Comparative evaluation of various immunodiagnostic tests for the diagnosis of Taenia solium cysticercosis in pigs, using fractionated antigens

The sensitivity and specificity of double immunodiffusion (DID), indirect haemagglutination test (IHA), immunoelectrophoresis (IEP), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA) were evaluated and compared using saline extracted of Taenia solium larval scolex and its Sephadex G-200 fractionated 1st and 2nd peak as antigens. Various immunodiagnostic tests gave different results with different antigens. Highest sensitivity (92.5%) was obtained with 84.6% sensitivity was obtained with IHA and CIEP respectively using scolex antigen. CIEP gave better results as compared to IEP. Crude antigen gave high sensitivity but less specificity. It was concluded that CIEP can be used as a field test for the anti-mortem diagnosis and ELISA can be employed for laboratory confirmation of T. solium cysticercosis in pigs using fractionated 1st peak antigen.     

Characterization of strain specific typing antisera for genetic monitoring of inbred strains of rats

Strain-specific typing antisera (SSTA) were prepared for six inbred strains of rats by using a pooled immunization protocol. The SSTA were used in both a haemagglutination assay and a complement dependent microcytotoxicity assay to compare the usefulness of the two test systems. Both assays were simple, reliable and repeatable, and each system had distinct advantages and disadvantages. The haemagglutination assay was faster and required less specialized equipment than the microcytotoxicity assay. On the other hand, interpretation of results in the microcytotoxicity assay was easier and more objective. It was concluded that SSTA could be used with the microcytotoxicity assay and/or the haemagglutination assay to provide a simple and effective genetic monitoring method for inbred strain of rats.     

ELISA法检测流行性腮腺炎病毒感染特异性IgM抗体的研究

用流行性腮腺炎(流腮)病毒Enders株接种鸡胚尿囊腔培养,尿囊液经聚乙二醇6000处理制备流腮病毒抗原,用ELISA法检测流腮患者血清中特异性IgM抗体,其敏感性,特异性、重复性和稳定性都很高。 79份流腮患者血清,检出特异性IgM72份,阳性率为91％,32例非流腮患者IgM全部阴性、两者有极显著差异(P<0.01)。 10份血清作血清倍比稀释至1∶3200测IgM仍全部阳性,1∶6400稀释仅1例阴性,1∶12800稀释5例中仍有2例阳性。 10份血清作流腮抗原特异性抗体阻断试验,光密度抑制率均大于50％,平均为87％,10份标本作2-ME和SPA阻断后检测IgM抗体,结果2-ME阻断标本全部阴转,而SPA阻断标本仍阳性,证实所检测为流腮特异性抗体。 24份标本2次重复检测流腮IgM,其阴、阳性结果一致,这期间抗原放4℃ 1个月,提示抗原的稳定性和方法的重复性都很好。本方法敏感性明显高于血凝抑制试验,其阳性率分别为91％和61％,两者有显著差异。而且所用试剂简单经济,操作简便,快速,适用于临床早期诊断,易于广泛推广应用。     

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.     

Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells.

Epstein-Barr virus-transformed human B cells expressed cell surface varicella-zoster virus (VZV) antigens after superinfection with VZV although they did not form infectious centers in a plaque assay. The VZV-superinfected cells were lysed by autologous VZV-stimulated T-cell lines and their derivative clones. The effector cells were specific for VZV and an HLA DR antigen and were T4+. The specificity of lysis of Epstein-Barr virus-transformed, VZV-superinfected targets by prestimulated mononuclear cells in this system contrasted with the unrestricted lysis seen when the targets were VZV-infected fibroblasts.     

去N端信号肽的水痘-带状疱疹病毒糖蛋白E的原核可溶性表达及其免疫原性评价

水痘-带状疱疹病毒(varicella zoster virus,VZV)糖蛋白E(glycoprotein E,gE)是VZV亚单位疫苗的主要候选蛋白,但目前原核表达系统制备的gE蛋白以包涵体形式为主,可溶性差。本研究采用去除第1～30氨基酸序列的VZV gE胞外域基因,将其与原核表达载体pET32a连接,并转化至感受态细胞BL21(DE3)中。使用异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-thiogalactoside,IPTG)诱导表达,His-tag柱纯化重组gE蛋白,蛋白质印迹法(Western blot,WB)检测其特异性。用该重组gE蛋白免疫BALB/c小鼠制备多克隆抗体,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和间接免疫荧光法检测多克隆抗体效价及特异性。结果显示,BL21/pET32a-VZV gE工程菌可以表达可溶性重组gE蛋白,纯化后纯度约为90%。WB鉴定该重组蛋白具有良好的免疫反应性。ELISA检测显示小鼠抗VZV gE多克隆抗体效价＞1∶10 000,间接免疫荧光实验结果显示该抗体特异性较高。结果表明,本研究在原核表达系统中成功表达可溶性重组VZV gE蛋白,同时该蛋白具有较强的免疫原性,这为VZV gE亚单位疫苗的研制和大规模生产奠定了基础。     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

Autoimmunity in amoebiasis.

Amoebic liver abscess (ALA) and symptomatic intestinal amoebiasis cases were assessed by indirect haemagglutination assay for auto-reactive IgG and IgA class of antibodies in response to healthy human serum IgG and IgA. The present results indicated the presence of autoreactive IgG and IgA class of antibodies in ALA and intestinal amoebiasis respectively.     

The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.     

水痘-带状疱疹病毒糖蛋白E在昆虫细胞中的表达、鉴定及免疫原性分析

目的:利用昆虫-杆状表达系统建立表达和纯化分泌形式的水痘-带状疱疹病毒(varicellazoster virus,VZV)糖蛋白gE的方法,并鉴定其理化性质及免疫原性。方法:利用Gibson assembly同源重组试剂盒快速构建重组质粒pFastbac-VZV gE。在sf9昆虫细胞中鉴定序列优化前后表达量的差异,并在High FiveTM细胞中大量表达。通过Ni-NTA亲和层析方法得到高纯度gE蛋白,之后通过酶联免疫吸附试验等验证了其理化性质,并进行小鼠免疫分析其免疫原性。结果:构建了pFastbac-VZV g E1/2重组质粒,经过PCR及双酶切鉴定后均为阳性克隆。使用BAC/PAC试剂盒提取Bacmid转染sf9细胞制备杆状病毒,经Western blot检测,sf9细胞开始表达gE蛋白且随着病毒代次升高g E蛋白表达量增加。序列优化后表达量明显增加,但是大部分以胞内形式存在。通过Ni-NTA一步亲和层析获得纯度较高的gE蛋白,并与VZV单抗9C8具有较好的反应性。通过免疫小鼠产生高滴度抗体,且免疫荧光结果显示其血清可以与天然病毒上的gE抗原结合。结论:成功获得了杆状表达系统表达的gE蛋白并且纯化和鉴定了蛋白质的性质及免疫原性,为进一步研发具有自主产权的VZV亚单位疫苗研究奠定了基础。     

圆形碘泡虫免疫原性的研究

间接红细胞血凝试验结果表明,自然感染圆形泡虫的鲫鱼血清中存在循环抗体,并且感染强度与抗体水平不相关。以圆形碘泡虫孢子的可溶性蛋白为抗原,制备多抗。ＥＬＩＳＡ和ＩＦＡＴ试验表明,不同发育时期的圆形碘泡虫存在共同抗原,并且粘孢子虫具有属特异性抗原。圆形碘泡虫的抗原成分主要集中在早体后部的一特异位点及四周的早壁上,两个极囊无抗原成分;而 营养体的抗原成分存在于整个虫体。关碘泡虫与兔抗圆形碘泡虫抗体的结合     

微量板细胞病变法滴定水痘疫苗的研究

建立了用微量板细胞病变法滴定水痘疫苗的实验方法。将适宜稀释度的病毒与2BS细胞混种于96孔板培养,种毒后第7天观察孔内细胞病变情况,以karber法计算疫苗滴度,并与蚀斑法进行比较。实验表明,微量板细胞病变法滴定水痘疫苗,结果稳定性好,精密度高,较现行的蚀斑法操作简便、误差小,能够较好的反映出水痘的真实滴度,可作为一种较为简便的滴定水痘疫苗的方法。     

Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.     

Comparison of Solid-Phase Radioimmunoassays for Baculoviruses

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.     

Evaluation of three serological tests for the detection of human plague in northeast Brazil.

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.     

Acanthamoeba culbertsoni and Naegleria fowleri: occurrence of antibodies in man

Serum samples of 1,054 inhabitants of Bohemia (Czechoslovakia) were examined by means of indirect haemagglutination test with antigens from Naegleria fowleri and Acanthamoeba culbertsoni. With N. fowleri antigen the frequency of positive reactions did not exceed 3.5 per cent in lowest serum dilutions only. N. fowleri could not be identified as a possible causative agent in any chronic form of disease in man. Significant accumulation of positive findings with A. culbertsoni antigen was observed in hepatitis A patients and convalescents (52 per cent). The potential interpretations of this phenomenon are discussed.     

A New Method for Strain Identification of Rhizobium trifolii in Nodules

An indirect haemagglutination test has been developed for the detection of strains of Rhizobium trifolii in nodules of subterranean clover plants. Preserved sheep red blood cells, coated with isolated specific rhizobial lipopolysaccharide, were used as the indicator of agglutination; these cells were agglutinated by specific antilipopolysaccharide antibody. Detection of lipopolysaccharide antigen in a suspension of nodular tissue was carried out by reacting the suspension with antilipopolysaccharide antibody prior to the addition of coated red blood cells. The presence of antigen in the suspension was indicated by an inhibition of agglutination. The test was more sensitive than agglutination and immunodiffusion in the detection of rhizobial lipopolysaccharide antigens, and could be used for the rapid screening of large numbers of nodules.     

The determination of the potency of human tetanus immunoglobulins by an enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay has been adapted to the quantitation of specific tetanus antibodies in commercial preparations of human immunoglobulins. The results of the assay are compared with those obtained from the same samples tested by seroneutralization tests in vivo. Statistical analysis of the data shows good correlation between the titres obtained with the two tests. Results obtained by indirect haemagglutination are also reported. It is proposed that all interested laboratories perform the described immunoenzymatic method in vitro for a given period in parallel with the in vivo test to gain sufficient experience of this technique with a view to its use as an alternative to the in vivo test.     

Anticomplement immunofluorescence for the titration of antibody to varicella-zoster virus

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.