Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli

A functional single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was produced at high level in Escherichia coli. The variable regions of heavy chain (V(H)) and light chain (V(L)) from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction. The construct of V(H)-linker-V(L) was inserted into a phagemid pCANTAB 5 E followed by selection with the Recombinant Phage Antibody System (RPAS). Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75% confirmed by spectrophotometer. The activity of refolded scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed anti-AIF scFv retains the specific binding activity to AIF with an affinity constant of 7.29 x 10(-8) mol l(-1). The overall yield of anti-AIF scFv with bioactivity in E. coli flask culture was more than 60 mg l(-1).     

Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins

A subset of lipophillic bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are thought to be biologically transformed into reactive intermediates forming covalently modified, "tissue-bound" bile acids that can exert several toxic effects. We have generated a single-chain Fv fragment (scFv) as a probe to monitor DCA residues anchored on proteins. DNA fragments encoding the variable heavy (V(H)) and light (V(L)) domains of a mouse antibody raised against a DCA hapten (Ab #88) were cloned by rapid amplification of cDNA 5'-ends. These sequences were combined via a common linker sequence coding (Gly(4)Ser)(3) to construct a single scFv gene with the gene segments in the following order: 5'-V(H)-linker-V(L)-3'. This construct was subcloned into an antibody-expression vector, pEXmide 5; soluble scFv protein was then expressed in the bacterial periplasm of the XLOLR Escherichia coli strain. In a competitive enzyme-linked immunosorbent assay using DCA-coated microtiter plates, the scFv provided a dose-response curve for free DCA ranging between 2 and 5000 pg/assay. The scFv reacts similarly with the l-lysine adduct of DCA (cross-reactivity, 72%), while bile acids having a modified DCA steroid skeleton were well-discriminated (cross-reactivity, <1%). This scFv could also monitor trace amounts of DCA residues anchored on a protein through DCA acyl adenylate reactions, the likely reactive intermediate. The present scFv may be a useful tool for trace characterization of tissue-bound bile acids; this usefulness may be significantly enhanced by fusion with signal-generating proteins, such as alkaline phosphatase or green fluorescent protein.     

Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli

Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V(H)) and light chain (V(L)) cloned from the hybridoma 3G7 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-DON V(H) was a member of the V(H) III gene family IA subgroup and the V(L) gene belonged to the Vlambda gene family II subgroup. Extensive efforts to express the functional scFv antibody in E. coli have been made by using gene fusion and chaperone coexpression. Coexpression of the molecular chaperones (DnaK-DnaJ-GrpE) allowed soluble expression of the scFv. The scFv antibody fused with hexahistidine residues at the C-terminus was purified by immobilized metal affinity chromatography (IMAC). Soluble scFv antibody produced in this manner was characterized for its antigen-binding characteristics. Its biological affinity as antibody was measured by surface plasmon resonance (SPR) analysis and proved to be significant but weaker than that of the whole anti-DON mAb.     

Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody. Affinity,specificity, and idiotype analysis

Single-chain Fv fragments (scFvs) against a corticosteroid, 11-deoxycortisol (11-DC), have been generated as a template antibody fragment from which a comprehensive mutated antibody library containing various anti-steroid antibodies could be constructed. The cDNAs encoding variable heavy (V(H)) and light (V(L)) domains of a mouse anti-11-DC antibody (CET-M8), were amplified by RT-PCR, combined via a common linker to construct the sequence of 5'-V(H)-(Gly(4)Ser)(3)-V(L)-3', and cloned into a phagemid vector, pEXmide 5. The phage clones exhibiting binding activity to 11-DC were isolated after single panning against a hapten-immobilizing immunotube. The scFv gene in one of these clones was reamplified to introduce the ochre codons, and then expressed in the bacterial periplasm as the soluble antibody fragment. Two different scFvs (#6 and #12) were cloned, whose binding characteristics were examined by a radioimmunoassay using a tritium-labeled 11-DC. Both of them showed high affinity (K(a)=1.3x10(10)M(-1)) and practical specificity (cross-reactivity: cortisol, <0.2%; cortisone, <0.3%) to 11-DC, and furthermore, strong reactivity with an anti-idiotype antibody which recognizes the paratope of CET-M8. These results suggest that the present scFvs retain the three-dimensional structure of the paratope of the original monoclonal antibody.     

Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support

Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

Bacterial expression and in vitro refolding of a single-chain fv antibody specific for human plasma apolipoprotein B-100

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V(L)-linker-V(H)) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15-20 mg per 1-liter flask cultivation.     

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.     

Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.     

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 Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V_H and V_L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V(L) and V(H) domains with linkers of fifteen, five, four, three, two, one and zero residues. These V(L)-V(H) constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V(H)-V(L) orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs. The result showed that as the linker length between V(L) and V(H) was reduced, different patterns of oligomerization were observed compared with those with V(H)-V(L) isomers. As was the case for V(H)-V(L) orientation, the scFv-15 V(L)-V(H) protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V(L)-V(H) proteins. In contrast to the V(H)-V(L) isomer, direct ligation of V(L) to V(H) led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody). Furthermore, the transition between dimers and higher order oligomers was not as distinct as for V(H)-V(L). Thus reducing the linker length in V(L)-V(H) from three to two residues did not precisely dictate a transition between dimers and tetramers. Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.     

Linker peptide and affinity tag for detection and purification of single-chain Fv fragments

The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs.     

Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display

A single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E(2) antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E(2)-carrier conjugate. DNA fragments encoding the variable heavy and light domains (V(H) and V(L)) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5'-V(H)-(GGGGS)(3)-V(L)-3'. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E(2) (K(a)=8.6x10(7)M(-1)) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvV(H) and V(L) genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0mM MnCl(2), the error frequency reached to 8.5% and 11% for the V(H) and V(L) respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 degrees C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I-->V) in CDR L1, isolated from this library, showed threefold higher affinity (K(a)=2.6 x 10(8)M(-1)) than that of scFv#4-4.     

Production,purification and characterisation of genetically derived scFv and bifunctional antibody fragments capable of detecting illicit drug residues

We have generated a single chain antigen binding protein (scFv) recognising morphine. Variable regions of heavy (V(H)) and light (V(L)) chain antibody genes isolated from a murine immune repertoire were connected via a glycine-serine linker and cloned into the expression vector pAK 400. The scFv was produced in Escherichia coli JM83 yielding a functional protein of approximately M(r) 30000. Immunoaffinity chromatography using M3G-BSA-Sepharose column proved most effective for scFv purification. Purity was monitored by SDS-PAGE and Western blotting and the scFv characterised using ELISA and BIAcore. The scFv was capable of specifically binding free morphine in solution and was applicable to real sample analysis in saliva. In order to express a bivalent "minibody" the scFv gene was recloned into a vector containing a gene encoding a helix for dimerisation. The scFv was expressed as a protein of M(r) 75000 and retained its antibody binding capabilities. Cloning the scFv gene into a vector containing the bacterial alkaline phosphatase gene produced a bifunctional molecule, which retained the binding activity of the parental scFv along with the enzymatic activity of alkaline phosphatase.     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Construction of multiform scFv antibodies using linker peptide

Multiform single chain variable fragments (acFvs) including different length linker scFvs and bispecific seFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON seFv and antizearalenone (anti-ZEN) scFv was also constructed through eormection by a short peptide tinker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecifie acFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgertic plants.     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.