Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.     

Selection,characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

Background

A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.

Results

An immune phage display library containing more than 10⁷ unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15–17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.

Conclusions

SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

     

Baculoviral display of functional scFv and synthetic IgG-binding domains

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG binding domain derived from protein A. Display on the baculovirus surface was achieved via fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In principle, the Z/ZZ IgG binding domain displaying baculoviruses could be targeted to specific cell types via the binding of an appropriate antibody. We envisage applications for scFv and Z/ZZ domain displaying baculoviral vectors in the gene therapy field.     

Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

Single‐domain antibodies (sdAbs), the variable domains of camelid heavy chain‐only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti‐methotrexate, anti‐triclocarban and anti‐cortisol sdAbs revealed unexpected contributions of the non‐hypervariable “CDR4” loop, formed between β‐strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15‐acetyl‐deoxynivalenol (15‐AcDON), and to carbohydrates. We constructed and panned a phage‐displayed library in which CDR4 of the 15‐AcDON‐specific sdAb, NAT‐267, was extended and randomized. From this library, we identified one sdAb, MA‐232, bearing a 14‐residue insertion in CDR4 and showing improved binding to 15‐AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage‐displayed libraries in which the CDR4 and other regions of three hapten‐ or carbohydrate‐binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan‐binding specificities, we panned the library against four tumor‐associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten‐specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V_L domain.     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.     

Generation of a phage‐display library of single‐domain camelid VHH antibodies directed against Chlamydomonas reinhardtii antigens,and characterization of VHHs binding cell‐surface antigens

Single‐domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain‐only variable V_H domain (V_HH) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and V_HH antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V_HH region contained at least 10⁶ individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for V_HH clones with specific recognition of cell‐surface epitopes. The lead candidate V_HH clones (designated B11 and H10) bound to C. reinhardtii with EC₅₀ values ≤0.5 nm . Treatment of cells with V_HH B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V_HH antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.     

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional V_H:V_L antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.     

Single-domain antibodies: a versatile and rich source of binders for breast cancer diagnostic approaches

Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. A crucial issue in this approach is the availability of relevant markers and corresponding monoclonal antibodies suitable for the development of effective immunodiagnostic modalities. The identification of such markers from human pathological lesions and the isolation of specific antibodies using conventional approaches remain major challenges. Camelids produce functional antibodies devoid of light chains in which the single N-terminal domain of the heavy chain is fully capable of antigen binding. When produced as an independent domain, these so-called single-domain antibody fragments (sdAbs) or nanobodies have several advantages for biotechnological applications owing to their unique properties of size (13 kDa), stability, solubility, and expression yield. In this work, we have generated phage display libraries from animals immunized with breast cancer biopsies. These libraries were used to isolate sdAbs against known and relevant antigens such as HER2, or several cancer-specific sdAbs against unknown targets. We describe the identification of one these targets, cytokeratin 19, using affinity purification in combination with mass spectrometry. Some of these sdAbs were used in several straightforward diagnostic applications such as immunohistochemical analysis of tumor samples, multiplexed cytometric bead array analysis of crude samples, or an immune enrichment procedure of rare cells. Here, we demonstrate that phage display-based selection of single-domain antibodies is an efficient and high-throughput compatible approach to generate binders with excellent characteristics for the fast development of diagnostic and prognostic modalities.     

Single domain antibodies: a new concept for epidermal growth factor receptor and EGFRvIII targeting

Epidermal growth factor receptor (EGFR) is one of the major molecular targets for cancer diagnosis and therapy. EGFR and EGFRvIII, mutated form of EGFR, have been identified as participating in pathogenesis of some forms of human cancers. Monoclonal antibodies (mAbs) targeting EGFR/EGFRvIII have been shown to suppress the signal transduction pathways controlling tumor cell growth, proliferation, and apoptosis. Until now, different types of mAbs or antibody fragments against EGFR family have been established. Some of these antibodies have been used clinically for treating various forms of human malignancies. More recently, a single domain antibody (sdAb) targeting this family of receptors has been introduced. The heavy chain antibodies (HCAbs) that made up variable regions of heavy chain, CH2, and CH3 domains are shown in camelids. SdAbs derived from camel HCAbs are the smallest known natural building parts for binding to antigen. They also possess a longer antigen recognizing region, which increases their capability for being more specific in target antigen enhancement. Camelid antibodies are highly valuable for their special characteristics, including heat resistance, small size, high solubility in an aqueous environment, and non-immunogenicity in a human environment. Due to these abilities, research on biotechnological production and treatment applications of recombinant smaller fragments of these only HCAbs is widely in progress. In this article, we will discuss the challenges and successes of different types of mAbs targeting EGFR/EGFRvIII in human cancer.     

Unique single‐domain antigen binding fragments derived from naturally occurring camel heavy‐chain antibodies

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy‐chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy‐chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy‐chain antibodies. The presence of the heavy‐chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen‐binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen‐binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti‐idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright © 1999 John Wiley & Sons, Ltd.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG₁) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.     

Expression of single-domain antibody in different systems

Applied Microbiology and Biotechnology - Camelid single-domain antibodies (sdAbs, VHHs, or Nanobodies®) are types of antibody fragments that are composed of the heavy-chain variable domain...     

Immunodetection of Venturia inaequalis Ascospores with Phage Antibodies

We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V‐gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non‐germinated ascospores. The binding of selected phage antibodies was monitored by enzyme‐linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross‐reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage‐derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers.     

A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain

Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrow's medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.     

Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.     

Synergistic capture of Clostridium botulinum type A neurotoxin by scFv antibodies to novel epitopes

A non‐immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (H_c)] with the goal of identifying scFv to novel epitopes. To do this, an antibody‐mediated labeling strategy was used in which antigen‐binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the H_c. Twenty unique scFv clones were isolated that bound H_c. Of these, 3 also bound to full‐length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep‐MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A‐specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep‐MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep‐MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456–2467. © 2011 Wiley Periodicals, Inc.     

特异结合人禽流感病毒H5N1的scFv分子性能鉴定

目的从人源化噬菌体抗体库（human single fold scFv libraries I＋J）中筛选到能高亲和性、特异结合人禽流感病毒H5N1的单链抗体,为建立H5N1快速筛查试剂和人源化治疗单抗奠定基础。方法以H5N1病毒的血凝素（hemagglutitin,HA）蛋白和核蛋白（nucleoprotein,NP）为目的蛋白,对上述单抗噬菌体文库以亲和性为原理进行筛选,经过3轮筛选富集后,随机挑选了96个噬菌体克隆扩增培养,ELISA法挑选能特异性、高亲和性结合目的蛋白的噬菌体克隆,并换用HB2151宿主菌对阳性单链抗体克隆进行可溶性表达,ELISA法鉴定可溶性单链抗体的结合活性,PCR扩增阳性克隆的轻、重链基因片段,并对阳性单链抗体分子测序和序列分析。结果经过3轮筛选,分别从96个噬菌体克隆中挑选到了两株能特异结合NP蛋白、3株能特异结合HA蛋白的单链抗体,PCR扩增都得到了长为300、302和935bp的轻链、重链和轻链-连接片段-重链的基因片段,测序结果分析发现上述5条单链抗体片段在轻链的47、49、50、51、53、54、56、96、97、98和99位的氨基酸组成不同,而特异结合NP蛋白的单链在重链区域氨基酸组成完全相同,而特异结合HA蛋白的单链在重链的44、47、85、86、87、88和89位氨基酸组成不同。结论从噬菌体抗体库中筛选到的特异结合HA和NP蛋白的单链抗体片段,可为进一步研发H5N1快速筛选试剂和人源性治疗抗体奠定基础,也可为鉴定HA和NP蛋白中的抗原决定簇提供结构信息。     

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