用硫氰酸盐洗脱法直接测定噬菌体抗体的相对亲和力

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

The Second Editorial Board of Chinese Journal of Biotechnology

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

一种定性比较抗体相对亲和力的酶联免疫吸附测定方法

目的:建立一种简便的对抗体相对亲和力进行定性比较的酶联免疫吸附测定(ELISA)方法,以便快速、简便地从大量抗体突变体中挑选高亲和力突变体。方法:将待测抗体倍比稀释后用直接ELISA方法进行定量,同时用相同浓度抗体作为一抗与抗原进行间接ELISA反应,以前者吸光度值为横轴、后者吸光度值为纵轴绘制散点图,通过拟和后的曲线判断抗体亲和力高低,并通过BIA-core法对该方法的准确性进行验证。结果:通过该方法获得的抗体亲和力高低情况与经测定抗体亲和力得出的结果一致。结论:该ELISA方法是一种简便可行、准确有效的抗体亲和力定性比较方法,可以应用于不同抗体的亲和力成熟比较研究。     

血管内皮生长因子人单链抗体--基因克隆、高效表达、亲和力成熟及生物活性鉴定

利用抗体噬菌体展示技术, 克隆了血管内皮生长因子人基因工程单链抗体, 抗体表达量达菌体总蛋白的45%. 用层析柱复性技术同步完成抗体的纯化和复性. 生物活性实验表明, 该抗体不仅特异结合人VEGF165, 而且竞争抑制VEGF165与其受体的结合. 为了提高单链抗体的亲和力, 采用错配PCR法, 随机突变抗体重链可变区基因, 建立次级抗体突变库, 并从中筛选出高亲和力突变株. 研究了抗体突变株蛋白质三维结构特点及其与亲和力的关系, 这些结果不仅为肿瘤血管靶向治疗奠定了基础, 而且为抗体的高效表达及其亲和力体外成熟提供了参考模型.     

具有高亲和力和稳定性的人源性抗PD-L1二硫键稳定Diabody的制备

目的：对天然噬菌体抗体库进行筛选并对抗体进行体外亲和力成熟,获得高亲和力人源性抗PD-L1抗体,然后对该抗体进行二硫键稳定改造,获得具有高亲和力和稳定性的人源性抗PD-L1的二硫键稳定Diabody。方法：首先以PD-L1重组蛋白为抗原在天然噬菌体Fab抗体库中筛选Fab抗体,其次分析结合能力较好的抗PD-L1的Fab抗体可变区基因中的热点,通过对轻链、重链CDR3区的7处热点随机突变构建噬菌体抗体突变库,从中筛选出亲和力得到提高的抗体。最后在抗体骨架区引入两个二硫键,构建二硫键稳定的抗PD-L1的ds-Diabody,并在毕赤酵母GS115中进行表达。结果：该方法筛选获得了6株特异性抗PD-L1噬菌体Fab抗体,对结合能力较好的其中一株抗体CDR3区的热点进行随机突变,成功构建库容为1.14×10⁸ CFU/mL的噬菌体抗体突变库,并从中筛选出亲和力提高约6倍的噬菌体抗体突变株。对该抗体骨架区进行二硫键引入,成功构建与表达二硫键稳定的ds-Diabody。结论：构建的ds-Diabody比Fab抗体与PD-L1结合亲和力高、稳定性好,为药物开发、肿瘤治疗等研究P...     

噬菌体展示抗体库筛选技术研究进展

噬菌体展示技术已成为制备高亲和力抗体的强有力的工具。而噬菌体抗体库的筛选是在获得高亲和力抗体过程中很关键的一个环节。总结了针对不同复杂程度的抗原所须采用的不同筛选方法,并简单介绍了高通量筛选的优势。     

链替换技术提高噬菌体抗体亲和力的研究进展

高亲和力抗体基因的克隆是噬菌体抗体在基础研究、临床诊断治疗研究等重要领域中应用的基础。链替换技术已逐步广泛用于噬菌体抗体的性能改造。该技术不仅可以促进噬菌体的体外亲和力成熟,提高抗体亲和力,有助于低剂量抗体达到实际应用所需要的抗原结合效应,拓宽抗体的应用开发前景,而且可应用于减少抗体人抗鼠抗体反应,分析重链或轻链基因对抗原抗体结合的影响,有助于较好的理解免疫化学。就链替换的原理、策略及其应用进行综述。     

抗P-选择蛋白人源性单克隆抗体的制备

目的:获得人源性抗P-选择蛋白(selectin)特异性抗体,为相关疾病治疗奠定基础。方法:在HEK293细胞中真核分泌表达人P-选择蛋白功能性片段,以此蛋白片段作为抗原,利用本室构建的大容量全合成人源性噬菌体抗体库进行筛选,经过3轮固相筛选后,阳性克隆得到富集;将其中富集效果最好的一株单链抗体A1改造成全抗体(IgG1),重组质粒转染H293细胞后,抗体得到表达;表达后在全抗体水平上用ELISA和Western印迹分别验证了A1抗体的特异性,并通过非竞争ELISA方法初步测定这株抗体的亲和力。结果:3轮筛选得到3株特异性噬菌体抗体,其中富集效果最好的单链抗体A1改造成全抗体形式后特异性良好,抗体亲和力Kd=2×10-8 mol/L。结论:筛选得到一株特异性较好的抗P-选择蛋白人源性单克隆抗体A1,其特异性和亲和力较好,有继续开发的价值。     

抗GM-CSF纳米抗体的筛选与鉴定

目的:制备抗粒细胞-巨噬细胞集落刺激因子(GM-CSF)纳米抗体,并测定其亲和力。方法:分离提取GM-CSF免疫后羊驼外周血淋巴细胞总RNA,PCR扩增得到纳米抗体基因片段,与载体pHEN1重组后克隆至大肠杆菌TG1以构建初始文库,经拯救构建得到噬菌体展示纳米抗体文库,并对其进行生物淘选;通过大肠杆菌BL21(DE3)原核表达阳性纳米抗体克隆,并测定其亲和力。结果:构建了多样性良好且库容量为1.37×10~9cfu的纳米抗体初始文库,3轮淘选后共筛选得到5株氨基酸序列差异性较大的纳米抗体,并对其中一株纳米抗体G1进行表达纯化,SDS-PAGE分析表明纳米抗体G1纯度较高,且有较高的亲和力(K_D=2.95×10~(-8)mol/L)。结论:制备了高亲和力的抗GM-CSF纳米抗体,可应用于相关炎症抗体药物研制和疾病监测等方面。     

人源抗转铁蛋白受体单链抗体的筛选及其构建与表达

目的:在人源单链抗体库中筛选抗人转铁蛋白受体(TfR)单链抗体(scFv)。方法:人源展示型抗体库质粒转染293T细胞,通过第一轮流式细胞分选得到比例为0.2%的阳性细胞,提取质粒并扩增,得到的质粒转染293T细胞作为下一轮筛选所需抗体库;第二轮分选时降低抗原浓度,最后得到2个候选的scFv序列。经序列分析,选择1号单链抗体基因,利用基因工程技术构建分泌型表达质粒,转染293E细胞并通过镍亲和层析纯化获得单链抗体蛋白,经Forte Bio Octet进一步测定单链抗体蛋白的亲和力。结果:经过2轮筛选获得了全新的抗TfR单链抗体序列,构建并表达了该单链抗体蛋白,该单链抗体与TfR的亲和力常数为5.57×10-7。结论:从展示型人源scFv抗体库中获得了全新的抗TfR单链抗体,该单链抗体与TfR具有较好的亲和力。     

Design of humanized antibodies: from anti-Tac to Zenapax

Since the introduction of hybridoma technology, monoclonal antibodies have become one of the most important tools in the biosciences, finding diverse applications including their use in the therapy of human disease. Initial attempts to use monoclonal antibodies as therapeutics were hampered, however, by the potent immunogenicity of mouse (and other rodent) antibodies in humans. Humanization technology has made it possible to remove the immunogenicity associated with the use of rodent antibodies, or at least to reduce it to an acceptable level for clinical use in humans, thus facilitating the application of monoclonal antibodies to the treatment of human disease. To date, nine humanized monoclonal antibodies have been approved for use as human therapeutics in the United States. In this paper, we describe procedures for antibody humanization with an emphasis on strategies for designing humanized antibodies with the aid of computer-guided modeling of antibody variable domains, using as an example the humanized anti-CD25 monoclonal antibody, Zenapax.     

The role of monoclonal antibodies and the recombinant DNA technology in studying autoantibody production

The hybridoma technology has made it possible to sample the B-cell repertoire and to generate monoclonal antibodies which can be analyzed for their specificity and idiotypy. Using the recombinant DNA technology, the structure of the genes which encode those antibodies can be analyzed. The knowledge gained from the application of these techniques has made it possible to pose specific questions about the origins of autoantibodies.     

噬菌体抗体库研究进展

治疗性抗体的发展经历了异源抗体、人源化抗体和人源性抗体几个阶段。目前,人源性抗体是治疗性抗体发展的主要方向,而噬菌体抗体库技术的出现为人源性抗体的制备提供了良好的技术平台,并且逐渐成为目前获得人源性抗体的主要手段之一。噬菌体抗体库技术是20世纪90年代初期抗体工程领域的一项重大进展,它是噬菌体展示和抗体库2种技术的集成,目前广泛应用于生物医学领域。本文对该技术的原理、类型、特点及研究进展进行了综述。     

Present status and future prospects for the hybridoma technology

Summary A somatic cell genetic technique has recently been developed that makes it possible to obtain very large amounts of homogeneous antibodies and to replenish the supply of the exact same antibodies whenever they are needed. This hybridoma technology has already contributed to major scientific advances and will surely improve the diagnosis and treatment of many diseases. Because the technology itself is relatively simple and inexpensive, it has captured the attention of basic scientists, clinicians, and industrial managers and investors. This work was supported by National Institutes of Health Grants AI 10702 and AI 5231, National Science Foundation Grant PCM77-25635, and American Cancer Society Grant NP-317.     

噬菌体抗体库构建技术在疾病防治中的应用

噬菌体抗体库技术(phage antibody library)是近年发展的一项分子生物学新技术,近年来越来越多的学者利用这项技术获得目标抗体以应用于医疗制药及安全检测等多个领域.噬菌体抗体库技术的发展和应用,为抗体技术领域带来了巨大的变化,极大地推动了各种性能优良基因工程抗体的开发和应用.针对噬菌体抗体库构建的原理、方法、构建过程中所需考虑的因素以及在不同领域的应用作了综述.     

A review: Cloning of human antibodies by phage display

Antibodies play a pivotal role in human health and disease. The application of phage display technology represents another milestone in the attempt to gain a better understanding of human antibodies. Immunoglobulin phage display permits human monoclonal antibodies for the first time to be readily available for analysis and for therapeutic use. Recent developments in molecular biology, in particular the polymerase chain reaction, have made it possible to amplify, clone, and express human antibody fragments in prokaryotic organisms. Phagemid display vectors have a distinct advantage over conventional cell culture technology used to immortalize human antibodies, in that one may quickly survey huge immunoglobulin repertoires for an antibody of desired specificity. Dual expression of immunoglobulin variable region light and heavy chain fragments permits combinatorial shuffling and thus an increase in diversity.The development of sophisticated computer algorithms, such as LINUS,⁵⁷ that can predict the three-dimensional structure of proteins from DNA sequences will have an enormous influence on the characterization and design of human antibodies. Future advances in computer software will be needed to aid in the identification of unique antibody sequence motifs expressed during disease and in the design of antibodies with defined functional epitopes.     

Generation of recombinant antibodies and means for increasing their affinity

Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.     

Human antibodies from transgenic animals

Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies (mAbs). However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity mAbs for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences. This technology has now been exploited by over a dozen different pharmaceutical and biotechnology companies toward developing new therapeutic mAbs, and currently at least 33 different drugs in clinical testing--including several in pivotal trials--contain variable regions encoded by human sequences from transgenic mice. The emerging data from these trials provide an early glimpse of the safety and efficacy issues for these molecules. Nevertheless, actual product approval, the biggest challenge so far, is required to fully validate this technology as a drug discovery tool. In the future, it may be possible to extend this technology beyond rodents and use transgenic farm animals to directly generate and produce human sequence polyclonal sera.     

Improved continuous-flow print head for micro-array deposition

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip™ protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.     

Protein microarrays for antibody profiling: specificity and affinity determination on a chip

Protein microarray technology facilitates the detection and quantification of hundreds of binding reactions in one reaction from a minute amount of sample. Proof-of-concept studies have shown that the set-up of sensitive assay systems based on protein arrays is possible, however, the lack of specific capture reagents limits their use. Therefore, the generation and characterisation of capture molecules is one of the key topics for the development of protein array based systems. Recombinant antibody technologies, such as HuCAL (human combinatorial antibody library; MorphoSys, Munich, Germany), allow the fast generation of highly specific binders to nearly any given target molecule. Although antibody libraries comprise billions of members, it is not the selection process, but the detailed characterisation of the pre-selected monoclonal antibodies that presents the bottleneck for the production of high numbers of specific binders. In order to obtain detailed information on the properties of such antibodies, a microarray-based method has been developed. We show that it is possible to define the specificity of recombinant Fab fragments by protein and peptide microarrays and that antibodies can be classified by binding patterns. Since the assay uses a miniaturised system for the detection of antibody-antigen interactions, the observed binding occurs under ambient analyte conditions as defined by Ekins (J. Pharm. Biomed. Anal. 1989, 7, 155-168). This allows the determination of a relative affinity value for each binding event, and a ranking according to affinity is possible. The new microarray based approach has an extraordinary potential to speed up the screening process for the generation of recombinant antibodies with pre-defined selection criteria, since it is intrinsically a high-throughput technology.