋白质含量的影响。结果表明,在电泳分析中,三种镰孢体外抗原及菌丝体可溶性蛋白质均具有各自菌种的特征,可作为菌种分类鉴定的重要指标。免疫印渍分析显示,体外抗原更适于用作免疫分类鉴定的指标,因为用体外抗原免疫动物所产生的抗体的特异性比菌丝体可溶性蛋白质要好。三种镰孢的体外抗原的抗体与种间菌株均有程度不等的交叉反应,但却不与谷物发生任何交叉反应,可用于谷物中镰孢的快速检测。在镰孢体外抗原中,能刺激机体产生抗体的抗原分子量在28000以上。葡萄糖酵母膏培养基比蔗糖硫酸铵培养基更适于体外抗原的产生。     

日本血吸虫重感染兔肝在生理盐水中冷藏24小时后的虫卵活性观察

本文探讨日本血吸虫重感染兔肝加生理盐水置冰箱浸泡24小时后分离的虫卵活性。1材料和方法感染1500条日本血吸虫尾蚴的26只家兔肝,用常规方法分离虫卵,按兔肝湿重等分为两份。一份即时分离虫卵称即时组,另一份加少量生理盐水(淹没肝组织为度)置冰箱24小时分离虫卵称24小时组。两组所获虫卵均分别制备加热超声虫卵〔1〕和冰冻干燥虫卵。加热超声的两组虫卵用玻片法作环卵沉淀试验以观察环沉率和反应强度。冻干卵两组则制备成1%虫卵抗原用对流免疫电泳(抗原稀释法)检测活性,并用凯氏微量定氮法测含氮量,依据含氮量稀释为10—15μg/ml致敏血球,…     

日本血吸虫14-3-3蛋白的表达与诊断价值研究

制备可溶性表达的重组日本血吸虫信号传导蛋白14-3-3(r Sj14-3-3)并优化表达条件,分析其免疫学特性和免疫诊断价值。成功构建了BL21/p ET30a-r Sj14-3-3表达菌株,最佳的表达体系为细菌生长至OD600为0.6~1.0时加入IPTG至最佳终浓度0.1 mmol/L,28℃诱导16 h。上清液中可溶性的r Sj14-3-3蛋白纯化后的浓度为2.804 mg/m L。以r Sj14-3-3为抗原,间接ELISA检测显示,48份急性与35份慢性血吸虫病患者血清的抗体阳性率分别为95.6%和92.0%;与27份华支睾吸虫和15份钩虫患者血清的交叉反应率分别为7.4%和6.7%;与30份健康人血清假阳性反应率为0%。抗r Sj14-3-3兔血清及所制备的多抗可被纯化出的r Sj14-3-3蛋白及血吸虫成虫抗原(AWA)和排泄分泌物(ESA)中的天然Sj14-3-3分子的反应,且多抗效价达1:1 280 000。本研究成功表达并纯化出r Sj14-3-3蛋白,优化了其诱导表达条件,且此蛋白具有较高的抗原特异性。     

牛肾上腺皮质LDL受体的纯化和抗体的制备

牛肾上腺皮质LDL受体经Triton X-100增溶,DEAE_(32)离子交换柱和LpB Sepharose亲和柱层析,在SDS-PAGE中有三条区带,分别在原点;Mr 160kD;Mr125kD处。进一步用8％SDS-PAGE纯化三个区带的蛋白质分别免疫新西兰大白兔所得的抗体,应用免疫印迹和ECL非同位素标记法可对牛肾上腺皮质和人皮肤纤维细胞膜上的LDL受体进行测定。     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

Immune responses and immunoregulation in relation to human schistosomiasis in Egypt. II. Cimetidine reversal of histamine-mediated suppression of antigen-induced blastogenesis

The effect of histamine on cell-mediated immune responses of chronic schistosomiasis patients was tested by peripheral blood mononuclear cell (PBMN) reactions to phytohemagglutinin-P (PHA) and soluble schistosomal antigenic preparations derived from eggs (SEA) or adult worms (SWAP). PBMN responses to PHA were suppressed by exogenous histamine (10(-5)M), and the addition of cimetidine (CIM) (10(-4)M), an H2-receptor antagonist, reversed this suppressive effect. Histamine primarily suppressed PBMN responses to suboptimal and optimal PHA concentrations. Exogenous histamine (10(-5)M) also suppressed PBMN responses of 27 schistosomiasis patients to SEA and SWAP, respectively. The addition of CIM (10(-4)M) to suppressed cultures reversed the effect of exogenous histamine. Most importantly, the addition of CIM to schistosomal antigen-induced cultures, without exogenous histamine, significantly increased patients' PBMN responses to SEA and SWAP. The mean optimal increase in SEA responses of 19 patients was 390%. With SWAP-induced responses of 21 patients this increase was 165%. The use of 10(-4)M diphenhydramine (DPH), an H1-receptor antagonist, resulted in general suppression of both PHA-induced and schistosomal antigen-induced PBMN responses. Lower concentrations of DPH lead to variable responses but did not result in consistent abrogation of the histamine-induced suppression. These data imply that an histamine-induced, H2-receptor-mediated suppressor circuit often helps modulate antigen-specific responsiveness of PBMN from patients with chronic schistosomiasis.     

Cytokine profile associated with chronic and acute human schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma (IFN-gamma) was evaluated in 43 schistosomiasis patients with different clinical forms. Whole-blood cultures cytokine production in response to soluble egg antigen (SEA), soluble worm adult preparation (SWAP), mitogens, neutralizing antibodies or recombinant IL-13 were measured by ELISA. After SWAP stimulation, chronic patients, particularly hepatointestinals, produced higher levels of IL-4 in comparison with acute patients, suggesting the presence of a type 2 cytokine profile in these patients. Following SEA and SWAP stimulation, hepatosplenic (HS) patients showed increased levels of IFN-gamma when compared with acute patients, indicating that HS disease in humans is associated with a type 1 cytokine response. The mechanisms of immune regulation are apparently different between the clinical stages of the disease, some of which are antigen-specific.     

Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.     

Immune responses during human Schistosomiasis mansoni. XI. Immunologic status of patients with acute infections and after treatment

Sixteen patients, 8 to 30 yr of age, with acute (toxemic) phase schistosomiasis mansoni were studied immunologically within 2 to 3 mo of their exposure to Schistosoma mansoni cercariae, and were monitored after chemotherapy. Total leukocyte levels and peripheral blood eosinophilias were higher in these patients than in similar individuals with chronic schistosomiasis mansoni. In contrast to chronic patients, the eosinophilias of the acute cases were decreased rather than elevated upon treatment. Total lymphocyte population (T and B cell) percentages were not altered during acute infection. Lymphoid subset (T3+, T4+, and T8+) analysis revealed elevated levels of both T4+ and T8+ cells. In vitro blastogenic responses of peripheral blood mononuclear cells (PBMN) to heterogeneous schistosome-derived antigens (eggs, SEA; adult worms, AW; and cercariae, CERC) were evaluated. SEA responsiveness was considerably higher than that of patients with chronic S. mansoni infections. The ratios of SEA to AW responses in acute cases gave a mean of 2.0, as opposed to 0.5 for a comparable group of chronically infected patients. The sera of most acute patients already contained suppressive factors that specifically decreased schistosomal antigen-induced PBMN blastogenesis. Chemotherapy of acute cases lead to a diminution of PBMN responsiveness to SEA and CERC. Treatment of patients with chronic infections lead to the elevation of such responses. PBMN from patients with acute infections produced lymphokine leukocyte inhibition factor upon exposure of the cells to SEA but not AW. A similar pattern was true for production of the lymphokine activity mitogenic factor. Levels of antibody in sera of acutely infected patients against SEA, CERC, and AW were considerably higher than levels in sera of chronically infected patients matched for age and intensity of their infections. These high antibody titers persisted for at least 6 mo after treatment, and were unrelated to the intensity of infection. The immunologic status of these patients with acute schistosomiasis mansoni differed considerably from patients with chronic infections. These findings re-emphasize the immunoregulatory events that apparently develop upon continued exposure to schistosomes and their products during chronic infection.     

Mapping of the conserved antigenic domains shared between potato apyrase and parasite ATP diphosphohydrolases: potential application in human parasitic diseases

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.     

Discovery and identification of anti-U1-A snRNP antibody in lung cancer

There are multiple reports of autoimmune response in patients with lung cancer. To investigate whether a novel autoantibody is present in patients with lung cancer and evaluate its clinical diagnostic and prognostic value, sera from 10 patients with lung cancer and 10 normal individuals were analyzed using immunofluorescence and Western blotting. It was found that one serum sample from the patients with squamous carcinoma gave a fine speckled pattern staining in nucleus and had a high titer antinuclear autoantibody which could recognize 31 kD of nuclear protein isolated from both cancer cells and normal cells. The same patient’s serum was further used to immunoprecipitate the target antigen. The protein bands were excised from the SDS-PAGE gels and were analyzed with a Qstar Pulser I Quadrupole time-flight mass spectrometer, and the 31 kD target antigen was identified as U1-AsnRNP. To test the prevalence of anti-U1-AsnRNP antibody, sera from 93 patients including 36 squmaous carcinomas (SCC), 26 adenocarcinomas (Ad), and 31 small cell carcinomas (SCLC) were screened by Western blotting. The results demonstrated that anti-U1-A snRNP antibody was present in 50% of SCC sera, 26.9% of Ad sera and 54.8% of SCLC sera. In this paper, we report for the first time that anti-U1-AsnRNP antibody could be detected in the patients with lung cancer.     

Discovery and identification of anti-U1-A snRNP antibody in lung cancer

Autoantibodies are frequently observed in sera of patients with malignancies and generally have been thought to be nonspecific and a reflection of can cer-related general immune system dysfunction. In the previous study, it was reported that autoimmune responses to cell cycle-regulatory proteins and nuclear proteins were present in cancer patients. For example, the antibody against human p53 protein was found in 20%―40% of esophageal carcinoma and oral squa- mous cell carcinoma[1], autoantibo…     

Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.     

Schistosoma mansoni: immunodiagnosis is improved by sodium metaperiodate which reduces cross-reactivity due to glycosylated epitopes of soluble egg antigen

ELISA with soluble egg antigen (SEA) from Schistosoma mansoni is widely used in the diagnosis of schistosomiasis, but cross-reactivity with other intestinal helminths, overestimating the true prevalence, represents a great limitation. The role of glycoproteins of SEA in cross-reactions was investigated. SEA was oxidized with sodium metaperiodate (SMP) in ELISA and immunoblot. One hundred schistosomiasis-negative individuals sera were submitted to SMP-ELISA improving the specificity from 73% without SMP treatment to 97% with SMP. On the other hand, 94 S. mansoni-positive sera were evaluated showing that 99% were positive in ELISA either with or without SMP treatment, indicating the maintenance of high sensitivity under SMP treatment. By immunoblot, 24 sera from persons with schistosomiasis and 10 sera from schistosomiasis-free persons were assayed under reducing and nonreducing conditions with or without SMP, looking for specific infection markers and cross-reactivity markers. Reactivity from positive sera showed that specific molecules were mainly low-molecular-mass antigens and seem to have predominant proteic epitopes. The unspecific molecules reacting with some schistosomiasis-negative individuals harboring other intestinal parasites (false-positive sera) were mostly larger than 60 kDa and seemed to be basically glycosylated. Glycosylated epitopes have an important role in cross-reaction and SMP can successfully be used to reduce the false reactivity of SEA with no decrease in sensitivity, especially in ELISA as an immunodiagnostic screening surveillance method, which is useful in areas of low schistosomiasis transmission.