Epitope Analysis Using Anti-Oligosaccharide (G4) Monoclonal Antibody

A murine monoclonal antibody recognizing (1→6)-β-d -glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.     

A Monoclonal Antibody to Rabbit Brain GABA Transaminase

A monoclonal antibody of class IgG (subclass IgG1) has been prepared to rabbit brain GABA transaminase (GABA-T). This antibody reveals a single band of molecular weight 52,000 on a nitrocellulose filter blotted with purified GABA-T. On a filter blotted with unfractionated rabbit brain supernatant a major band of molecular weight 58,000 is revealed. An immunoaffinity column was prepared by coupling proteins from ascites fluid containing anti-rabbit GABA-T antibody to Bio-Rad Affi-Gel 15. This column bound purified GABA-T and extracted from unfractionated rabbit brain supernatant a protein of molecular weight 58,000, which was almost homogeneous and which had GABA-T enzyme activity. Using immunoaffinity chromatography, therefore, a high degree of purification of GABA-T may be achieved in a single step. Further, this technique may preserve an authentic form of the enzyme that is lost during the conventional purification procedure. The antibody inhibits GABA-T enzyme activity, up to a maximum of 35%.     

Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus''s restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.     

Flow Cytometric Analysis of Circadian Changes in Platelet Activation Using Anti-Gmp-140 Monoclonal Antibody

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-α-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

经蛋白A连接于琼脂糖的单克隆抗体亲和层析柱用于抗独特型抗体的纯化

 以Sepharose CL-4B-Pro A吸附胃癌单克隆抗体(McAb)PD4,继之以交联剂二甲基庚二亚胺二盐酸盐(dimethyl pimelimidate dihydrochloride)处理,形成亲和介质(柱Ⅰ)。该亲和介质用于纯化抗PD4独特型抗体(aIdAb)具有明显的优点。与Sepharose CL-4B-PD4(柱Ⅱ)相比,前者的结合容量为后者的1.78～1.94倍。采用柱Ⅰ纯化的aIdAb,当其与McAb PD4的分子比分别为1:1及4:1时可50％或100％地抑制McAb PD4与靶细胞的结合,但采用柱Ⅱ获得的aIdAb,只有当分子比达到2:1及8:1时,才能达到同等的抑制效应。这可能是由于Pro A与McAb PD4的Fc片断结合,使后者的Fab端得以充分暴露,因而有更多的机会与aIdAb结合。     

应用HTS-ELISA筛选方法制备抗黄曲霉毒素M1单抗

摘要：【目的】 确立基于高通量酶联免疫（HTS-ELISA）筛选方法制备高亲和力抗黄曲霉毒素M1单克隆抗体的方法体系。【方法】 采用AFM1-BSA （Aflatoxin M1-bovine serum albumin）免疫Balb/C小鼠,利用HTS-ELISA方法筛选分泌抗黄曲霉毒素 M1单抗的杂交瘤细胞株,并分析抗体的特性。【结果】筛选到14株具有分泌高活性抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株,纯化的最佳抗体的亲和力为5.5×10-10 mol/L。与黄曲霉毒素M1及其结构类似物黄曲霉毒素M2、B1、B2、G1、G2以及其他物质脱氧雪腐镰刀菌烯醇和BSA的交叉反应率分别为100%、4.5%、21.5%、1.0%、16.6%、1.0%、0%、0%。间接竞争ELISA检测最低检测限可达0.01 μg/L,线性范围为0.1-10 μg/L,竞争性抑制抗体反应50%的抑制浓度IC50为0.82 μg/L,添加0.25-5 μg/L黄曲霉毒素的牛奶间接竞争ELISA检测回收率在60.3%-152.8之间。【结论】HTS-ELISA方法可以制备具有高亲和力的抗黄曲霉毒素M1单克隆抗体,可为黄曲霉毒素M1免疫检测体系的建立提供优质抗体材料。     

抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备

[目的]小麦赤霉病菌产生的毒素不仅在病害发展过程中具有加重赤霉病的作用,而且污染谷物导致严重的食用安全性问题.由于赤霉病的普遍发生,有必要建立快速、灵敏、有效的毒素检测方法,本试验旨在制备可用于检测被脱氧雪腐镰刀菌烯醇污染的粮谷类特异性单克隆抗体.[方法]本实验首先将脱氧雪腐镰刀菌烯醇(DON)的衍生物3-半琥珀酰-脱氧雪腐镰刀菌烯醇(3-HS-DON-OVA)与卵清蛋白(OVA)采用碳化二亚胺法进行偶联得到人工抗原,以此人工抗原免疫BALB/C小鼠,取该鼠脾细胞与SP2/O鼠骨髓瘤细胞融合,经筛选和克隆,得到了1株能稳定分泌DON抗体的单克隆细胞株(382),并制备单克隆抗体腹水.[结果]经检测382的抗体类型及亚类均为IgG1,其轻链为κ链.腹水通过间接酶联免疫吸附测定效价在1×10-7以上.该单克隆抗体与脱氧雪腐镰刀菌烯醇特异性结合反应的50%抑制质量浓度为29 μg/L,除与3-acetyldeoxynivalenol(3-Ac-DON)的交叉反应率为78.38%,与其他脱氧雪腐镰刀菌烯醇结构类似物无交叉反应.[结论]本实验所制备的单克隆抗体有较高的灵敏度和特异性,具有较好的应用价值.     

A Monoclonal Antibody that Binds Preferentially to Human Suppressor T Cells

A monoclonal antibody termed JM3-3-3A was produced by somatic cell hybridization. The reactivity was assessed by indirect immunofluorescence. JM3-3-3A was reactive with 41% human peripheral blood T cells and 48% non-T cells. Among various human lymphoblastoid cell lines (MOLT 4F, JM, and TALL-I), TALL-I was found not to be reactive with JM-13-3-3A. Human peripheral blood Tcells fractionated by JM3-3-3A-coated dish panning were submitted to functional studies. JM3-3-3A positive T cells responded to mitogens, concanavalin A and phytohemagglutinin-P much better than JM3-3-3A negative T cells in the presence or absence of adherent cells. JM3-3-3A positive T cells showed suppressor activity and JM3-3-3A negative T cells showed helper activity in pokeweed mitogen-induced Ig production. More than fifty percent of the JM3-3-3A positive T cells were reactive with OKT8 which binds to suppressor/ cytotoxic T cells, whereas 18% of JM3-3-3A negative T cells were reactive with OKTIl. In addition, immunoprecipitation experiments identified a protein with an approximate molecular weight of 43,000 as a cell surface antigen for JM3-3-3A. Thus, the reactivity of JM3-3-3A showed a wide distribution but human peripheral blood T cells could be dissected functionally by this antibody.     

Analysis of X-Ray-Induced Cell-Cycle Perturbations In Mouse Osteosarcoma Cells: A Two-Signal Cell-Cycle Model

ABSTRACT The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. the first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. the X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cellcycle-dependent. A qualitative model to account for the observations is presented.     

Detection of Anti-tetanus Toxoid Monoclonal Antibody by Using Modified Polycarbonate Surface

In recent years, CD surface modification methods are employed for immunoassay techniques that is called BioCD technology. In this research, first polycarbonate surface was activated with UV ozone and a hydrophilic surface was obtained. Contact angle measurements and atomic force microscopy technique confirmed the hydrophilic property of surface. After that, tetanus toxoid was immobilized on modified CD surface then specific monoclonal antibody, gold nanoparticles conjugated antibody, silver salt, and hydroquinone were added on modified CD surface. So a sandwiches complex as tetanus toxoid, tetanus toxoid monoclonal antibody, and gold nanoparticles conjugated antibody was obtained on CD surface. ATR result showed the immobilization of tetanus toxoid on modified CD surface. Localized surface plasmon resonance (LSPR) and DLS results confirmed the complex formation. Silver salt and hydroquinone were added for signal amplification. Detection limit of anti-tetanus toxoid IgG monoclonal antibody was obtained 0.005 IU/ml by LSPR and DLS techniques. The presented method increases the assay’s sensitivity. BioCD-based immunoassay for detection of anti-tetanus toxoid IgG monoclonal antibody could be applicable in development and fabrication of biomedical devices.     

利用hTM表达细胞株制备抗人血栓调节蛋白单克隆抗体

为了研制抗人血栓调节蛋白(hTM)的单克隆抗体(mAb),给经CHO细胞免疫的BALB/c小鼠腹腔注射环磷酰胺诱导其对CHO和CHO-TM5细胞的共同抗原表位产生免疫耐受,再用高效表达hTM的CHO-TM5常规免疫小鼠．细胞融合后,ELISA筛选分泌抗hTM的特异性mAb阳性克隆,并将其腹腔注射BALB/c小鼠诱生腹水．腹水中的mAb经亲和层析纯化后,采用ELISA、流式细胞术、免疫组织化学染色法及Western blotting对其进行特异性鉴定．结果显示,共获得了5株阳性克隆,其中2F7可稳定分泌IgG1亚型的mAb,腹水抗体效价为1×10_-6,含量为19.56 g/L．2F7在体内与正常组织的交叉反应极少,对HUVEC、CHO-TM5有特异性结合,并可识别细胞裂解液还原条件下分子质量为105 ku左右的蛋白质多肽．2F7的解离常数K_d约为1.22×10_-9 mol/L．实验结果表明,通过采用新的技术路线,直接应用hTM表达细胞株免疫小鼠,成功制备了具有高度特异性与高亲和力的抗hTM mAb,为其他来源困难的蛋白质mAb制备提供了可借鉴的技术方案,同时2F7的研究鉴定为进一步应用抗hTM mAb进行hTM生物学功能及临床意义研究提供了新的物质基础．     

单克隆抗体仿制药物的结构分析策略

市场高达数千亿美元的单克隆抗体药物即将结束专利保护期,这对发展中国家产业升级,提高国民医疗水平,都是一次难得的战略机遇。然而,单抗类蛋白仿制药的研发与质控工作难度巨大,必须使用液相色谱-质谱联用技术进行分析。针对单抗仿制药结构必须进行的分析项目(氨基酸序列表达正确性、糖基化修饰形态相似性、以及高级结构统一致性)的液质分析方法进行了系统介绍。此外还就分析工作效率及质控过程的法规依从问题予以讨论。     

A Monoclonal Antibody that Specifically Recognizes m'A Nucleoside

Abstract

A hybridoma against the nucleoside m⁶A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m⁶A. The competition assays showed that the monoclonal antibody was highly specific for m⁶A nucleoside.     

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.     

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 spec...     

治疗性单克隆抗体研究进展

杂交瘤技术使鼠源单克隆抗体（鼠单抗）被广泛用于人类疾病的诊断和研究,建立了治疗性抗体的第一个里程碑。但随后出现的人抗鼠抗体等副作用极大地限制了鼠单抗的临床应用。随着生物学技术的发展和抗体基因结构的阐明,应用DNA重组技术和抗体库技术对鼠单抗进行人源化改造,先后出现了嵌合抗体、改型抗体和全人抗体,同时也涌现了各种单抗衍生物,它们从不同角度克服了鼠单抗临床应用的不足,未人类疾病治疗带来新的曙光。我们就上述治疗性抗体人源化的研究进展做简要综述。     

变速上样在MabSelect填料上分离WLB303

随着抗体表达量的提升和生产规模的扩大,Protein A亲和层析不仅需要高载量填料,也需要提高工艺效率。变速上样的方法可以在满足载量要求的同时大大缩短工艺耗时。通过测定WLB303单克隆抗体在GE MabSelect填料多个保留时间的动态载量,建立一元三次方程拟合载量和保留时间的关系。以该方程计算获得填料在快、中、慢速下不同保留时间的动态载量表,并以此作为变速上样组合的参考依据。在2.7ml层析柱上使用精纯样品测试最快的变速上样组合的可行性;然后用常规纯化工艺在7ml层析柱上使用细胞培养澄清液,对最快的变速上样组合和恒速上样的两种工艺周期进行了比较,证实变速上样的方式能明显提升整体工艺效率。     

Development and Characterization of a Monoclonal Antibody to Phytosiderophores

Mugineic acid-family phytosiderophores (MAs) are low molecularweight chelators that are secreted by graminaceous plants, formcomplexes with soil Fe(III) and are essential for plant growth.Methods to detect MAs which include HPLC and radio-immunoassaywith polyclonal antibody require sophisticated equipment orradio-labelled MAs which are difficult to synthesize. Our objectivewas to develop a detection and quantitation system for MAs basedon monoclonal antibody specificity and technology. A monoclonalantibody was produced which reacts with nicotianamine (NA),deoxymugineic acid (DMA), mugineic acid (MA) and epi-hydroxymugineicacid (epi-HMA) in a competitive ELISA. Azetidine-2-carboxylicacid (A-2-C) was not reactive while N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid (A-2-C dimer) was partially reactive.The range of detection using the competitive ELISA is from 2x 10^–6 to 2 x 10^–7 M MAs. Besides detection andquantification of MAs, the potential uses for the monoclonalantibody are numerous and include affinity chromatography andimmunocytochemistry. (Received September 26, 1991; Accepted December 16, 1991)     

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of the...