从人源性噬菌体抗体库中筛选人抗HBsAg的Fab噬菌体抗体

目的从人源性噬菌体抗体库中筛选人抗HBsAgFab噬菌体抗体。方法用固相化HBsAg对所构建的噬菌体抗体库进行三轮淘筛,从中筛选人抗HBsAgFab噬菌体抗体。结果第三轮淘筛后洗脱下来的噬菌体,较第一轮增加80倍,含有抗体轻链基因和重链基因的克隆,也由淘筛前的17%增至100%,经ELISA证实,筛选到的抗HBsAg噬菌体抗体对HBsAg具有良好的结合活性,而与牛血清白蛋白和HAVAg等无关抗原均不反应。结论HBsAg对抗体库的淘筛,富集了表面呈现抗HBsAg的单克隆抗体的噬菌体。     

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.     

噬菌体抗体库的构建及人源抗内毒素类脂A抗体的筛选

目的寻找一种治疗内毒素血症及其并发症较为有效的途径.方法从感染革兰阴性杆菌J7患者体内含有内毒素类脂A抗体的淋巴细胞中提取mRNA,经反转录再从IgG重链Fd两端及轻链通用引物分别扩增Fab基因片段,依次插入到噬菌体载体pCOMR3中,电穿孔转入大肠杆菌XL-blue,经辅助病毒VCSM13感染,重组噬菌体溶源裂解.结果Fab抗体表达于噬菌体CPⅢ的N端,噬菌体抗体库的容量为4.8×106,筛选出抗内毒素类脂A抗体,经LPS蛋白对噬菌体抗体库进行3轮淘选,使抗内毒素类脂A的特异性抗体得到100倍的富集.结论通过直接和竞争ELISA实验筛选出3株结合活性好的特异性抗体,为下一步应用研究奠定了基础.     

肝细胞癌人源抗c-Met抗体Fab的筛选及鉴定

目的 从大容量人源Fab抗体库中筛选全人源抗c-Met抗体,并对抗体与肝癌细胞的结合活性进行初步鉴定.方法 利用Met-Fc融合蛋白对大容量人源Fab抗体库进行固相筛选,经过5轮固相筛选,随机挑选30个克隆经酶联免疫吸附法差减鉴定,阳性克隆进行可溶性表达,用c-Met表达阳性的人肝癌细胞株鉴定抗c-Met抗体Fab的结合活性.结果 Western blot、免疫荧光结果显示,c-Met分子表达于SMMC721、BEL7402人肝癌细胞膜上;从大容量人源Fab抗体库中筛选出1株抗c-Met抗体Fab(AM2-26),经免疫共沉淀、荧光激活细胞分类术、免疫荧光分析,结果显示AM2-26与人肝癌细胞表面c-Met分子有较好的结合活性.结论 从人源Fab抗体库中筛选的AM2-26能够与肝癌细胞表面c-Met分子特异性结合,为研制用于肝癌生物治疗的靶向药物,提供了候选分子.     

Study of a humanized inhibitory anti-platelet glycoprotein VI phage antibody from a phage antibody library

Objective: The aims of the study were to study the effect of anti-platelet glycoprotein (GP) VI auto-antibodies on platelet aggregation and use phage surface display technology to produce anti-platelet GPVI phage antibody fragment, which may be developed to inhibit platelet aggregation in the treatment of cardiovascular disease.

Methods: Plasma samples from patients with immune thrombocytopenia (ITP) were screened by monoclonal antibody immobilization of the platelet antigen assay and the platelet aggregation test for anti-platelet GPVI auto-antibody with an inhibitory effect. The humanized anti-platelet GPVI phage antibody was produced by phage surface display technology. The function of the phage antibody fragment against platelet aggregation was examined by the platelet aggregation test.

Results: Of 726 ITP patients, 2 (0.27%) patients’ plasma significantly inhibited platelet aggregation induced by collagen-1. After five rounds of selection, enrichment, and purification, a soluble phage antibody fragment was produced, which can inhibit platelet aggregation induced by collagen-1. The results demonstrate that only a few of the screened anti-platelet GPVI auto-antibodies showed an inhibitory effect on platelet aggregation.

Discussion: A completely humanized anti-GPVI soluble phage antibody can be produced by phage surface display technology. The antibody was able to specifically block collagen-induced platelet aggregation without influencing the aggregation responses to other agonists.

Conclusions: Results of the present study suggest that very few anti-platelet GPVI auto-antibodies inhibit the aggregation function of platelet. The humanized anti-platelet GPVI produced by phage surface display technology is promising to be used to inhibit platelet aggregation in the treatment of cardiovascular disease.     

Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.     

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

The enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P <.001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P <.005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.     

Characterization of a monoclonal anti-idiotype antibody to human anti-factor VIII antibodies.

Approximately 15% of individuals with hemophilia A develop antibodies (inhibitors) to therapeutically infused factor VIII that interfere with F.VIII coagulant activity. By using isoelectric focusing and immunospecific detection of anti-factor VIII antibodies, inhibitor plasma showed varied patterns of reactivity characteristic of a polyclonal response. Inhibitor plasma from patient Bt was observed to have an isolated banding pattern, or spectrotype, at pI 8.4 (SP8.4) distinct from his remaining anti-factor VIII antibodies. SP8.4 antibodies from this patient were partially purified and used to prepare monoclonal anti-idiotype antibodies. Monoclonal antibody Mab20-2H was found to detect a spectrotype in isoelectric-focused Bt plasma identical to SP8.4 and to bind anti-factor VIII antibodies. Furthermore, Mab20-2H binding could inhibit the binding of these anti-factor VIII antibodies to antigen, indicating that Mab20-2H recognizes an idiotope associated with antigen binding. Mab20-2H was also found to recognize antibodies from another inhibitor patient. This and other anti-idiotype reagents will be useful for defining genetic factors involved in the human immune response to factor VIII and in designing approaches to prevent or ameliorate this response.     

Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library

BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens. We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens. MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen. Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells. Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA). RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated. CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens.     

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10⁹ members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.     

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.     

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM:To construct the natural immune Fab antibody phagedisplay libraries of colorectal cancer and to select antibodiesrelated with colorectal cancer.METHODS:Extract total RNA from tissue of local cancermetastasis lymph nodes of petients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and lightchain k and the amplification products were insertedsuccessively into the vector pComb3 to construct the humanlibraries of Fab antibodies.They were then panned by phagedisplay technology.By means of Dot immunoblotting andEUSA,the libradse were identified and the Fab phageantibodies binding with antigens of colorectal cancer wereselected.RWSULTS:The amplified fragments of Fd and k gained byRT-PCR were about 650bp.Fd and k PCR products weresubsequently inserted into the vector pComb3,resulting in arecombination rate of 40% and the volume of Fab phagedisplay library reached 1.48×10~6.The libraries wereenriched about 120-fold by 3 cycles of adsorption-elution-multiplication(penning).Dot immunoblotting showed Fabexpressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activitieswith antigens of colomctal cancer.CONCLUSION:The nstuml immune Fab antibody phagedisplay libraries of colorectal cancer were constructed.Theycould he used to select the relstive antibodies of colorectalcancer.     

人源性大肠癌自然致敏噬菌体抗体Fab呈现库的构建与筛选

目的构建大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,初步筛选相关抗大肠癌抗体。方法取大肠癌病人转移淋巴结,提取淋巴结总 RNA,逆转录 PCR 扩增重链 Fd 和 k 轻链 cDNA。依次将 PCR 产物插入载体 pComb3的相应部位,构建人源性大肠癌噬菌体 Fab 基因库,并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。结果所选2种 Ig 亚类的重链 Fd 片段、2种 k 轻链 cDNA 得到扩增。Fd 片段和 k 轻链均插入 pComb3的重组率为40%,Fab 噬菌体表达库容达1.48×10~6。经3轮淘选,抗体库得到约120倍的富集。3轮抗体库的点印记免疫染色均显示有 Fab 表达:酶联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,利用噬菌体抗体库技术,可筛选相关抗大肠癌抗体。     

噬菌体抗体库技术及其研究进展

抗体工程经历过多克隆血清 ,单克隆抗体阶段 ,现发展到了基因工程抗体时代。目前 ,抗体工程领域最突出的进展就是噬菌体抗体库技术的建立。噬菌体抗体库技术实际上是丝状噬菌体展示技术 (phagedisplaytechnology)与抗体组合文库技术 (combinatorialimmunoglobulinlibrarytechnology)相结合而成 ,该技术的出现开创了一条简便快捷的基因工程抗体生产路线 ,为人源抗体的制备提供了新途径 ,是抗体工程史的里程碑〔1,2〕,现将噬菌体抗体库技术及其研究进展综述如下。1　噬菌体抗体库技术1 1　噬菌体抗体库技术的操作路线〔3〕　噬菌体抗体库技术…     

丙型肝炎病毒非结构蛋白NS3抗原模拟表位的筛选和鉴定

目的 筛选丙型肝炎病毒(HCV)非结构蛋白NS3(HCV NS3)特异性噬菌体模拟表位，为抗—HCV的疫苗研究探索新途径。方法 以抗—HCV NS3的单克隆抗体作为固相筛选分子，对人工合成的噬菌体随机七肽库进行3轮“吸附—洗脱—扩增”的筛选过程，随机挑取60个克隆，经噬菌体酶联免疫吸附法(ELISA)鉴定并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV NS3抗原的模拟表位。结果 经噬菌体富集后，从随机筛选的60个克隆中得到13个阳性克隆，确定氨基酸序列SRNTxKL为HCV NS3的模拟表位。结论 用噬菌体七肽库成功筛选得到HCV NS3的模拟表位。     

丙型肝炎病毒非结构蛋白NS5抗原模拟表位的筛选和鉴定

目的 筛选丙型肝炎病毒非结构蛋白NS5（HCV NS5）特异性噬菌体模拟表位，为抗HCV的疫苗研究探索新途径。方法 以抗－HCV NS5的单克隆抗体作为固相筛选分子，对人工合成的噬菌体随机七肽库进行5轮“吸附－洗脱－扩增”的筛选过程，随机挑取30个克隆，经噬菌体酶联免疫吸附法（ELISA）鉴定并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV NS5抗原的模拟表位。结果 经噬菌体富集后，从随机筛选的30个克隆中得12上阳性克隆，确定氨基酸序列QIRPTRQ为HCV NS5的模拟表位。结论 用噬菌体七肽库成功筛选得到HCV NS5的模拟表位，为用HCV模拟表位探索HCV感染的研究创造了条件。     

Rapid isolation of monoclonal antibodies specific for cell surface differentiation antigens.

Two immunization procedures were compared for their ability to yield monoclonal antibodies that react with plasma membrane-bound differentiation antigens of Dictyostelium. In the first method, hybridomas prepared from BALB/c mice immunized with aggregating amoebae produced monoclonal antibodies that recognized antigens present on both growing and aggregating Dictyostelium amoebae. None of the monoclonal antibodies reacted with only the injected aggregation-stage cell type. In contrast, monoclonal antibodies that reacted with differentiation antigens were easily obtained by primary immunization of BALB/c mice with living aggregation-stage cells, followed by secondary immunization with a preparation of plasma membrane from aggregating cells or intact aggregating cells mixed with polyclonal BALB/c antiserum raised against undifferentiated cells. By this method, approximately 20% of all anti-Dictyostelium monoclonal antibodies obtained in a fusion are specific for differentiation antigens. The properties and developmental regulation of several of these antigens are described. The possible uses of this immunological method to detect unique determinants on other kinds of cells and the likely immune mechanisms that make it successful are discussed.     

Isolation of a vascular cell wall-specific monoclonal antibody recognizing a cell polarity by using a phage display subtraction method

Using a strategy consisting of (i) the isolation of cell walls from synchronously differentiating cells of Zinnia, (ii) the generation of mAbs with an antibody phage display method, and (iii) screening with a subtraction method, we isolated mAbs recognizing vascular development-specific cell wall components without prior antigen identification. One of the isolated mAbs, designated CN 8, recognized a cell wall component contained in the hemicellulosic fraction. Immunohistochemical analyses showed that the CN 8 epitope was localized to the cell wall of immature tracheary elements and xylem parenchyma cells. In immature tracheary elements, the CN 8 epitope had a polarized localization pattern regardless of whether the cells are formed as parts of vessels in situ or as single tracheary elements in vitro, suggesting that cell polarity autonomously formed on the cell wall may function in tracheary element differentiation.     

大肠癌噬菌体抗体库的构建及初步鉴定

目的 构建及初步鉴定大肠癌噬菌体抗体Fab呈现库.方法 分离大肠癌患者外周血淋巴细胞,提取淋巴细胞总RNA,逆转录成cDNA.用相应引物进行PCR,扩增出轻链和重链Fd段基因,经酶切后分别和噬粒载体pComb3连接,再经电穿孔转化大肠杆菌XLl-Blue菌株.将轻链和重链Fd基因先后克隆人pComb3中.结果 从分离出的淋巴细胞中提取到高质量RNA,RT-PCR分别扩增出约680 bp大小的κ、λ和Fd基因.PCR 产物和载体经纯化、双酶切后进行连接转化,成功地构建了人源性Fab抗体基因库,库容量达2.1×107,轻链K、κ、λ和重链Fd基因均插入pComb3的重组率为50%.结论 构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库,为筛选大肠癌相关抗体打下基础.