蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果：混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果:混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

蛋白质芯片技术

以前对蛋白质的研究集中在一次研究一种蛋白质 ,通常费时费力 ;而蛋白质芯片技术是研究蛋白质组的新技术 ,是高通量、微型化和自动化的蛋白质分析技术。它可以用来研究蛋白质的亚细胞定位和蛋白质与蛋白质之间的相互作用 ,以及对蛋白质的功能进行生物化学分析 ,将对蛋白质组研究及医学生物学的发展有很大的推动作用。较系统地介绍了蛋白质芯片的概念、制作及检测方法 ;同时也讨论了蛋白质芯片的两种功能形式、存在问题和应用前景。     

蛋白质芯片技术进展

人类基因组测序工作的完成 ,引起人们对蛋白质组研究的热忱。蛋白质作为生命活动的执行者 ,种类繁多 ,结构复杂 ,并且其活性与空间结构密切相关 ,需要更为先进的技术去研究和探索。近来出现的蛋白质芯片以并行、高通量检测、分析和处理蛋白质样品 ,发展迅速 ,应用前景广泛。介绍蛋白质芯片的种类、蛋白质固定的表面化学以及不同的检测方法 ,简述蛋白质芯片在不同领域的应用 ,并讨论蛋白质芯片目前存在的问题。     

蛋白质芯片技术研究进展

蛋白质芯片技术是近年发展起来的一项新技术,这项技术是将蛋白质的分析微缩到小型芯片上进行,利用荧光或酶显色进行探测,最后用特 计算机软件加以分析。该技术在基因表达、抗原抗体检测、药物开发、疾病诊断等研究方面显示出快速、高效、高通量处理信息的能力。     

蛋白质芯片

随着人类基因组计划 (ＨＧＰ)顺利实施 ,以生命活动的执行者———蛋白质为研究对象的蛋白质组学越来越显得重要[1] ,并构想和发展了快捷、高效、并行、高通量的蛋白质组检测新技术———蛋白质芯片技术。1.蛋白质芯片的基本构成蛋白质芯片是高通量、微型化和自动化的蛋白质分析技术。目前蛋白质芯片主要分两种[2 ] :一种类似于ＤＮＡ芯片 ,即在固相支持物表面高密度排列的探针蛋白点阵 ,可特异地捕获样品中的靶蛋白 ,然后通过检测器对靶蛋白进行定性或定量分析 (如图1)。另一种就是微型化的凝胶电泳板。在电场作用下 ,样品中的蛋白质通过芯…     

蛋白质芯片技术研究进展

蛋白质芯片是继基因芯片后的又一种用于生命科学研究的技术平台。目前这一技术已经被广泛应用到生命科学研究的多个领域,如蛋白质组学研究,新药的开发以及疾病的临床诊断等。就蛋白质芯片的基本原理、研究进展及应用状况做一介绍。     

蛋白质芯片构建技术进展

蛋白质芯片是一项实现蛋白质高通量分析和检测的新型技术,在生命科学的各个领域具有重要的应用前景。但是由于蛋白质本身的敏感性质和芯片的独特结构,蛋白质芯片技术的发展面临许多难点,这些正是蛋白质芯片研究者需要共同讨论的问题。本文从芯片构造、探针的固定方法和检测方法三个方面对芯片的构造进行分析和讨论,并对其相关技术的进展进行综述。     

蛋白质芯片及其应用

人类基因组计划的实施和大量珍贵的功能基因组数据的获得,使得蛋白质的研究显得越发重要。蛋白质芯片技术的出现为我们提供了一种较传统的凝胶电泳技术更为方便和快速的研究方法。     

蛋白质点阵/芯片技术的新进展

蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具.     

Monoclonal antibody higher order structure analysis by high throughput protein conformational array

The elucidation of antibody higher order structure (HOS) is critical in therapeutic antibody development. Since HOS determines the protein bioactivity and chemo-physical properties, this knowledge can help to ensure that the safety and efficacy attributes are not compromised. Protein conformational array (PCA) is a novel method for determining the HOS of monoclonal antibodies. Previously, we successfully utilized an enzyme-linked immunosorbent assay (ELISA)-based PCA along with other bioanalytical tools to elucidate the structures of antibody aggregates. In this study, applying a new multiplex-based PCA with 48-fold higher throughput than the ELISA-based one we revealed structural differences between different antibody molecules and antibody structure changes affected by various processing conditions. The PCA analysis of antibody molecules clearly demonstrated significant differences between IgG1 and IgG4 subclasses in epitope exposure and folding status. Furthermore, we applied small angle X-ray scattering to decipher mechanistic insights of PCA technology and validate structural information obtained using PCA. These findings enhance our fundamental understanding of mAbs' HOS in general. The PCA analysis of antibody samples from various processing conditions also revealed that antibody aggregation caused significantly higher exposure of antibody epitopes, which potentially led to a “foreign” molecule that could cause immunogenicity. The PCA data correlated well with protein stability results from traditional methods such as size-exclusion chromatography and protein thermal shift assay. Our study demonstrated that high throughput PCA is a suitable method for HOS analysis in the discovery and development of therapeutic antibodies.     

Generation and application of a monoclonal antibody that detects a wide variety of protein tyrosine kinases

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

鼠抗衣原体属抗原单克隆抗体的研制

本文利用杂交瘤技术,成功地建立了３株稳定分泌小鼠抗衣原体属脂多糖抗原单克隆抗体的杂交瘤细胞。实验结果表明,３株单抗均为ＩｇＭ类,特异性强,识别相似抗原位点,为今后的应用打下了基础。     

抗牙龈卟啉单胞菌血凝素2单克隆抗体的制备

目的制备抗牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-2,HA-2)的单克隆抗体(monoclonal antibody,mAb)。方法用重组HA-2(recombinant HA-2,rHA-2)免疫BALB/C小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,间接ELISA方法筛选杂交瘤细胞.用ELISA方法测效价。结果获得1株能够高效识别rHA-2的mAb,命名为4F11。此单克隆抗体的免疫球蛋白亚类为IgG1,效价达1?106。结论成功制备了重组牙龈卟啉单胞菌血凝素2的单克隆抗体mAb.将进一步用于牙龈卟啉单胞菌的诊断,并为牙周疾病的治疗研究奠定基础。     

An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus

Protein detection is a common yet time-intensive task in many laboratories. Here we report a protocol that makes use of cold microwave technology to reduce the total processing time to less than 1 h with dot and Western blot applications while yielding lower background noise at similar signal strength when compared with conventional protocols. With dot blots, the time savings was accompanied by a decrease in reagent use. With Western blots, the visibility of prestained markers was maintained, in stark contrast to conventional procedures. Experiments kept at a constant temperature of 21 degrees C support the existence of a microwave radiation effect, whereas an additional thermal effect is noted when the temperature is increased to 37 degrees C from ambient. Microwave-assisted dot blotting is suggested as an effective way of facilitating large-scale screening of expressed proteins.     

Interaction of a monoclonal antibody with the urease of Ureaplasma urealyticum

The monoclonal antibody (mAb) U.u. 5B2 against the urease of U. urealyticum (U.u) serotype 8, was affinity purified and was found to be an IgG 1 type, with an apparent dissociation constant of 2.9 × 10⁻¹⁰ M. Immunoblot analysis of the cytoplasmic proteins of U.u electrophoresed under non-denaturing conditions showed a reaction with a major and a minor band corresponding to the urease activity. The mAb, U.u.5B2 inhibited the urease activity up to 93% and precipitated a protein from the cytoplasm with a molecular weight of 75 kDa, corresponding to the purified urease subunit. This mAb also reacted with six other U.u serotypes but not with Jack bean urease, other urease containing bacteria or genital mycoplasma.     

基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础,以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L,反应时间为60 min。当菌浓在104 107cells/mL范围时,检测值可信度较高,可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测,变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。     

Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella

Abstract Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG ( P < 0.01), and specific anti-flagellar antibodies were induced.     

Comparative protein profiling of serum and plasma using an antibody suspension bead array approach

In the pursuit towards a systematic analysis of human diseases, array‐based approaches within antibody proteomics offer high‐throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor‐specific rather than preparation‐dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL‐4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.