人源性肺癌噬菌体展示单链抗体库的构建

目的:构建人源性肺癌噬菌体单链抗体库,为筛选肺癌相关抗原的抗体奠定基础。方法:提取肺癌转移淋巴结总RNA,用RT-PCR技术扩增人抗体重链可变区(VH)和轻链可变区(VL)基因,在体外将VH和VL连接成单链抗体(ScFv)基因,并克隆到噬菌粒载体pCANTAB 5E中,电转化至感受态的大肠杆菌TG1,经辅助噬菌体超感染,形成噬菌体单链抗体库,采用限制性内切酶鉴定其多样性。结果:从肺癌转移淋巴结中成功提取RNA,逆转录PCR扩增出人可变区基因,连接形成单链抗体,最终构建了库容为1.2×108的抗人肺癌单链抗体库。BstNⅠ酶切法证明构建的抗体库具有良好的多样性。结论:成功地构建了噬菌体展示的抗人肺癌单链抗体库,为进一步筛选肺癌相关蛋白的可溶性抗体奠定了基础。     

噬菌体抗体库的构建及抗肿瘤单抗的筛选

鼠源抗肿瘤单抗通常用杂交瘤技术制备。作者首次尝试以噬菌体抗体库技术替代杂交瘤方法制备抗肿瘤鼠单抗。以ＲＴ－ＰＣＲ方法直接从免疫Ｂａ１Ｂ／ｃ小鼠脾脏淋巴细胞扩增出全套免疫球蛋白重链Ｆｄ段及轻链基因，克隆到原核表达载体并将抗体片断Ｆａｂ表达于线状噬菌体表面，构建了噬菌体抗体库（１．０×１０￣７）。以人乳腺癌细胞系Ｂｃａｐ３７活细胞为靶抗原对噬菌体抗体库进行的４轮亲和筛选显示了噬菌体抗体的特异性富集。从第４轮筛选后选取１８２个克隆进行ＥＬＩＳＡ检测发现１７４个具有与肿瘤细胞结合活性。用１２种人肿瘤细胞系及人淋巴细胞和成纤维细胞对所获克隆进行了进一步鉴定。我们所应用的方法为抗肿瘤单抗的制备提供了一条更为有效的新途径。     

噬菌体抗体库的筛选策略及其进展

噬菌体抗体库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.随着现代分子生物技术的迅速发展,抗体库的筛选工作也在传统的亲和淘选的基础上不断改进,涌现出越来越多新的有效筛选方法,显示出噬菌体抗体库技术在抗体开发应用中极为广阔的前景.     

噬菌体抗体库的筛选策略及其进展

噬菌体抗体库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.随着现代分子生物技术的迅速发展,抗体库的筛选工作也在传统的亲和淘选的基础上不断改进,涌现出越来越多新的有效筛选方法,显示出噬菌体抗体库技术在抗体开发应用中极为广阔的前景.     

抗人肝癌血管内皮功能性单抗的筛选与鉴定

目的:筛选、鉴定抗人肝癌血管内皮功能性单抗,为治疗肝癌提供靶向治疗剂,并为分离获得肝癌相关的分子靶标打下基础。方法:采用活细胞荧光、MTT细胞增殖实验、成管实验和动物体内治疗实验,筛选鉴定抑制肝癌内皮细胞的功能性单抗。结果:从能与肝癌内皮细胞膜反应的119株克隆中,筛选出16株单抗显著地抑制肝癌内皮增殖其抑制率达21%~46%,46株能显著抑制肝癌内皮细胞的成管,3株单抗1F9、12B5和1B11能显著抑制肝癌移植瘤的生长抑制率分别为50.8%、48.7%和47.0%。选择1株体内抑瘤效果最好的1B11单抗,通过抗体抗原亲和层析法进行纯化,并对纯化的单抗的相关抗原蛋白进行免疫组织化学实验鉴定,结果显示1B11抗原在肝癌血管组织较高的表达而在正常肝血管组织极少表达。蛋白质印迹法显示其抗原相对分子质量约46×103。结论:采用大容量功能性抗体库技术成功获得了多株具有抑制肝癌内皮恶性生物学行为的功能性单抗,体内外抑制肺癌的生长,具有成为肺癌靶向治疗剂的潜力。其中1株可能是一个靶向治疗肝癌的新靶位。     

全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的：对全人源肝癌噬菌体单链抗体库进行鉴定，筛选肝癌抗体，同时对抗体的活性及特异性进行鉴定。方法：PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC-7721为抗原对所建抗体库进行3轮“吸附-洗脱-扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定；通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果：ScFv基因插入率为70％。在亲和筛选过程中，肝癌噬菌体单链抗体得到富集，收获率逐轮得到提高，第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定，均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC-7721呈阳性反应，阳性率为90％，15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明，以正常胎肝细胞L-02为对照，ScFv与肝癌结合比率为41．3％。特异性鉴定表明，其与肝癌细胞结合活性明显高于正常细胞。结论：利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因，且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。     

抗人小细胞肺癌独特型单克隆抗体的制备

抗人小细胞肺癌独特型单克隆抗体的制备杨瑶琴胡晶莹莫忠根张中华李斯德作者单位：上海市肿瘤研究所免疫及细胞生物学研究室（上海２０００３２）关键词抗体，单克隆肺肿瘤癌，小细胞抗独特型抗体的应用是近年来肿瘤免疫治疗的一种新方法［１］，抗肿瘤特异性抗体（Ａｂ１...     

抗内毒素噬菌体抗体库的构建及筛选

目的构建一个鼠源性的抗内毒素单链噬菌体抗体库,从中筛选出对内毒素具有较高亲和力的单链抗体。方法从小鼠脾细胞中提取总RNA,通过RT-PCR技术扩增出小鼠抗体重链、轻链可变区基因（VH,VL）,用Linker将VH,VL交联形成单链抗体可变区片段（ScFv）。经NotⅠ,SfiⅠ双酶切后与经同样双酶切的pCANTAB5E载体相连,转化入大肠杆菌TG1以构建鼠抗内毒素单链噬菌体抗体库。在援救噬菌体抗体库后,用内毒素淘筛特异性的ScFv,富集的噬菌体阳性克隆重新感染TG1。在96孔板分别援救单个含特异性ScFv的TG1菌落,最后随机挑选出190个菌落经ELISA检测抗内毒素ScFv。结果小鼠血清中抗内毒素的效价为1∶12800。提取的总RNA浓度为12.3813μg/ml,纯度较好。扩增出的VH长约340bp,VL约320bp,ScFv约800bp。转化入TG1后有约1.9×107个菌落。淘筛一轮过后即有3×104阳性菌落长出,190个菌落经ELISA检测有2个阳性克隆。结论成功地构建了一个库容量为1.9×107的鼠抗内毒素单链噬菌体抗体库,并从中筛选出了2株抗内毒素ScFv。     

抗人肺腺癌单抗5F-11噬菌体抗体库的构建及表达

目的　构建抗人肺癌单抗 5F 11的噬菌体显示文库。方法　利用RT PCR方法从 5F 11杂交瘤细胞扩增抗体轻重链可变区基因 ,用连接子连接成为ScFv基因后克隆至噬菌体载体pCANTAB5E。以肺腺癌细胞系A2 为抗原进行 4轮筛选 ,得到富集的次级噬菌体表达文库。结果　制备了库容为 8× 10 7pfu/ml的噬菌体展示文库。随机抽取未经筛选的克隆进行酶切 ,酶切图谱呈多样性 ,富集后的酶切图谱则显示特异构型的噬菌体得到富集。检测 30个噬菌体克隆上清 ,其中 2 3个与A2 细胞有较强反应。结论　本研究获得了抗人肺腺癌单抗 5F 11的单链抗体 ,为拓展抗体的应用创造了条件。     

全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的对全人源肝癌噬菌体单链抗体库进行鉴定,筛选肝癌抗体,同时对抗体的活性及特异性进行鉴定。方法PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC7721为抗原对所建抗体库进行3轮“吸附洗脱扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定;通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果ScFv基因插入率为70%。在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮得到提高,第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定,均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC7721呈阳性反应,阳性率为90%,15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明,以正常胎肝细胞L02为对照,ScFv与肝癌结合比率为41.3%。特异性鉴定表明,其与肝癌细胞结合活性明显高于正常细胞。结论利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。     

Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo.     

Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system

A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.     

Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.     

Production and characterization of an active single-chain variable fragment antibody recognizing CD25

The alpha subunit of the interleukin-2 receptor (IL-2Ralpha, CD25) is a potential target in therapeutic approaches for hematolopoietic malignancies expressing CD25 on their cell surface, such as adult T cell leukemia/lymphomas. Recent reports have demonstrated that depletion of CD4+CD25+ regulatory T cells with anti-CD25 antibodies may enhance host tumor immunity. We previously raised a mouse monoclonal antibody (mAb), Ta60b mAb (IgG1kappa), specifically recognizing CD25, and an attempt was made here to produce a single chain Fv fragment (scFv) from this mAb as an initial step to development of scFv-based therapeutics. cDNA fragments encoding for the variable regions of the light and heavy chains of the Ta60b mAb were thus isolated by polymerase chain reaction-mediated cloning, and, an expression vector constructed to express Ta60b scFv fused with the maltose binding protein (MBP) in the periplasm of Escherichia coli. The soluble form of MBP-Ta60b fused scFv could be extracted and affinity-purified with an amylose/agarose column, allowing its immunoreactivity to be analyzed by enzyme-linked immunosorbent assay (ELISA), mixed hemadsorption assay, and fluorescence activated cell sorting. In addition, binding activity was studied by competitive ELISA and surface plasmon resonance. The results showed that Ta60b scFv obtained from periplasm retains good reactivity, although its KD value was 4-fold lower than that of the whole Ta60b antibody, suggesting possible clinical use for treatment of patients with CD25-expressing tumors and also for enhancing anti-tumor immunity.     

结肠癌单抗MC5的噬菌体呈现型单链可变区片段的制备

背景与目的：MC5是一种特异性良好的针对人结肠癌的鼠源性单克隆抗体,而将鼠源性抗体小型化可使其用于在体研究时引起人抗鼠抗体反应的可能性大大降低。本研究的目的是制备MC5的噬菌体呈现型单链可变区片段（ScFv)。方法：从分泌MC5的杂交瘤细胞分离mRNA,RT-PCR分别扩增抗体的重,轻链可变区DNA（VH和VL DNA）,两者经linker DNA连接形成ScFvDNA,将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7辅助噬菌体感染后,获得重组噬菌体抗体ScFv,以高表达MC5结合抗原的细胞株SW480对重组噬菌体抗体ScFv进行两轮筛选后,随机挑取克隆经ELISA筛选呈现MC5 ScFv的噬菌体单克隆,经竞争ELISA对阳性克隆结合抗原的能力进行鉴定。结果：VH,VL和ScFvDNA分别约为340bp,320bp和750bp,在随机筛检的25个克隆中得到10个呈现MC5ScFv的噬菌体单克隆,其中结合抗原能力强的克隆有3个,结论：用噬菌体呈现技术成功地制备了单抗MC5的ScFv,为拓展该抗体的应用范围奠定了基础。     

Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein.

The Fab gene of anti-CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system. The variable heavy and light chain genes of HBJ127 were found to be derived from VOx-1 and IgVk8-30 germline, respectively. Extensive somatic mutation was found in the heavy chain complementarity determining region 2. rFab fragment was purified homogeneously from crude bacterial lysates by Ni-chelate chromatography in a yield of 71.4 mg from 100 ml of culture. rFab fragment was reactive with the cell surface of CD98-positive cells irrespective of tissues of origin, but not with CD98-negative cells. The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS(3) cells was completely inhibited by pretreatment with an excess of mAb. The relative affinity values of rFab fragment and mAb were found to be 0.11 x 10(8) and 0.35 x 10(8) M(-1), respectively. Three-fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation. Cell growth inhibition in vitro by rFab fragment preincubated with anti-Fab suggests that the rFab fragment produced by cloned gene-bearing Escherichia coli was identical to the Fab part of HBJ127 mAb. These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system. To our knowledge, this is a first report of the production of anti-CD98 h.c. rFab fragment.     

Production and characterization of IgA monoclonal antibody against ovalbumin

We established a hybridoma clone secreting an immunoglobulin A (IgA) monoclonal antibody (MAb) against ovalbumin (OVA). The MAb was produced using nasal-associated lymphoid tissues (NALT) of BALB/c mice that had been intranasally immunized with OVA together with cholera toxin. The isotype of the MAb was determined to be IgA, kappa. The established IgA MAb exhibited saturable and dose-dependent binding to immobilized OVA on ELISA. The majority of the antibodies formed a dimer on immunoblot analyses. To determine the affinity of each binding site, we performed surface plasmon resonance analysis, in which the binding of soluble OVA to immobilized IgA was measured. The results revealed a slow association rate and relatively low affinity of each binding site. Despite this, the stable binding of the MAb to the immobilized OVA suggests that IgA may gain high avidity through formation of the dimer. This hybridoma will provide a unique source of genuine IgA MAb, not an IgG-IgA chimeric one, against food allergens.     

Epitope mapping of PR81 anti-MUC1 monoclonal antibody following PEPSCAN and phage display techniques

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.     

Preparation and characterization of anti‐tissue factor single‐chain variable fragment antibody for cancer diagnosis

Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10⁻⁸ (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.     

Production and characterization of monoclonal antibody against human serum albumin

Hybridomas secreting monoclonal antibodies (MAbs) producing stable, specific and high affinity against human serum albumin (HSA) have been established. The aim of the present study was the production of MAbs that will be potentially used in designing immunoassay methods especially immunochromatography assay kit for screening of microalbuminuria (MAU) in the early detection of diabetic and nondiabetic nephropathy. The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2). After limiting dilutions three clones producing antibodies were designed as EMRC1-3, which displayed different pattern of fine specificity for HSA and low cross reaction with other proteins as elucidated by inhibition enzyme-linked immunosorbent assay (ELISA). These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all three MAbs secreted IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA were expanded in 50-mL flasks for large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg per well covering up to 100 ng per well. The high binding activity to HSA antigen and having no cross reactivity with other related molecules illustrated the potential application of these antibodies as an immunodiagnostic reagent in designing an immunochromatography assay kit for screening of MAU in diabetic and nondiabetic patients.