Generation and characterization of a recombinant single chain Fv antibody to von Willebrand factor A1 domain from phage display library

In this study, cDNA of von Willebrand factor domain A1 (VWF-A1) was cloned from human umbilical vein endothelial cells, and expressed in E. coli. The expressed VWF-A1 was used as antigen to immunize Balb/c mice. Reverse transcribed-polymerase chain reaction (RT-PCR) was used to amplify VH and VL gene segment from the RNA isolated from the immunized mouse spleens. Single chain Fv (ScFv) genes were generated by splicing overlap extension and subcloned into a phagemid vector pHEN-1. Phage display library of repertoire single chain antibodies of anti-VWF-A1 was then constructed and screened for ScFvs that interact with VWF-A1. The ScFv gene of phage clone 28 that has best binding activity to VWF-A1 was cloned into pET20b vector for higher expression in E. coli strain BL-21(DE3) pLysS. The recombinant ScFv specifically reacted with VWF, rVWF-A1 and rVWF-A1/A3, but not with rVWF-A3, P-selectin and BSA. It inhibited ristocetin-induced platelet aggregation with an IC50 of 0.75 micromol/l, and its maximal extent of inhibition is 62.5%. The ScFv had no effect on thrombin-induced platelet aggregation. These data show that the ScFv is specific against VWF-A1 and could have a potential to be used as an antithrombotic agent.     

Human single chain Fv antibodies and a complementarity determining region-derived peptide binding to amyloid-beta 1-42

A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Abeta42). Two new anti-Abeta42 phage-displayed scFvs antibodies were obtained, and the sequences of their V(H) and Vkappa genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V(H)) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Abeta42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Abeta42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Abeta42 but also synthetic peptides based on the V(H) CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.     

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient's plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.     

Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library

A novel single-chain fragment variable (scFv) antibody was developed directed against human fibrin clots by using the human single fold scFv libraries I+J (Tomlinson I+J). Three positively binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing. Then the positive scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC) with a yield of about 1 mg/l, the expression of soluble scFv was verified by Western blot analysis. The purified scFv could specifically recognize human fibrin clots and indicate no binding ability with human fibrinogen shown by ELISA. Furthermore, we will amplify the gene of the positive scFv by polymerase chain reaction (PCR) for future study of its role in diagnosis and therapy of thrombus-correlated diseases.     

Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage display library

Current Alzheimer's disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study this phenomenon we developed monoclonal antibodies to acetylcholinesterase synaptic peptide (ASP), a synthetic peptide with the C-terminal sequence unique to the human synaptic variant AChE-S. Screening of a phage display human antibody library allowed the isolation of single-chain Fv (scFv) antibodies that were highly specific for ASP, and displayed closely related third complementarity determining regions of the variable heavy chain domain (V(H)-CDR3). BIAcore analysis demonstrated dissociation constants at the micromolar range: 1.6 x 10(-6) and 2.0 x 10(-6) M for ASP and the complete AChE-S protein, respectively. The anti-ASP antibodies provide a novel tool for studying the synaptic AChE-S variant, the expression of which is altered in ageing and dementia.     

Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibalpha

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibalpha was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbalpha. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH-VL orientation, being joined together with a 15-amino-acid (Gly(4)Ser)(3) linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.     

Generation and brain delivery of anti-aggregating antibodies against beta-amyloid plaques using phage display technology

Antibodies towards the N-terminal region of the beta-amyloid peptide bind to beta-amyloid fibrils, leading to their disaggregation. We generated anti-aggregating beta-amyloid antibodies using filamentous phages displaying the only four amino acids EFRH found to be the main regulatory site for beta-amyloid formation. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' mAbs to the betaA plaques in the brain, we applied antibody engineering methods to minimize the size of the mAbs while maintaining their biological activity. We found that single-chain antibodies displayed on the surface of the phage are capable of entering the central nervous system (CNS). The feasibility of these novel strategies for the production and targeting of anti-aggregating antibodies against beta-amyloid plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease.     

Single chain Fv antibodies against neural cell adhesion molecule L1 trigger L1 functions in cultured neurons

The neural cell adhesion molecule L1 plays important roles in cell adhesion, neuronal migration, neurite outgrowth, fasciculation and pathfinding, neuronal survival, and synaptic plasticity. Many of these functions have been identified and characterized by using antibodies. Because of the need for reproducible and functionally active antibodies, we have generated two single-chain variable fragment antibodies against mouse L1 from a human synthetic phage display library. The complementarity determining region 3 of the variable heavy chains of the two antibodies differed in length and sequence. Both antibodies recognized mouse, but not human L1, by enzyme-linked immunosorbent assay, Western blot, and immunofluorescence staining of cultured neurons. Epitope mapping showed reactivity with the fibronectin type III repeats 1-2 of mouse L1. The antibodies stimulated neurite outgrowth from cerebellar dorsal, root ganglion and motor neurons when offered in substrate-coated form in a dose-dependent manner with maximal effects at approximately 32 nM. Furthermore, substrate-coated antibodies enhanced survival of cerebellar neurons. Peptides comprising 8 and 11 amino acids derived from the complementarity determining region 3 of the variable heavy chains of the two single-chain variable fragment antibodies also promoted neurite outgrowth. The combined observations indicate that single-chain variable fragment antibodies against L1 and peptides derived from their binding domains can mimic some beneficial functions of homophilically binding L1 in vitro and may thus serve to trigger these functions in vivo.     

胶质瘤患者人源性噬菌体抗体基因库的构建

目的 构建胶质瘤患者人源性噬菌体抗体基因库。方法 从脑胶质瘤患者外周血淋巴细胞（PBL）中提取细胞总RNA,经逆转录后用套式多聚酶链反应（PCR）分别扩增抗体轻链VK和重链VH基因。先构建VK基因库,并使linker与VK基因连接,再将VH基因克隆人VK基因库,即构建成功单链抗体（ScFv）基因库。随机挑取菌落鉴定后测序。结果 从PBL中扩增出人源性VK和VH基因,并构建了库容量约为2×106的ScFv噬菌体抗体基因库。随机挑取克隆的测序结果表明,测定的VH基因与Kabat的抗体基因库中人抗体VH基因有较高的同源性,证明所克隆的基因属于人抗体可变区基因。结论 本实验构建成胶质瘤患者人源性噬菌体抗体基因库,为进一步筛选入源性抗脑胶质瘤抗体奠定了基础。     

Platelet integrin alpha2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.     

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.     

Cloning the antibody response in humans with inflammatory CNS disease: isolation of measles virus-specific antibodies from phage display libraries of a subacute sclerosing panencephalitis brain.

We have developed a strategy to identify the disease-relevant antigens in a chronic inflammatory CNS disease exhibiting intrathecally expressed oligoclonal IgG. Using subacute sclerosing panencephalitis (SSPE), a chronic inflammatory measles virus infection of the brain as a model system, we constructed a phage display antibody Fab library from the amplified products of IgG expressed in the brain. Selection of the library against measles virus-infected cell lysates yielded four distinct Fabs which, by ELISA and by immunostaining, reacted specifically with measles virus-infected cells. Three Fabs immunoprecipitated a 72 kDa protein from infected cell cultures corresponding to the measles virus phosphoprotein. The fourth Fab immunoprecipitated and recognized by immunoblotting a 60 kDa protein corresponding to the measles virus nucleoprotein. The results demonstrate that functional antibodies from an inflammatory CNS disease can be expressed in bacteria and used to identify disease-relevant antigens. This approach could be applied to chronic inflammatory CNS diseases of unknown cause such as multiple sclerosis.     

Multiple sclerosis immune complexes: an analysis of component antigens and antibodies

Serum immune complexes were isolated from 21 patients with multiple sclerosis and 23 control subjects, who were normal or had other neurological diseases, and then probed for neurotropic viral and brain-reactive components. Multiple sclerosis complexes contained antibody to herpes simplex (13 cases), measles (8 cases), cytomegalovirus (5 cases), and rubella virus (5 cases). In some complexes, herpes simplex or cytomegalovirus antigen was detected along with antibody. Myelin basic protein or antibody to myelin basic protein was found in the complexes of 7 patients with multiple sclerosis and 2 patients with other neurological diseases. Serum complexes containing antibody reactive with galactocerebroside and ganglioside were present in 12 patients with multiple sclerosis and 3 with other neurological diseases. Over 60% of the multiple sclerosis group had IgM and IgA serum complexes, including 5 patients with very high IgA complex levels. This study suggests that patients with multiple sclerosis have ongoing systemic virus production with resultant immune complex formation. In addition, they often have serum complexes that contain brain-reactive components.     

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.     

单链抗体对重症肌无力致病性抗乙酰胆碱受体抗体的抑制

目的 探讨单链抗体(ScFv)在体外对重症肌无力(MG)的治疗作用。方法 从分离自MG患者胸腺的抗乙酰胆碱受体(AChR)抗体构建单链抗体ScFv637，应用免疫荧光法、竞争性ELISA和竞争性放射免疫测定法，对ScFv637与人肌肉冰冻切片中AChR结合活性以及抑制致病性抗AChR抗体和MG患者血清与人AChR结合活性进行测定。结果 ScFv637能与人肌肉中神经肌肉接头处的AChR结合，对致病性抗AChR抗体与人AChR结合的抑制率可达73．0％，对MG患者血清与人AChR结合的抑制率为27．8％～45．5％。结论 单链抗体在体外能抑制致病性抗AChR抗体与AChR的结合，对AChR具有保护作用。     

Specificity of recombinant antibodies generated from multiple sclerosis cerebrospinal fluid probed with a random peptide library

We generated recombinant antibodies (rAbs) from over-represented IgG sequences expressed by single plasma cells from multiple sclerosis (MS) cerebrospinal fluid (CSF). Panning of a phage-displayed random peptide library with the rAbs revealed several specific peptide sequences. Inhibition assays confirmed specific binding of the peptides to the antigen-binding site of the antibody. The native IgG of MS CSF from which the recombinant antibody was cloned also recognized these peptides. Our data demonstrate that MS rAb reflects the specificity of IgG in the CSF. Thus, the epitopes/mimotopes identified by MS rAb may provide clues to disease-relevant antigens.     

Edrophonium-induced inward membrane current in single neurons physically isolated from Aplysia californica

The action of edrophonium on Aplysia neurons was studied using a concentration clamp technique which combines internal perfusion and a rapid drug application. Edrophonium elicited a dose-dependent inward current in the concentration range 10(-6) to 10(-4) M. At higher concentrations (10(-3) and 10(-2) M), the amplitude of the current often decreased and there was a rapid decay of the current. At these high concentrations, the current increased immediately after washing the neuron with normal solution. These results suggest that edrophonium blocks the ion channel which it opens. Removal of Na+ from the external solution greatly reduced the current amplitude by more than 90%. Removal of Ca2+ also reduced the amplitude of the response; however an increase of Ca2+ did not augment the response. These results suggest that Ca2+ does not carry the current, but is necessary for generation of an Na+-dependent inward current. Edrophonium, 10(-2) M, which completely blocked the current it induced within 20 s, did not significantly affect the voltage-dependent Na+ current. Tetrodotoxin, 1 x 10(-6) M, did not affect the edrophonium response. Hexamethonium, 1 x 10(-4) M, did not change the response elicited by edrophonium, while it significantly reduced the ACh response mediated by Na+. In some neurons edrophonium elicited an inward current, but ACh induced an outward current. Therefore the Na+ channels opened by edrophonium appear to be distinct from both the voltage-gated and ACh receptor-operated Na+ channels.     

Ultrastructural observations on nodes of Ranvier from isolated single frog peripheral nerve fibres

A preparative procedure is described by which well-preserved nodes of Ranvier from isolated frog peripheral nerve fibres may be obtained. The following steps are crucial: careful and gentle dissection and isolation of a single nerve fibre, mounting in a chamber limiting lateral movements of the fibre during the fluid exchange, simultaneous glutaraldehyde-OsO4 fixation and embedding in the same chamber used for fixation. Serial sectioning of individual nodes from both motor and sensory fibres made it possible to reconstitute three-dimensional models of several nodes and to study their morphology extensively. In addition to well-known ultrastructural features of the nodal and paranodal architecture, evaginations of a nodal membrane containing mitochondria and outpouchings of the paranodal axoplasm containing vesicles are described.