抗HBs mAb的研制及其对野生与免疫逃逸变异HBsAg的交叉反应特征

目的:抗HBs G6 mAb制备及其对重组野生与免疫逃逸变异HBsAg结合能力与特点的评价.方法:常规制备并纯化抗HBs mAb,以纯化抗HBs mAb IgG包被,采用ELISA对17种野生及"a"决定簇替代性变异全基因重组表达HBsAg进行检测,并与几种市售HBsAg检测试剂进行比较.结果:该杂交瘤细胞生长与分泌特性稳定,其培养上清与腹水效价分别为2048及4096×103;对野生HBsAg检测(ELISA)敏感性不低于0.125 μg/L.该抗体可与15种变异抗原中12种发生反应(P/N≥2.5),反应信号强度分别为低反应组(2份,与野生株A值比较,下同)平均7.55%;中反应组(1例)为59.40%;高反应组(9种)分别为野生株的92.1%～109.4%,低反应或无反应表达产物的免疫逃逸变异部位集中在HBV"a"决定簇I环的起始部即120～124序列之间.部分表达产物采用市售试剂作同步检测,平均显色强度高于或明显高于几种国内应用最为普遍的HBsAg ELISA(P<0.05).结论:抗HBs G6 mAb对多数免疫逃逸变异HBsAg有很强的结合能力,所针对的变异类型亦表现出某种特殊的规律.     

金黄色葡萄球菌的免疫逃逸策略

金黄色葡萄球菌(S.aureus)是一种常见的条件性致病菌,可引发多种疾病,如皮肤和软组织感染(SSTIs)、肺炎、脑膜炎和败血症等.S.aureus也是引发奶牛乳房炎的重要病原菌之一.S.aureus感染的重要特征是重复感染.S.aureus可产生大量毒力因子,逃避先天性和适应性免疫应答.本文重点就S.aureus如...     

肿瘤免疫逃逸的分子机制研究

本文介绍了肿瘤免疫应答过程中参与MHCⅠ类途径和MHCⅡ类途径的多种免疫分子发生丢失 ,导致肿瘤细胞逃避免疫应答 ,同时也介绍了肿瘤微环境中抑制免疫应答的分子过度表达 ,进一步增加肿瘤的免疫逃逸作用     

结核分枝杆菌免疫逃逸机制的研究进展

结核分枝杆菌是一种烈性、强毒、传染性病原菌。由其引发的结核病极难治愈,普通的抗生素药物对其根本不起作用。究其原因是该菌通过一系列目前仍尚未阐明的逃逸机制逃脱了巨噬细胞溶酶体对其的融合杀伤作用,因而得以存活并寄生在巨噬细胞内的吞噬体中。对此,近年来发表了一系列极有价值的研究成果,主要集中在可溶性N-乙基顺丁希二酰亚胺敏感因子连接蛋白受体（SNARE）融合核心复合体的形成,Rab7、Rab5及其效应蛋白EEA-1在晚内体成熟中的作用以及自噬在免疫逃逸机制中的作用等几个方面,这对于进一步丰富和阐明结核分枝杆菌的免疫逃逸机制理论具有重要意义。     

肿瘤的免疫逃逸机制研究进展

大量研究证实,肿瘤病人体内存在广泛的免疫逃避现象。目前所知的肿瘤逃逸机制很多,比如产生免疫抑制因子,改变表面抗原,通过Fas/Fasl反击机体的免疫系统而逃避机体的免疫监视,从而发生免疫逃逸。本文就肿瘤发生免疫逃逸机制的研究现状作一综述。     

IMC在肿瘤免疫逃逸中作用机制的研究进展

未成熟骨髓细胞(immature myeloid cell,IMC)是一个异质造血细胞群体,它主要存在于骨髓,并少量存在于外周血中。在健康个体中,IMC在骨髓内产生并分化为成熟的粒细胞、巨噬细胞或DC。然而,在病理条件下,例如癌症,IMC的分化途径受到部分阻滞而导致其大量增殖,IMC的大量增殖大大促进了肿瘤的生长和转移,引发了肿瘤的免疫逃逸。本文就IMC在肿瘤免疫逃逸中作用机制的研究进展进行综述。     

衣原体持续性感染宿主的免疫逃逸机制

全球范围内衣原体感染病例不断增加,且超过半数患者表现为持续性感染或无症状感染,这可能归因于衣原体的免疫逃逸机制.诱发衣原体持续性感染的外界因素包括抗生素的作用与病毒共感染等,同时衣原体感染可抑制免疫细胞应答并引起宿主细胞凋亡而影响免疫细胞的功能,如促进巨噬细胞的极化、抑制中性粒细胞杀菌作用、促进T细胞的耗竭等.深入研究...     

胞内寄生菌对巨噬细胞免疫逃逸的研究进展

病原微生物在感染宿主后,巨噬细胞作为主要的免疫哨兵细胞首先识别并吞噬入侵的病原体,而入侵的病原体可发展多种策略逃避巨噬细胞杀伤,并适应宿主细胞内环境,在胞内复制和存活。现对胞内感染常见细菌和真菌对巨噬细胞免疫逃逸的策略进行概述。     

Fas/FasL系统与肿瘤的免疫逃逸

最近的研究揭示肿瘤细胞通过表达FasL反击免疫系统 ,消除体内抗肿瘤T细胞 ,并且抵抗Fas/FasL系统介导的凋亡 ,从而使肿瘤成为机体的免疫豁免部位而逃逸免疫系统的攻击。Fas反击作为肿瘤免疫逃逸新机制的阐明为临床工作及肿瘤的免疫治疗提供了新策略     

027 Fas/FasL系统与肿瘤的免疫逃逸

最近的研究揭示肿瘤细胞通过表达FasL反击免疫系统,消除体内抗肿瘤T细胞,并且抵抗Fas/FasL系统介导的凋亡,从而使肿瘤成为机体的免疫豁免部位而逃逸免疫系统的攻击.Fas反击作为肿瘤免疫逃逸新机制的阐明为临床工作及肿瘤的免疫治疗提供了新策略.     

ELISA方法进行婴幼儿HBsAg弱阳性标本检测的方法学评估

目的 评价ELISA方法在检测婴幼儿HBsAg时的适用性.方法 用弱阳性昆合血清评价ELISA方法测定HBsAg时的精密度;使用经化学发光法定值的混合血清系列稀释,分析ELISA方法测定HBsAg时的检出限;使用添加350μmol/L胆红素或5g/L血红蛋白的系列浓度血清评价该方法的抗干扰能力;收集临床1岁以下患儿化学发光方法检测弱阳性(＜ 5ng/mL)的样品,进行化学发光和ELISA方法的比对,并以化学发光确证试验为参考方法评价ELISA方法的诊断灵敏度、特异性和诊断效能.结果 在弱阳性水平上(1 ng/mL,S/CO=4.97) ELISA方法测定HBsAg的变异系数为18.91％;检出限为0.125ng/mL;350μmol/L胆红素或5g/L血红蛋白对检验结果准确度无显著影响;经比对,ELISA法与化学发光法在小于0.25 ng/mL区间一致率为30.3％,而在大于0.25ng/mL区间一致率达95.6％;以二者一致和化学发光法中和试验为参考方法,ELISA方法的灵敏度为87.14％,阴性预测值为79.07％.结论 ELISA方法尽管分析灵敏度低于化学发光法,然而它在测定婴幼儿弱阳性标本时,分析性能尚可接受,考虑到其方法的易得和低廉的成本,ELISA方法仍不失为一种好的筛查方法.     

重组乙型肝炎病毒变异s基因的表达及其抗原性的分析

目的 构建乙型肝炎病毒（HBV）变异s基因真核表达载体。研究变异所致乙肝表面抗原（HBsAg)免疫学性状的改变。方法 利用Dra Ⅲ和XhoI双酶切含有突变位点的HBV DNA 1．2拷贝的质粒P．38Ⅱ，获得904bp带有突变点的HBV－S基因片段。将其置换含有HBV DNA（adr亚型）S和S2片段的真核表达载体pCMV-S2．S的相应片估，从而构建s基因nt587G→A突变的真核表达载体CMV-S2.S 145R；并转染人肝癌细胞系Hep G2，以获得分泌变异型HBsAg的细胞系。利用EIA及细胞免疫染色法，探讨变异抗原与抗－HBs结合力的变化。结果 构建了HBsAg的第145位甘氨酸－精氨酸突变体的真核表达载体。将其转染哺乳动物细胞后第3天，培养上清中用EIA检测用HBsAg呈阳性，A值随时间延长而上升，但变异型的A值明显低于野生型。变异型和野生型HBsAg细胞免疫化学检测均有部分细胞呈阳性，但差异不显著。结论 HBsAg第145位甘氨酸－精氨酸的突变体的真核表达载体产物具有良好的抗原性，能够与抗－HBs结合，但与野生型相比结合力明显降低。     

Improved ELISA for the detection of HBsAg

A sandwich ELISA for hepatitis B virus surface antigen (HBsAg) was developed using monoclonal anti-HBs for the solid phase and horse-radish peroxidase labelled sheep anti-HBs.

The sensitivity was approx. 0.3 U/ml HBsAg, in the standard test procedure,comprising two incubation steps of 1 h at 37°C, or in a shortened procedure comprising two incubation steps of 30 min at 50°C. A slightly reduced sensitivity (approx. 0.5 U/ml) was obtained when the two incubations were combined in a one-step incubation for 1 h at 37°C. All three procedures were completed by an incubation for 30 min at room temperature with peroxide and tetra-methylbenzidine.

The number of false positives obtained when donor sera were screened was below 0.5%. False positive reactions occurred more frequently, but still to a limited extent, when previously selected sera containing rheumatoid factor or other possibly interfering factors were tested with the standard procedure. Most sera containing factors that interfere with a commercial ELISA for HBsAg using sheep anti-HBs coated plates, were negative. Rheumatoid factor positive sera seldom gave false positive results.

The lower detection limit became approx. 0.1 U/ml when the cut-off was reduced, while the number of false positives in a donor population only increased to 1.5%. The results obtained with reagents from four different batches indicate a stability of up to 4 wk at 37°C and for at least 26 wk at 4°C.     

检测可溶型TRAIL的ELISA的建立和初步应用

目的 :建立检测可溶型TRAIL的ELISA ,并评价其临床应用价值。方法 :采用本室制备的鼠抗TRAIL的单抗 (mAb)FMU1.1作为包被抗体 ,兔的抗TRAIL高效价多抗为夹心抗体 ,建立检测sTRAIL的ELISA ,并测定了 9例肾综合征出血热 (HFRS)患者和 4 0例银屑病患者血清sTRAIL的水平。结果 :建立了由单抗和多抗组成的检测人sTRAIL的夹心ELISA ,灵敏度为 0 .0 8μg/L。 9例肾综合征出血热 (HFRS)患者中有 3例急性期患者血清sTRAIL水平升高 ,4 0例银屑病患者中 8例升高。结论 :成功地建立了一种可用于检测血清sTRAIL的双夹心ELISA ,为判定某些相关疾病患者的病情、疗效及预后提供有用的工具     

Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes

A sensitive method which permits analysis of IgG containing circulating immune complexes without detailed knowledge of the nature of the antigens and the specificity of the antibodies involved is described. Soluble BSA: anti-BSA were used as model immune complexes and isolated from serum. The procedure involves the use of gel chromatography for the separation of the high molecular weight fraction containing the immune complexes as measured by binding to 125I-labeled Clq, followed by absorption of the immune complex fraction to immobilized protein A-Sepharose CL-4B. After desorption from protein A-Sepharose the complexes were dissociated and separated into free antigen and antibody by chromatofocusing in the presence of urea. The isolated free antigen and antibody retained their immunological activity as shown by immunodiffusion, binding after their recombination to 125I-labeled Clq, and by recombining antigen and antibody with much enhanced sensitivity using a microplate ELISA system. By means of the ELISA recombination technique it is possible to analyze less than 1 microgram of BSA:anti-BSA model complexes. Application of this technique may provide more information about the nature of immune complex like material associated with diseases.     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

双抗体夹心ELISA检测SEB方法的建立及在不同基质中检测的应用

目的:建立检测SEB的双单克隆抗体(mAb)夹心ELISA,并检测在多种基质中SEB的敏感性。方法:制备、纯化抗SEBmAbB4和D6。D6mAb标记辣根过氧化物酶(HRP)作为检测抗体,B4mAb作为包被抗体。结果:成功建立了敏感、特异检测SEB的ELISA方法,检测抗体稀释液和小牛血清中SEB,检测灵感度0.2μg/L,定量线性范围0.78～12.5μg/L,r2=0.992,批间变异系数(CV)<10%,回收率80%～110%。检测50g/L脱脂牛奶、尿液和自来水中的SEB,灵敏度0.39μg/L,定量线性范围0.78～25μg/L,r2=0.997。与SEA和SECl无交叉反应。结论:本方法测量准确,灵敏度高,特异性好,在食品工业和临床标本检测中有广阔的应用前景。     

Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen

The heterobifunctional reagent 3-(2-pyridyl-dithio)propionate (SPDP) was used to prepare defined conjugates composed of horseradish peroxidase (HRP) and goat anti-HBs IgG. The modification of HRP and IgG with SPDP was dependent on both the SPDP: protein molar ratio and the pH of the buffer. Conjugates were separated by a single affinity chromatographic step using concanavalin A-Sepharose 4B equilibrated with 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl and 1 mM EDTA. The conjugate was eluted with 10 or 100 mM alpha-methyl-D-mannoside and appropriate pools were made reflecting various HRP/IgG molar ratios. Each pool was examined for performance in an enzyme-linked immunosorbent assay for HBsAg. Conjugate composed of an HRP/IgG molar ratio of 2.5-4 yielded the greatest sensitivity.     

重组HBsAg免疫逃逸突变体的表达及其抗原性分析

为了深入研究乙型肝炎病毒S基因变异所致的包膜抗原抗原性的改变，从含ＨＢＶＤＮＡ双拷贝的质粒载体ｐＥｃｏｂ６，获得一个８３７ｂｐ的ＨＢＶ－Ｓ基因片段，将其插入至载体ｐＢｌｕｅｓｃｒｉｐｔ ＫＳ~＋的ＳｍａⅠ位点，通过体外寡核苷酸介导的人工定点突变分别获得第１４５位、１２６位和第１４５位＋１２６位氨基酸三种Ｓ基因变异型。然后将这些Ｓ基因变异片段克隆到真核表达载体ｐＭＥｐ４上，从而构建了含ＨＢＶ－Ｓ基因及其突变型的表达载体ＰＭＥｐ４ＨＢＶＳＭ。用其转染人肝癌细胞系ＨｅｐＧ２，获得稳定分泌ＨＢｓＡｇ及其变异体的抗性细胞系。经体外初步研究表明，ＨＢｓＡｇ １４５位氨基酸变异体可影响ＨＢｓＡｇ的“ａ”抗原决定簇的结构。