Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10⁻⁸ M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.     

从scFv噬菌体库分离特异性的人源化抗D-dimer抗体

目的 从scFv(单链Fv)噬菌体抗体库分离出对D-dimer有特异性的人源化单克隆scFv.方法 对Tomlinson scFv噬菌体文库进行3轮淘洗,富集特异性的抗D-dimer抗体并进行ELISA 验证.通过酶联免疫检测和双脱氧终止法基因测序,获取特异性的人源化单克隆抗体.结果 3轮淘洗选择出38个抗D-dimer噬菌体抗体,酶联免疫和基因测序分析后,20个不同的全长单克隆抗D-dimer scFv噬菌体抗体被筛选出来,3轮选择后阳性克隆获取率为100%;分泌性抗体ELISA结果显示单克隆anti-D-dimer噬菌体顺利表达了抗体蛋白;5个A450值较高的单克隆中,3个显示了对D-dimer的高特异性和亲和力.结论 抗体噬菌体展示技术是分离获取人源化特异性anti-D-dimer抗体的高效快速方法.

Abstract：

Objective To isolate specific humanized anti-D-dimer scFv(single chain Fv) antibody from scFv phage libraries. Methods Isolate anti-D-dimer positive clones from Tomlinson I + J phage libraries by three rounds of panuing, then sequence monoclonal genes by bideoxy-mediated chain termination and express soluble scFv antibody; Pick out anti-D-dimer antibodies with high specificity and affinity by ELISA.Results After three rounds of selection from human scFv phage libraries Tomlinson I and J, 38 monclonal specific anti-D-dimer scFv fragments were selected. By polyclonal and monoclonal phage ELISA and gene sequencing, 20 different full-length monoclonal scFv phages were identified, the result of soluble scFv ELISA showed that 20 full-length monoclonal scFv were expressed smoothly. According to the result of soluble scFv ELISA, in 5 scFv antibodies with high value of A450 selected, 3 scFv antibody fragments showed high specific and affinity. Conclusion Antibody phage display was an effective, rapid method to isolate anti-D-dimer antibodies with high specificity and affinity.     

Analysis of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunizations

BACKGROUND: Generation and cloning of antibodies against cell surface antigens can be simplified by combining DNA immunization which enables generation of antibodies against a protein in its natural configuration without the need for any protein purification step and antibody phage display which due to its immense screening power and physical coupling between the phenotype and genotype of antibodies simplifies the cloning of antibody genes. OBJECTIVES: Since DNA immunization is expected to elicit antibodies against a protein in its natural configuration, we wanted to see if it can mimic the antibody response generated by cell immunization. STUDY DESIGN: A phage display library made from splenic mRNA of a mouse immunized with mesothelin cDNA was panned on mesothelin-positive cells. The single-chain Fvs (scFvs) selected were then analyzed. RESULTS: We obtained several anti-mesothelin scFvs. One of these Fvs is almost identical to the Fv of a monoclonal antibody that was previously obtained from a hybridoma in which the mice were immunized with a mesothelin-positive ovarian cancer cell line. Another Fv was found to be specific for mesothelin present on human cells. CONCLUSION: Our results indicate that an antibody phage display library made from spleens of DNA-immunized mice is a rapid and efficient alternative to cell immunization for obtaining antibodies against different epitopes of a membrane antigen that is very difficult to purify in a native form.     

An ELISA for detection of infectious bursal disease virus and differentiation of very virulent strains based on single chain recombinant chicken antibodies.

Two chicken single-chain variable antibody fragments (scFv) designated scFv154 and scFv88, previously shown to react with either all or very virulent (vv) infectious bursal disease virus (IBDV) strains, respectively, were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for differentiation of vvIBDV. Specificity and sensitivity of the vvIBDV ELISA was assessed when scFv154 and scFv88 were expressed as soluble antibodies (sAb), phage antibodies (pAb) or hyper-phage antibodies (hpAb). The highest test sensitivity and specificity was obtained using hpAb154 to detect all IBDV and pAb88 to differentiate vvIBDV strains. Such an ELISA was eight to 16 times more sensitive for IBDV antigen detection than the mouse monoclonal antibody ELISA. Using field samples, the scFv ELISA was able to differentiate between flocks infected with vvIBDV and those infected with classical or variant IBDV. In one instance IBDV was detected in a flock found to be negative by the monoclonal antibody ELISA. The results showed that scFv can be utilized as highly specific and sensitive ELISA reagents for the detection and discrimination of avian pathogens.     

中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.     

Sheep monoclonal antibody fragments generated using a phage display system

Monoclonal sheep antibodies have great potential for biomedical, veterinary and agricultural purpose. Although conventional sheep monoclonal antibodies can be generated by a modified hybridoma technology, the procedures are not routine. Here, we describe a method to generate recombinant sheep antibody fragments from immunised animals using a modified phage display system. Total RNA from pooled spleens of sheep immunised with the model antigens human serum albumin and conalbumin were used to amplify immunoglobulin V gene repertoires and an efficient two-step cloning method was employed to rapidly construct a phage display single-chain Fv (scFv) library. A total of 14 different scFvs were isolated and characterised. Sequence analysis indicated typical ovine immunoglobulin characteristics. Thirteen Vlambda and 11 VH genes were identified that could be grouped into the sheep Vlambda families I, II, VI and a single VH family. Soluble monomeric scFvs, produced in the periplasm of Escherichia coli, were subjected to affinity measurement via surface plasmon resonance analysis and affinities typical of the secondary immune response were observed. The method described here should be of value for the study of sheep immunology as well as for biorecognition in general.     

Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.     

人源性抗乳腺癌单链抗体库的筛选与初步鉴定

目的:本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定。方法:以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况。结果:成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白。结论:本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库。研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础。     

全人源抗肝癌噬菌体单链抗体的筛选与鉴定

目的: 构建人源抗肝癌噬菌体单链抗体库, 从中筛选抗肝癌单链抗体(scFv), 并对其特异性进行鉴定.方法: 采用体外致敏法和EBV转化技术联合噬菌体展示技术构建噬菌体抗体库.对初级抗体库进行亲和富集及ELISA筛选, 获得的阳性克隆进行免疫组化鉴定并测序.结果: scFv基因与载体连接后得到库容量为1.0×108的初级噬菌体抗体库.对抗体库进行3轮正负淘选和富集后, 随机挑取2 798个克隆进行ELISA, 发现3个克隆对HepG2呈强阳性反应, 而与QSG-7701等人正常细胞系呈弱阳性或不反应.对克隆SA3进行免疫细胞化学鉴定, 结果与ELISA一致.免疫组织化学鉴定表明SA3与肝癌组织和肝组织阳性率的差别有统计学意义.结论: 构建了库容量达1×108的全人源抗肝癌噬菌体抗体库.通过淘选富集、 ELISA和免疫组织化学鉴定获得特异性较强的噬菌体克隆, 为肝癌的临床诊断及导向治疗奠定了基础.     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具     

基于基因免疫制备抗人绒毛膜促性腺激素β亚基单克隆抗体及其特性研究

用真核表达人绒毛膜促性腺激素β亚基((human chorionic gonadotrophinβ,hCGβ)的重组质TR421-hCGβ免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,ELISA法筛选阳性细胞株,经过多次的克隆化培养,最终获得10株持续、稳定分泌抗hCGβ单克隆抗体的杂交瘤细胞株。随机抽取6H1杂交瘤细胞进行抗体的制备、纯化及免疫学特性的分析。间接ELISA法证明6H1单抗属于IgG2a亚类。Western blot证明6H1单克隆抗体可以特异性结合hCGβ,间接免疫荧光和流式细胞仪等结果表明,6H1单克隆抗体能够不同程度的结合不同来源的肿瘤细胞膜上表达的hCGβ分子,为进一步研究其生物学功能奠定了物质基础。     

Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients

Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved. In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10-250copies/cell. CMVpp65(+)surfaceIgG(+) B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (β-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development.     

Chicken single-chain variable fragments against the SARS-CoV spike protein

The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5x10(7) and 9x10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750-1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.     

Production and characterization of a human monoclonal anti-idiotype to anti-ribosomal P antibodies

Autoantibodies to ribosomal P protein (anti-P) are a specific hallmark of systemic lupus erythematous (SLE). Several authors found significant associations of anti-P antibodies with neuropsychiatric, hepatic, and renal disease. We now report the isolation by phage display of human anti-idiotype (Id) monoclonal antibody fragments as single-chain Fv fragment (scFv) against anti-P antibodies. The V gene repertoires were derived from the RNA obtained from the B cells of a SLE patient. Affinity-purified anti-P antibodies were used for the selection of bacterial clones producing anti-P-specific scFv antibody fragments and little reactivity with normal IgG and other IgG antibodies. The anti-Id antibody recognizes a public idiotope broadly cross-reactive with polyclonal anti-P antibodies and inhibited binding of anti-P to ribosomal P antigen in immunoassays and on Jurkat cells. The anti-Id scFv antibody fragment may have therapeutic implications in SLE. They may also be used as probes in the study of the structure of the idiotype.     

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.

To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.     

Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma

A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.     

Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine

Use of phage display of recombinant antibodies and large repertoire naïve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naïve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naïve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1–3×10⁻⁷ M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.     

In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library

We have isolated a monoclonal antibody binding to oestradiol with high affinity (3.7 nM), and exhibiting a better than 1000-fold selectivity in binding to other steroids. A high affinity antibody with good specificity is essential for the accurate determination of circulating oestradiol levels. To date, conventional hybridoma technology has not yielded a reagent of sufficiently high affinity and specificity for this ligand. The aim of this study was to investigate whether such a reagent was accessible through the engineering of antibodies on the surface of filamentous phage. Antibodies were isolated from a large repertoire of single chain Fv fragments (scFv) derived from non-immunised human donors, with selection and screening procedures biased to favour those binding to free oestradiol. This resulted in an antibody with nanomolar affinity for oestradiol, while affinities for related steroids are in the micromolar range. The relative lack of reactivity for steroids substituted at either end of the molecule suggests that this antibody is unique among anti-steroid monoclonal antibodies in lacking a 'blind-spot'. Our results demonstrate that phage display can provide solutions to problems that have so far proved intractable using conventional hybridoma technology.     

Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus

A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized. Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2. The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc). The RS10B5huFc antibody was expressed in E. coli and purified by affinity chromatography as a 70-kDa protein. The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs). Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers. The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody. The Fc domain was capable of binding to protein G and human complement. The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies.     

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.