Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants

Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.     

Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.     

Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the US, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.Key words: anti-IgE, E. coli expression, scFv, antibody engineering, human antibodies, allergy diseases, antibody therapeutics     

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.     

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ～27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.     

Expression of a single-chain antibody against indole-3-acetic acid in Escherichia coli

A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.     

Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody.

A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme.     

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).     

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast

We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single‐chain variable fragment (scFv) antibody library was constructed in a yeast two‐hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin‐8 (hIL8) into the yeast two‐hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error‐prone PCR of the scFv sequence followed by additional rounds of yeast two‐hybrid screening. The scFv antibodies of both primary and affinity‐matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus

Severe acute respiratory syndrome (SARS) brought aglobal outbreak in spring of 2003 [1–3], and more andmore attention has been paid on it when a new caseresurfaced in Singapore last September [4]. By the endof May in 2003, WHO reported a cumulative total of 8202infected cases with 725 deaths from 28 countries.Because of the high transmission and morality rate ofSARS, scientists in many countries have made theirefforts in studying SARS coronavirus (SARS-CoV)[5, 6]. Several genomes of…     

Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid

Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.     

Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.     

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.     

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.