Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.     

Production of Monoclonal Antibody against Scrippsiella trochoidea Cysts and Its Application to Analysis during Cyst Formation and Enzyme-linked Immunosorbent Assay

A monoclonal antibody was produced by the fusion of mouse myeloma cells with spleen cells from a mouse immunized with the cysts of Scrippsiella trochoidea, marine phytoplankton. Immunofluorescence micro-scopic observation showed that the antibody reacted with the spines on the cyst but not with the vegetative cells and cyst walls. An enzyme-linked immunosorbent assay could be used to measure the cysts in muddy bottom sediments using the purified antibody conjugated with horseradish peroxidase.     

Explaining the Non-Newtonian Character of Aggregating Monoclonal Antibody Solutions Using Small-Angle Neutron Scattering

A monoclonal antibody solution displays an increase in low shear rate viscosity upon aggregation after prolonged incubation at 40°C. The morphology and interactions leading to the formation of the aggregates responsible for this non-Newtonian character are resolved using small-angle neutron scattering. Our data show a weak repulsive barrier before proteins aggregate reversibly, unless a favorable contact with high binding energy occurs. Two types of aggregates were identified after incubation at 40°C: oligomers with radius of gyration ∼10 nm and fractal submicrometer particles formed by a slow reaction-limited aggregation process, consistent with monomers colliding many times before finding a favorable strong interaction site. Before incubation, these antibody solutions are Newtonian liquids with no increase in low shear rate viscosity and no upturn in scattering at low wavevector, whereas aggregated solutions under the same conditions have both of these features. These results demonstrate that fractal submicrometer particles are responsible for the increase in low shear rate viscosity and low wavevector upturn in scattered intensity of aggregated antibody solutions; both are removed from aggregated samples by filtering.     

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)     

单克隆抗体在植物研究中的应用

单克隆抗体是现代生命科学研究的重要工具。随着分子生物学的发展,单克隆抗体在植物研究中发挥着越来越重要的作用。本文综述了单克隆抗体在蛋白表达、蛋白定位、蛋白相互作用、植物成分的定性与定量、植物成分纯化、植物病害检测、标签抗体等方面研究中的应用。     

肺炎衣原体单克隆抗体的研制和应用

目的:以杂交瘤技术制备抗肺炎衣原体(Cpn)单克隆抗体,用于衣原体感染的诊断及相关疾病的研究。方法:以进口Cpn抗原免疫BALB/c小鼠,将免疫小鼠的脾脏细胞与SP2/0细胞融合,用间接ELISA法筛选抗体阳性杂交瘤细胞。收集接种过杂交瘤细胞的小鼠腹水,分别用ELISA法检测抗体效价、用免疫琼脂扩散试验鉴定单抗的类别、用微量荧光免疫试验(MIF)检测单抗的种属特异性。用克隆表达的主要外膜蛋白(MOMP)通过Dot-ELISA法分析单抗的特异性。通过建立直接免疫荧光法(DIF)检测病人和正常人外周血单核细胞(PBMC)标本,并进行统计处理。结果:小鼠脾脏细胞与SP2/0细胞的融合率为61.46%(236/384),最终获得4株稳定分泌Cpn单抗的细胞株。用ELISA法检测小鼠腹水,效价高者可达1∶100000。免疫琼脂扩散试验鉴定为IgG类单抗,扩散效价达1∶128。自制单抗能与重组MOMP发生结合反应,表明其为抗CpnMOMP抗体。自制单抗与进口单抗类似,即与鹦鹉热衣原体(Cps)出现一定程度的交叉反应,而与沙眼衣原体(Ct)则无交叉反应。对240份PBMC标本用自制单抗和进口单抗同时检测Cpn抗原,2种单抗检测均阳性的共86份,经SPSS软件分析两者具有较好的一致性。DIF检测显示,心血管疾病和呼吸道疾病Cpn抗原阳性检出率分别为69.34%(95/137)和72.06%(49/68),与正常人标本Cpn抗原阳性率相比,均具有显著性差异。结论:获得IgG类抗CpnMOMP单抗,自制Cpn单抗的特异性和敏感性均与进口单抗具有较好的一致性。PBMCCpn抗原检测的统计分析证实,对于动脉粥样硬化等某些疾病的发生和发展,Cpn感染可能是重要的原因之一,但其中的因果关系还有待深入研究。     

消减免疫法在单克隆抗体制备中的应用

单克隆抗体是现代生命科学研究的重要工具。常规的杂交瘤技术通常并不能获得特定目的抗原的单克隆抗体,而消减免疫法可在一定程度上弥补这一缺陷。消减免疫利用特殊的免疫耐受途径来大幅增加目的单抗的产生数目。它建立在宿主动物对免疫显性抗原或非目的抗原（耐受原）耐受的基础上,宿主动物可通过高区带耐受、新生期耐受和药物介导的耐受等3种方法获得耐受,然后再对已耐受的动物接种目的抗原（免疫原）,特异性抗体便随之产生。近20年来,用消减免疫法成功地制备了多种独特抗体。简要阐述了可用消减免疫法获得的单克隆抗体的制备。     

Studies of a Murine Monoclonal Antibody Directed against DARC: Reappraisal of Its Specificity

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is ²²FEDVW²⁶, with ²²F and ²⁶W being the most important residues. In addition, we demonstrated that ³⁰Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that ²⁵V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.     

单克隆抗体及其在节肢动物捕食作用研究中的应用

血清学方法是目前研究节肢动物捕食作用的较为理想的方法,特别是酶联免疫吸附试验(ＥＬＩＳＡ),由于其灵敏度高,特异性强,适于大规模检测田间样本,已被国外许多研究者采用[5]。但是所有这一切都依赖于高效价和高度特异性的抗血清,而传统的抗体制备方法常常不能满足这一要求。特别是早期使用的抗体,特异性甚至只在科的水平,不能满足准确确定猎物种类的要求[3]。而单克隆抗体不仅具有高度的特异性,而且其特异性水平可以根据需要在制备过程中进行筛选。近年来单克隆抗体在国外已开始应用于节肢动物捕食作用的研究,但在国内还未见报道。为此,我…     

Structure and Circular Dichroism of DNA in Concentrated Polymer Solutions

DNA driven into a compact state by excluded volume interactions undergoes a spontaneous rearrangement to an ordered tertiary structure characterized by a circular dichroism spectrum greatly differing from that of DNA in solution.     

P—选择素凝集素—EGF功能域的克隆、表达及其单克隆抗体制备

制备特异性抗人P 选择素的凝集素 表皮生长因子 (L EGF)功能域的单克隆抗体。利用特异引物 ,通过RT PCR从外周血血小板中扩增出人P 选择素的L EGF功能域基因 ,将其克隆至pET42b( )载体中 ,测序验证后转染大肠杆菌BL2 1,经诱导表达了C端融合 6×His的蛋白质。融合蛋白质经分离纯化后 ,免疫Balb/c小鼠 ,应用杂交瘤技术 ,通过间接ELISA筛选阳性克隆。获得 3株可稳定分泌抗L EGF功能域单抗的杂交瘤细胞株 (B10、F3和H5 )。其亚型分别为IgG2 、IgG1和IgG3;轻链均为κ型。所获的单抗对LPS刺激活化的人脐静脉内皮细胞均有特异性结合反应 ,并可在体外阻断经凝血酶激活的血小板与中性粒细胞间的粘附。表明所获的单抗可特异性识别结合天然P 选择素 ,具有体外抗活化血小板与中性粒细胞粘附的功能 ,为进一步应用此单抗进行P 选择素结构和功能及抗粘附治疗研究提供了实验基础。     

河豚毒素中和性单抗的制备、鉴定及TTX检测方法的建立

目的研制河豚毒素中和性单抗,建立基于河豚毒素单抗的河豚毒素检测方法。方法用TTX-KLH免疫Balb/c小鼠,用TTX-BSA间接ELISA筛选,建立杂交瘤细胞系,腹腔接种Balb/c小鼠诱生腹水,Protein A Sepharose CL4B亲和柱纯化,SDS-PAGE、间接ELISA鉴定;用常规法确定TTX对昆明小鼠的LD50;将单抗和TTX混合物注入小鼠腹腔,检测单抗对TTX的中和能力;建立检测TTX的竞争ELISA法。结果获得了2株TTX中和性单抗,腹水用Protein A Sepharose CL 4B纯化后抗体纯度大于95%;常规间接ELISA检测,显示单抗5E7的结合能力高于5E4。单抗对2 LD50 TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。结论获得了TTX中和性单抗,对致死剂量TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。     

单克隆抗体在呼吸道合胞病毒感染诊断和防治中的应用

呼吸道合胞病毒 (RSV)是世界范围内婴幼儿下呼吸道感染的重要病原 ,目前尚无成熟疫苗应用于临床。展开对RSV被动免疫的研究显得尤为重要。单克隆抗体成为深入研究和防治RSV感染的有力武器。本文综述了单克隆抗体在RSV感染的诊断、预防和治疗中的应用     

A Fibrin-Specific Monoclonal Antibody from a Designed Phage Display Library Inhibits Clot Formation and Localizes to Tumors In Vivo

Fibrin formation from fibrinogen is a rare process in the healthy organism but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones) for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal amino acids of fibrin with high affinity (K_d = 44 nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, small immune protein and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors but did not exhibit any detectable staining toward normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.     

单克隆抗体技术在水生动物病毒研究中的应用

单克隆抗体技术作为病毒病检测与防治的有力武器,在人类、畜牧兽禽等的病毒病控制与预防中发挥出日益显著的作用.从二十世纪八十年代中期Chinchar等研制蛙虹彩病毒(Frog virus 3, FV3)单抗开始[1],这一技术及其研究成果也运用于水生动物病毒学研究中.尤其是近年,人们对水产品蛋白质不断增长的需求,使水产养殖业呈现突飞猛进的发展势头,但普遍流行的水生动物病毒病已造成巨大经济损失.滥用药物不仅防治效果差,而且严重污染水体及生态系统.利用生物技术和环保方式对水生动物病毒病进行防治,就成为人们关注的热点.因此,单抗技术在水生动物病毒病研究中得到越来越广泛的运用,并有新的进展.本文就此作一综述.     

A Study of DNA Melting in Concentrated Water-Alcohol Solutions

Abstract

The DNA melting profiles with high resolution have been studied for conditions corresponding to the B and A conformations of DNA in water-alcohol solutions. The melting profiles of the A-form and B-form DNA, their mean melting temperatures and melting range width were found to differ. DNA was shown to be heterogeneous in respect of the B-A transition, the GC-rich regions more readily converting into the A form than AT-rich ones. The presence of boundaries between the A and B sections within the transition zone did not smooth off the fine structure of melting profiles.