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。     

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.     

大肠癌转移淋巴结构建人源性抗大肠癌噬菌体Fab抗体库

目的 构建人源性抗大肠癌噬菌体Fab抗体库.方法 术中剥取老年大肠癌患者系膜内转移淋巴结,提取细胞总RNA,逆转录合成cDNA,进行PCR,克隆于pComb3 载体,再经电转化大肠杆菌XLI-Blue菌株,形成噬菌体抗体库,最后通过免疫印记法鉴定初级抗体库中噬菌体上有Fab段的表达.结果 从系膜内转移淋巴结中扩增出650 bp重链Fd和轻链基因产物,轻链基因产物插入测得转化菌数为4.7×10~6,鉴定插入率为70%;重链Fd基因产物测得转化菌数为3.5×10~6,插入率为60%;免疫印迹法鉴定成功建立噬菌体Fab抗体库,库容量达1.4×10~6.结论 筛选大肠癌特异性抗原和相应噬菌体抗体,对大肠癌发病机制的研究以及探讨早期诊断方法有重要意义.     

人源性促甲状腺激素受体抗体Fab段抗体库的构建

目的 通过噬菌体表面展示技术,构建人源性促甲状腺激素受体抗体(TRAb)Fab片段抗体库.方法 从甲状腺功能亢进症患者外周血单个核细胞抽提总RNA,PCR法扩增免疫球蛋白分子轻链K、λ基因及重链Fd基因.构建轻链文库及组合文库.一个随机的组合文库在噬菌体表面表达.结果 从外周血单个核细胞中抽提得到总RNA,并反转录获得cDNA文库.PCR法扩增了大小约为680 bp的轻链K、λ基因及重链Fd基因,并构建了库容为1.32×105基因抗体库(轻链库)和库容为2.28×105 Fab抗体库(组合文库).Fab组合文库转化到大肠杆菌XL1-Blue感受态细胞,在辅助噬菌体M13K07的辅助下,扩增得到噬菌体抗体库.结论 噬菌体表面展示技术可成功构建人源性TRAb Fab片段组合文库.     

人源抗EV71病毒中和表位SP70 的Fab抗体研制

目的构建人源抗EV71病毒Fab噬菌体抗体库,筛选具有中和活性的人源单克隆抗体,为EV71感染引起的手足口病临床诊断及治疗奠定基础。方法从成人志愿者抗凝血中分离外周淋巴细胞,提取总RNA并逆转录为cDNA。以cDNA为模板,用人源IgG Fab段基因引物扩增轻链及重链Fd区基因,分步克隆至pComb3载体,构建Fab噬菌体抗体库。以SP70为抗原对Fab噬菌体抗体库进行富集筛选,将获得的重组噬菌体感染XL1-Blue菌并诱导表达,用SP70对诱导表达的菌上清进行筛选。结果共获得3株轻、重链基因组合序列不同的克隆,且这3株Fab抗体可特异性结合SP70多肽。结论成功构建人源抗EV71病毒Fab噬菌体抗体库,并从中筛选出3株针对EV71病毒结构蛋白VP1上中和线性表位SP70的特异性人源Fab抗体,为研发新的手足口病诊断和治疗工具奠定基础。     

人天然Fab段噬菌体抗体库的构建

目的通过构建人天然F ab段噬菌体抗体库为恶性肿瘤的免疫治疗提供新方法。方法取4个健康献血员新鲜血液各200m l,分离淋巴细胞,提取总RNA,经逆转录合成cDNA,设计多对引物,以PCR扩增抗体轻链和重链Fd段cDNA。轻链PCR产物和质粒载体pCom b3经SacⅠ和X baⅠ酶切,由T 4 DNA连接酶连接,电穿孔转化感受态大肠杆菌XL 1-B lue,扩增后提取质粒,构建轻链库。轻链库和重链Fd段PCR产物经X hoⅠ和SpeⅠ酶切,同样方法连接转化XL 1-B lue,加入辅助噬菌体VCSM 13产生噬菌体抗体全库。酶切鉴定轻链库和全库有无插入片段。结果提取的淋巴细胞总RNA质量好,部分重链Fd片段、部分κ轻链和λ轻链cDNA得到扩增。F ab全库库容达5.4×106,酶切证实有插入片段。结论本研究构建的人天然F ab段噬菌体抗体库可为进一步筛选特异性抗体及从肿瘤组织中提取肿瘤抗原用于免疫治疗奠定基础。     

人源性抗日本血吸虫肌球蛋白Fab噬菌体抗体体外杀童虫作用

目的观察人源性抗日本血吸虫肌球蛋白Fab噬菌体抗体体外杀伤血吸虫童虫作用。方法利用日本血吸虫肌球蛋白(Sj myosin)对人源性抗日本血吸虫Fab噬菌体抗体库进行富集筛选,获得抗Sjmyosin的特异性噬菌体抗体,将该抗体和巨噬细胞或补体与日本血吸虫童虫共培养,测定体外杀伤日本血吸虫童虫作用。结果将抗Sj myosin噬菌体抗体和巨噬细胞及补体与血吸虫童虫共培养时,在培养后24h即出现杀伤作用(童虫死亡率为4.69%),48h童虫死亡率达84.06%,96h童虫死亡率达89.38%。结论人源性抗Sj myosin Fab噬菌体抗体具有体外杀伤日本血吸虫童虫活性。     

抗TNF-α蛋白Fab抗体的制备和鉴定

目的制备人源性抗肿瘤坏死因子（TNF）-α蛋白的可溶性Fab抗体。方法用固相化的TNF-α蛋白从人天然Fab噬菌体抗体库中筛选出表达抗TNF-α蛋白Fab抗体的阳性克隆,经酶切及连接反应构建可溶性表达噬菌粒并转化至大肠杆菌BL21（DE3）pLysS中,IPTG高效可溶性表达,SDS-PAGE及Western blot分析鉴定抗体表达情况,ELISA鉴定其抗原结合活性和特异性。结果筛选并表达了人源性抗TNF-α蛋白的Fab抗体,在非还原SDS-PAGE中形成相对分子质量47 kD的条带;Western blot分析证实表达的蛋白即Fab抗体;ELISA筛选出的2株抗体（TA-04、TA-13）具有良好的抗原特异性和抗原结合活性,与小牛血清白蛋白无交叉反应。结论成功制备并鉴定了人源性抗TNF-α蛋白的可溶性Fab抗体。     

结肠癌患者淋巴结构建抗结肠癌噬菌体Fab抗体库

目的　构建 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库。方法　取 2个结肠癌患者转移淋巴结 ,提取淋巴结总RNA ,逆转录PCR扩增重链Fd和κ轻链cDNA。依次将PCR产物插入载体 pComb3的相应部位 ,噬菌体VCSM13辅助感染。以点印迹检测噬菌体表面Fab的表达。 结果　所选 2种Ig亚类的重链Fd片段、2种κ轻链cDNA得到扩增。Fd片段和κ轻链均插入 pComb3的重组率为 40 %,Fab噬菌体表达库容量达 1.48× 10 6。噬菌体悬液的点印迹免疫染色显示有Fab表达。结论　构建了 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库 ,为筛选结肠癌相关抗体奠定了基础。     

抗-HBs的人源性噬菌体抗体库的构建

目的构建含抗－ＨＢｓ的人源性噬菌体抗体库。方法用ＲＴ－ＰＣＲ技术,从自然感染的抗－ＨＢｓ阳性的两名献血员的外周血淋巴细胞中,扩增出人抗体轻链Ｋ基因和重链Ｆｄ基因,将二基因先后克隆人ＰＣｏｍｂ３Ｈｓｓ载体中,转化大肠杆菌,再用辅助噬菌体感染。结果构建了库容量为１５×１０５的轻链基因抗体库,轻链基因插人率为５７％和库容量为５×１０５的人Ｆａｂ体库,轻、重链基因插人率为５０％。经辅助噬菌体感染,得到噬菌体滴度为５×１０１４ＣＦＵ／ｍｌ的人源性噬菌体抗体库。结论成功构建了合抗－ＨＢｓ的人源性噬菌体抗体库,为进一步筛选ＨＢｓＡｇ的人Ｆａｂ噬菌体抗体奠定了基础。     

输血前11606例患者HIV和HCV及TP抗体检测结果分析

目的为避免因输血传播疾病引起医疗纠纷和防止职业感染。方法采用酶联免疫吸附试验对2010-01～2011-03 11 606例就医患者进行输血前人类免疫缺陷病毒(HIV)抗体、丙型肝炎病毒(HCV)抗体、梅毒螺旋体(TP)抗体三项指标筛查。结果共检出阳性例数856例。结论患者输血前进行三项感染性指标检测,对诊断和预防医患交叉感染、减少医疗纠纷的发生具有重要意义。     

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.Human cytomegalovirus (HCMV) is an important pathogen in transplant patients (1–5), and its infection can lead to invasive end-organ diseases, such as pneumonitis and hepatitis, as well as vascular pathology contributing to graft failure (4, 6, 7). HCMV is also the most common cause of in utero viral infections in North America and Europe, affecting 0.5–2% of newborns annually (8–10). Congenital HCMV infection can lead to symptomatic diseases at birth and also cause developmental disabilities in children (10, 11). Maternal seropositivity before conception protects against congenital transmission (12, 13), and both maternal humoral and cellular immunity are likely to contribute to the protection (14–16). Antibodies in particular are important for preventing congenital infection, serving as the first line of defense against maternal infection. It may also play a role in preventing transmission to the fetus, supported by the results of a small, nonrandomized study in pregnant women with primary HCMV infection, in which the passive immunity of monthly infusions of HCMV hyperimmune human IgG (HCMV-HIG) (200 mg/kg maternal weight) was ∼60% effective in protecting against congenital HCMV infection (17, 18). These studies suggest that it is feasible to develop a vaccine for preventing congenital HCMV infection and its sequelae. However, despite the fact that the Institute of Medicine has identified development of an effective vaccine for prevention of congenital HCMV as a top priority since 1999 (19), progress toward this goal has only been incremental (8, 20, 21). One of the hurdles to the efforts is our limited understanding of component of natural immunity associated with protection against HCMV infection.HCMV is a large, complex virus, with a genome capable of encoding >150 proteins (22–26). Because of the strict species specificity, options of animal models for HCMV research are limited (27). Thus, the functions of most HCMV antigens in viral infection in vivo and their roles as targets for host immunity are poorly understood. Furthermore, culture systems of single cell types have limitations for studying HCMV pathogenesis. Immunohistochemistry studies showed that HCMV can infect varieties of cells in vivo, including endothelial, epithelial cells, fibroblasts, and leukocytes (28–36). Many HCMV end-organ diseases, such as pneumonitis and gastroenteritis, are due to infection of the epithelial/endothelial cells in the affected organ (35–39). However, common laboratory strains, such as AD169 and Towne, were culture-adapted in fibroblast cells, with genomic mutations (22, 24, 40) and, more importantly, have lost their tropism to endothelial and epithelial cells, in contrast to pathogenic clinical isolates (32, 33, 41, 42).Loss of viral tropism to endothelial and epithelial cells was mapped to various mutations in the viral UL131-128 locus, and these mutations abrogated the expression of the pentameric glycoprotein H (gH) complex, composed of gH, gL, UL128, UL130, and UL131 proteins, a determinant for viral tropism to endothelial and epithelial cells (42–44). Because the pentameric gH complex is missing in common laboratory strains (42, 43), its importance in viral tropism, viral pathogenesis, and vaccine design was not fully appreciated until recently (42, 45). With this understanding, it is not surprising that Towne virus and recombinant glycoprotein B (gB) vaccines, although with ∼50% efficacy against primary infection in the clinic (46–49), induced poor neutralizing titers against viral infection of epithelial cells, in contrast to immune sera from HCMV-seropositive donors (50, 51). Thus, missing the pentameric gH complex is likely a deficiency in antigen composition for both vaccines (50). Studies of monoclonal antibodies (mAbs) isolated from HCMV-seropositive donors or polyclonal IgG enriched for antigen specificity supported the hypothesis that the pentameric gH complex, not gB, appears to be important for neutralizing activity in human subjects with natural infection (52).We recently described an experimental vaccine virus in which expression of the pentameric gH complex was restored (53). Unlike the parental AD169 virus and the recombinant gB vaccine, this virus can elicit high levels of neutralizing antibodies in rabbits and rhesus macaques (53). To support clinical development of this vaccine centered its concept on the pentameric gH complex, we established a comprehensive panel of 45 mAbs from a single rabbit that received vaccination. Of the 45 mAbs, 25 had neutralizing activity against viral entry in epithelial cells, including 11 elite neutralizers with ≥10-fold greater potency than HCMV-HIG. Biochemical analysis demonstrated that all elite neutralizers preferentially bound to the virus expressing the pentameric gH complex, and the majority of elite neutralizers (8 of 11) specifically recognized a recombinant form of the pentameric gH complex. Interestingly, binding affinity for intact virions was not correlated with neutralizing activity. Moreover, genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. In addition, neutralizing antibodies had longer complementarity-determining region 3 (CDR3) for both heavy and light chains than those of antibodies with no neutralizing activity. These data establish the importance of the pentameric gH complex as the primary target for potent neutralizing antibodies by vaccination, and support development of an experimental HCMV vaccine featuring the pentameric gH complex.     

Anti-endothelial cell antibodies to the endothelial hybridoma cell line (EAhy926) in systemic lupus erythematosus patients with antiphospholipid antibodies

The endothelial hybridoma (EAhy926) cell line was employed to clarify whether antiphospholipid antibodies (aPA) [lupus anticoagulant (LA), antiprothrombin antibody (aPT) and/or anticardiolipin antibody (aCL)] and anti-endothelial cell antibodies (AECA) are identical, and establish whether β₂-glycoprotein I (β₂-GPI) is needed for reactivity of aPA to endothelial cells. Ig-G AECA was positive in 9/30 SLE patients with aPA (30.0%) and 10/22 SLE patients negative for aPA (45.5%). Ig-M AECA was positive in one SLE patient with aPA and one SLE patient without aPA. AECA-positivity was not significantly different among unfixed, TNF-stimulated and fixed EAhy926. The influence of β₂-GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin-liposome/β₂-GPI or phosphatidylserine-liposome/prothrombin gave little evidence of cross-reactivity of aPA and AECA. The results of our study suggest that aPA and AECA may have partially cross-reacted, but were different antibodies. However, further study is needed to clarify the clinico-pathological significance of AECA.     

Determination of antiplatelet antibody activity in sera cytotoxic for human lymphocytes

Pooled serum aliquots obtained from sensitized potential renal allograft recipients on chronic hemodialysis were evaluated for their lymphocytotoxicity titers against the lymphocytes and then for alloantibodies against the platelets of 7 random donors by 5 methods. Platelet donor specific lymphocytotoxicity was present in 93% of 42 combinations. Of the positive combinations, 57% had a positive test for antiplatelet activity by the 14C serotonin release assay, 16% by the platelet aggregation method, and 19% as judged by acid phosphatase availability on the platelet membrane. No serum tested released beta-glucoronidase or lactic dehydrogenase. No correlation of the height of the titer of antiplatelet activity with that for lymphocytoxicity was detected. Thus, even in sera demonstrating significant activity against donor lymphocyte antigens, detection of associated platelet antibody activity is not uniform. Thus, a positive lymphocytoxic titer does not necessarily predict detectable antiplatelet activity. Therefore, additional tests for detection of antiplatelet activity should also be considered. This study shows that of the tests evaluated, the 14C serotonin release assay is the most sensitive for detection of antiplatelet antibodies.     

单克隆抗独特型抗体NP30用于日本血吸虫病血清学诊断的研究

本研究用单克隆抗独特型抗体NP30与日本血吸虫肠相关抗原(GAA)和可溶性虫卵抗原(SEA)检测了702份不同病期及正常人群中的血清抗体,结果显示,在急性感染时,NP30抗体的检出率为98％,与GAA(94％)和SEA(98％)的无差别。在慢性感染时NP30抗体的检出率为87％,与GAA(86％)的无差别,但低于SEA(98％)的。在正常人群中,上述3种的抗体假阳性率均为3％左右,无差别。NP30的抗体滴度几何均数在急性血吸虫感染时高于GAA的,低于SEA的,在慢性感染时低于后两者,提示NP30的抗体在血吸虫感染期间出现比较早,消退较快。上述结果提示,NP30可以替代虫源性抗原,用于日本血吸虫病诊断。     

From the Cover: Construction of a rationally designed antibody platform for sequencing-assisted selection

Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ∼10¹²-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform.     

The influence of anti-endothelial/antiphospholipid antibodies on fibrin formation and lysis on endothelial cells

The prothrombotic mechanisms associated with antiphospholipid antibodies remain incompletely defined. Antibody binding to endothelial cells in vitro is a feature of antiphospholipid antibody-positive sera. We hypothesised that impairment of endothelium-dependent fibrinolysis by antiphospholipid/anti-endothelial antibodies is a contributory factor in the pathogenesis of thrombosis. We also aimed to confirm the displacement of annexin-V from endothelial cells and enhanced fibrin formation. Binding of immunoglobulin (Ig) from antiphospholipid antibody-positive sera to endothelial cells was examined using a cell-based enzyme-linked immunosorbent assay. Effects on fibrin formation and lysis were examined on cultured endothelial cell monolayers. Plasminogen activator inhibitor-1 (PAI-1) was assayed in supernatants. We confirmed antibody binding to endothelial cells. With four of 14 antiphospholipid antibody-positive sera there was some prolongation of fibrin clot lysis time, consistent with impairment of endothelial fibrinolytic activity. Secretion of PAI-1 was significantly correlated with clot lysis time on endothelial cell monolayers incubated with antiphospholipid/anti-endothelial antibody-positive sera, but not with control sera. IgG from antiphospholipid antibody-positive sera had little effect on endothelial cell surface annexin-V expression. We conclude that impaired endothelial fibrinolysis is a potential prothrombotic mechanism in subjects with antiphospholipid antibodies. We were unable to confirm enhanced displacement of annexin-V from endothelium by antiphospholipid antibodies.     

自身免疫性肝炎患者自身抗体的测定及意义

目的:探讨自身抗体测定对诊断自身免疫性肝炎的临床意义．方法:采用间接免疫荧光法(IIF)检测47例自身免疫性肝炎患者、158例非自身免疫性肝炎患者及40例健康体检者体内抗核抗体(ANA)、抗平滑肌抗体(SMA)、抗中性粒细胞胞质抗体(ANCA)、抗线粒体抗体(AMA)等自身抗体,ELISA法检测抗MPO抗体,并对结果进行回顾性分析．结果:ANA、SMA及ANCA检出率的比较,结果显示AIH中阳性率最高为SMA(66．0％, 31／47),而非AIH中则为6．3％(10／158),两组差异有非常显著性意义(P<0．01)．经X2检验, SMA、AMA和MPO抗体检测在AIH与PBC中,均有非常显著性意义(P<0．01)．AIH各型自身抗体检测结果表明,AIH-Ⅰ与ANA、SMA和ANCA相关,AIH-Ⅱ与LKM相关,而AIH=Ⅲ与SLA和ANCA相关．结论:血清自身抗体的检测对诊断、治疗和阻止自身免疫性肝炎的发展有着十分重要作用,对提高AIH在临床上同其他肝病鉴别诊断和治疗有着非常重要的意义．