应用酶联A蛋白检测流行性出血热抗原抗体的研究

目前,流行性出血热(EHF)抗原抗体的检测方法颇多,虽然都有一定使用价值,但也各有不足之处。为了探求更特异、敏感、快速、简便、易于普及推广的有效方法,作者利用酶联金葡菌A蛋白(HRP—SPA)代替二抗,对EHF抗原抗体检测进行了研究,并与间接免疫荧光进行了比较。结果表明,两法效果相同。现将方法与结果摘要如下: 本实验采用EHFV感染Vero—E_6细胞制作点片抗原。经冷纯丙酮固定后,以0.3%H_2O_2和1%牛血清盐水去除组织内源酶;再用EFF病人恢复期血清作第一抗体、酶联     

SPA菌体协同ELISA及其在检测血清抗HCV·IgM中的初步应用

采用HCV_1合成抗原多肽,SPA 菌体及酶标记抗人IgM 等建立血清抗HCV.IgM 检测SPA菌体协同ELISA(SPA-ELISA)。以多重替代试验、阻断试验及其2-巯基乙醇破坏试验证实其方法的特异性.将其用于一组临床标本的检测,并对该法检测血清抗HCV-IgM 的优点进行了探讨。     

固相血球吸附试验检测iRNA致敏鼠低水平EHFV抗体

在研究抗流行性出血热病毒免疫核糖核酸(EHFV—iRNA)传递体液免疫的活性中,我们建立了检测EHFV—iRNA致敏小白鼠特异性IgM和IgG抗体的固相血球吸附试验(SPARC).该方法简便、快速、特异、敏感,在检测iRNA诱导正常小鼠产生抗体实验研究中取得了满意结果.     

家兔感染EHFV后抗原的分布和抗体应答反应

<正> 近年来,国内外报道了家兔感染流行性出血热病毒(EHFV)后可产生高效价的特异性抗体,朱智勇等首先用家兔作为研究EHFV的易感动物模型,并成功地在动物脏器中找到EHFV抗原。然而,系统地观察动物感染病毒后,抗原在不同脏器中的分布和抗体反应的动态,迄今尚未有详细报道。本文应用EHFV-A9株组织培养抗原,分别在感染前及感染后的第7～20天,用直接和间接免疫荧光技术,检测了兔脾、肝、胰、肺、肾、小肠、大肠等组织切片中的病毒抗原,并在感染后第1～13周内动态观察了血清特异性IgG抗体,现将实验结果报告如     

应用酶标记金葡萄球菌A蛋白在尸检乙脑病毒组织中的免疫定位

金黄色葡萄球菌A蛋白具有与人和多种哺乳动物IgG的Fc段结合的特性,它容易与荧光素、辣根过氧化物酶、铁蛋白、~(125)I和胶体金等结合。Dubois—Dalcq和Falini等以辣根过氧化物酶标记葡萄球菌A蛋白用于免疫组化的研究,获得了满意的结果。国内丁宗武等首先成功地将辣根过氧化物酶标记在葡萄球菌蛋白A上,建立了一种新的酶联免疫方法(ELISA)。1984年凌静萍等将酶标记金葡菌A蛋白(HRA—SPA)应用于乙脑与狂犬小鼠脑组织的病毒抗原定位获得敏感性不低于酶标抗体法的结果。本文是将HRA—SPA应用在尸检乙脑病毒     

“免疫标记”和“放射免疫标记”结合试验:筛选杂交瘤上清的简单方法

为了筛选杂交瘤上清液,需要一种快速简便的方法。许多把杂交瘤技术用于检出肿瘤特异性和肿瘤相关抗原的研究者,已经将洗涤的靶细胞提取物用于他们的筛选试验中,然而,洗涤的提取物可能改变抗原的结构形态,或/和暴露了细胞内的抗原,另一方面未受损的细胞存在一层未破的细胞膜,这和在活体条件下更相似。作者的标准筛选试验是用完整的活细胞,这些细胞首先被杂交瘤细胞上清液激活,然后经过洗涤再和~(125)I SPA起反应,这些SPA     

酶联SPA-斑点-ELISA检测体液(痕)ABO血型抗原

金黄色葡萄球菌A蛋白(SPA)具有与人和多种哺乳动物IgG的Fc段非特异性结合,稳定性好,易于长期保存等特点,是一种很好的免疫学试剂。本研究是将SPA用于斑点-ELISA的一种检测技术。基本原理与间接斑点-ELISA相同,只是以酶标记的SPA代替间接法中的酶标记第二抗体。直接、间接斑点-ELISA对20份唾液标本的研究结果表明,三种方法的灵敏度经统计学处理无显著差异,均能检出0.0001μl分泌型唾液的血型抗原。     

酶标A蛋白检测40例EHF病人血清结果报道

流行性出血热(EHF)的临床实验诊断,已有多种方法。本文利用酶标A蛋白(HRP—SPA)对EHF病人血清进行检测。检测结果如下。将东风试剂厂生产的SPA,按改良的过碘     

链球菌G蛋白用于组织化学染色的实验研究

用理化方法制得链球菌G蛋白(SPG)-辣根过氧化物酶及SPG-胶体金探针,并将其用于常规病理组织切片、电镜包埋后、包埋前及冰冻超薄切片的抗原定位。组织化学染色显示,SPG定位准确、标记密度高,背景清晰,显著优于经典的葡萄球菌A蛋白(SPA)。研究结果表明,SPG的一个最重要的优点就是在免疫组化染色条件下仍有着比SPA更稳定、更可靠、更强的IgG亲合力,尤其适用于单克隆抗体,可获得更为理想的标记结果,因而是一种能用于多种不同条件组织化学染色的新型、高效免疫学检测剂。     

着色SPA的制备及在免疫斑点试验中的应用

<正> 金葡菌A蛋白(SPA)能与人或某些动物的IgGFc段结合,故己广泛用于协同凝集试验和替代第二抗体作ELISA等免疫分析。Shoham首先报道用苏木素染色的 SPA在Sepharose柱上检测HCG,敏感性达0.9IU/ml(Clin Chem 1987;33(6),800)。我们在该法启示下,试制了着色SPA菌体试剂(SSPA),并用于免疫斑点试验测定人IgG,初步结果满意。     

EHF病毒感染家兔后血清特异性抗体水平的动态观察

本文报告了流行性出血热病毒(Epidemic haemorrhagic fever virus,EHFV)J10、A9株分别感染实验家兔后,用反向间接ELISA(RIELISA)和间接免疫荧光(IFA)方法,动态观察了家兔特异性IgG抗体水平的变化。发现特异性IgG于感染后第1周开始出现,以后抗体水平稳步上升,于第5—7周便达到最高水平,高滴度的抗体可以维持较长时间。结果还表明;RIELISA检测家兔抗体的敏感性明显优于IFA法。用IFA进行的交叉反应还证实;EHFV-J10、A9两种毒株的抗血清能与不同来源的数株病毒发生特异性结合反应。     

Study of the cultured human endothelial cells infected by epidemic hemorrhagic fever virus]

Virus antigen could be detected in the cytoplasm of infected human endothelial cells (HEC) by immunofluorescent assay (IFA) 2 to 10 days after the inoculation of epidemic hemorrhagic fever virus (EHFV), but no apparent histologic changes could be found by phase contrast light microscopy, as well as no mature virus particles could be detected under the transmission electron microscope. Reinoculation of the freeze-melt supernatant of HEC 8 days after the inoculation of EHFV to EHFV susceptible Vero E-6 cells, viral antigen could be detected in most of these cells and mature EHFV particles or viral inclusion bodies could also be obtained in the cytoplasm under transmission electron microscope. The results show that HEC is a susceptible target cell to EHFV and infection by this virus may not give apparent cytopathogenic effect in HEC.     

以ABTS为底物的双抗体夹心间接ELISA法的建立及应用

以多-单克隆抗体与正常鼠腹水平行包被固相载体,并用一种新的底物——ABTS作指示系统,建立了EHFV的双抗体间接夹心ELISA法。结果表明:1)ABTS与酶的反应平稳,不需加终止剂终止酶的反应,结果易于肉眼观察;2)对EHFV的定量测定较IFA客观、准确;3)对EHFV抗原的检出范围为351.65±122.82～2091.87±31.27ng/ml;4)对EHFV感染组织和细胞内EHFV的检出率为100％。以上结果说明该法敏感、特异、快速、简便、实用。并指出ABTS作为辣根过氧化物酶的底物的换代产品,应用前景广泛。     

Coagglutination (COA) test for the rapid diagnosis of cryptococcal meningitis

Cryptococcus coagglutination (COA) test reagent was prepared locally and showed no cross reactions with different species of bacteria or yeasts or with 75 control sera including 25 that gave positive results for RA factor. We used the COA test to detect cryptococcus antigen in the CSF and we could confirm the diagnosis of 11 out of 115 suspected cases of fungal meningitis; the titre varied from 4 to 128. A four-fold rise in titre confirmed the diagnostic value and a steady fall in titre in three patients on therapy indicated the prognostic value of the test. The earliest confirmation was in a renal transplant patient on the eighth day after onset of symptoms. The COA test was negative with the CSF of 118 patients with chronic meningitis. Cryptococcal colony forming units (cfu) in CSF varied from 100 to greater than 100,000/ml and correlated well with microscopy and with the COA antigen titre in CSF. Four out of the 11 patients who had cryptococcaemia, had 50,000-100,000 cfu/ml in the CSF. Cryptococcus antigen was detected by COA in the serum of all 11 patients, even in those with only 100 cfu/ml in CSF. In the three post-renal transplant patients, who were being monitored regularly, the diagnosis was made early and all three recovered on antifungal therapy with no relapse to date (1-2 years). All the others, including the two primary CNS infections, succumbed to the disease because they presented late for diagnosis and therapy. The cryptococcus COA test is a simple and specific test that can be used as a rapid test to confirm early diagnosis and permit prompt therapy, which should improve the prognosis in CNS and other forms of systemic cryptococcosis. Moreover, it is reproducible and cost-effective, particularly in countries where the latex and other expensive test reagents are not generally available.     

Improved immunodiagnosis of human cysticercosis with scolex protein antigens

Crude antigenic extract (CAE) and a scolex protein antigen (SPA) of Taenia solium cysticerci were used in indirect haemagglutination (IH) and enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies in cysticercosis patients. Complement fixation (CF) was used for comparison with antigens obtained as an alcoholic extract of CAE (ACAE) and SPA (ASPA). For each test, a dilution was chosen which showed no cross-reactivity with sera from patients with other parasitic diseases such as taeniasis, schistosomiasis, ancylostomiasis, ascaridiasis, strongyloidiasis, Chagas' disease and syphilis. For the CF, 14 was the discriminative dilution determined; for IH, 116 and for ELISA, 1256. The CF could detect serum antibodies to cysticerci in 45% of patients when ACAE antigen was used and 73% using ASPA. In the IH, serum antibody was detected in 73% of patients when CAE was used and 86% using SPA. In ELISA 63% of patients were positive when CAE was used and 91% using SPA. The use of SPA improved the serological distinction between infected and uninfected patients in all tests and the ELISA showed a significantly higher capacity to detect infected patients.     

免疫酶斑点法检测汉坦病毒抗原的研究

目的 建立一种新的诊断肾综合征出血热(HFRS)的实验手段。方法 采用R22株、陈株和湖北株3种汉坦病毒接种家兔，制备兔抗汉坦病毒多克隆抗体(抗HTV-IgG)。然后采用混合的3种抗体进行免疫酶斑点法实验(IEDA)，检测患者血清和尿液中的汉坦病毒抗原。同时采用间接免疫荧光法(IFA)检测患者血清中抗汉坦病毒-IgM作对照。结果 经用相关分析，IEDA与IFA检测的结果高度相关。血中汉坦病毒抗原检出率为73．68％，尿中为65．00％。发病5d内，血中检出率为94．34％，尿中检出率为83．33％。5病日内的早期诊断率前者明显高于后者。结论 IEDA与IFA相比，阳性率比后者有较大提高，特别是5病日内的早期阳性率的提高明显，值得用于临床HFRS的早期诊断。     

Identification of Group a Streptococci by Reverse Passive Hemagglutination

A method for preparing sensitized human 0 erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble antibody globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic streptococci or antigen produced during colony formation. Antibody purified-by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or “pseudo-immune” agglutination occurred when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.     

A functional role for Fcμ receptors on human lymphocytes

The essential findings in this paper are that ox erythrocytes (ORBC) sensitized with human heterophile IgM antibody failed to detect Fcμ receptors on human lymphocytes. These results contrast to those obtained when the ORBC were sensitized with mouse hybridoma IgM antibody (SCC I). It is therefore suggested that the functional role of Fcμ receptors on human lymphocytes is not to bind IgM/antigen complexes.     

Identification of Group a Streptococci by Reverse Passive Hemagglutination

A method for preparing sensitized human 0 erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble antibody globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic streptococci or antigen produced during colony formation. Antibody purified-by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or “pseudo-immune” agglutination occurred when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.     

Advantages and limitations of staphylococcal protein A-Sepharose for isolating soluble immune complexes from goat, rabbit and human sera.

Staphylococcal protein A (SPA), bound to CNBr-activated Sepharose, was evaluated as a selective adsorbent for soluble immune complexes (ICs) prepared in antigen (Ag) excess. Goat antibody (Ab) to human serum albumin (HSA) and rabbit and human antisera to diphtheria toxoid (DT) were utilized for complex formation. Monomeric goat IgG did not bind SPA. However, HSA-goat anti-HSA complexes which were greater than 12S by sucrose density-gradient ultracentrifugation and had molar Ab:Ag ratios greater than 1.5 were adsorbed, and could subsequently be eluted with acidic phosphate-buffered saline, pH less than or equal to 3.8. Elution with 3.5 M MgCl2 enhanced recovery, but also resulted in hydrolysis of the bound Ab. Ninety per cent of the DT-anti-DT ICs prepared with rabbit Ab and 55% of those prepared with human Ab, in the presence of excess free Ag, bound to the SPA columns. However, only 42% of the DT-rabbit anti-DT complexes, and 32% of those prepared with human antisera were isolated in the acidic phosphate-buffered eluate, free of contaminating proteins. Recovery of ICs by SPA affinity chromatography was significantly decreased when the ICs were partially purified by PEG or ammonium sulphate precipitation before application to the SPA-Sepharose columns. These studies indicate that SPA can be used to isolate ICs prepared in far Ag excess and with Abs which, by themselves, do not bind to this absorbent. They also demonstrate that recovery of ICs from sera using this adsorbent is invariably incomplete.