Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa...Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.展开更多
BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody...BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.展开更多
In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human an...In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.展开更多
BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a...BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.展开更多
Grass carp （Ctenopharyngodon idella） is an important species of freshwater aquaculture fish in China. However, grass carp reovirus （GCRV） can cause fatal hemorrhagic disease in yearling populations. Until now, a s...Grass carp （Ctenopharyngodon idella） is an important species of freshwater aquaculture fish in China. However, grass carp reovirus （GCRV） can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic ca- pacity of the virus＇s structural proteins for preparing an effective vaccine has not been available. In this study, some sin- gle-chain variable fragment antibodies （scFv）, which could specifically recognize grass carp IgM, were selected from a con- structed mouse naive antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from vi- rus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.展开更多
Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibod...Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment （scFv） phage display library consists of approximately 1 ~ l0s clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Topl0F＇ and then purified by affin- ity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.展开更多
Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies fo...Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.展开更多
目的探讨碎裂QRS波群与陈旧性心肌梗死患者心率变异性分析(heart rate variability,HRV)及室性心律失常发生情况的关系。方法2018年8月至2019年10月首次就诊于河北北方学院附属第一医院心脏功能检查科的陈旧性心肌梗死患者200例,依据患...目的探讨碎裂QRS波群与陈旧性心肌梗死患者心率变异性分析(heart rate variability,HRV)及室性心律失常发生情况的关系。方法2018年8月至2019年10月首次就诊于河北北方学院附属第一医院心脏功能检查科的陈旧性心肌梗死患者200例,依据患者首次就诊于我院的病例库资料及常规十二导联心电图诊断分为陈旧性心肌梗死碎裂QRS波群组99例与陈旧性心肌梗死无碎裂QRS波群组101例,之后回顾性分析患者出院后1年内复查的24 h动态心电图,采用χ^(2)检验比较两组室性心律失常的发生情况,秩和检验比较两组心率变异性的差异,采用多元Logistic回归分析心率变异性不同指标的对陈旧性心肌梗死碎裂QRS波群的评估价值,绘制受试者工作特性曲线(receiver operating characteristic,ROC),通过曲线下面积(area under the curve,AUC)分析心率变异性不同指标的对陈旧性心肌梗死碎裂QRS波群的诊断准确性。结果依据室性期前收缩Lown分级,Lown分级室性期前收缩Ⅰ级、室性期前收缩Ⅲ-Ⅴ级患者室性心律失常发生率陈旧性心肌梗死碎裂QRS组均高于陈旧性心肌梗死无碎裂QRS组[室性期前收缩Ⅰ级:54.5%(54/99)与39.6%(40/101)(χ^(2)=4.484,P<0.05;室性期前收缩Ⅲ-Ⅴ级:34.3%(34/99)与9.9%(10/101)(χ^(2)=17.406,P<0.05)]。室性期前收缩0级陈旧性心肌梗死碎裂QRS组发生率为8.1%(8/99),低于陈旧性心肌梗死无碎裂QRS组48.5%(49/101)(χ^(2)=37.995,P<0.05)。室性心律失常陈旧性心肌梗死碎裂QRS波群组发生率为91.9%(91/99)高于陈旧性心肌梗死无碎裂QRS波群组51.5%(52/101)(χ^(2)=57.146,P<0.05)。室性期前收缩Ⅱ级陈旧性心肌梗死碎裂QRS组室性心律失常阳性发生例数与陈旧性心肌梗死无碎裂QRS组之间比较差异无统计学意义(P>0.05)。陈旧性心肌梗死碎裂QRS波群组较陈旧性心肌梗死无碎裂QRS波群组HRV时域指标全部窦性心搏间期的标准差(standard diviation of NN intervals,SDNN)、窦性心搏间期均值的标准差(standard diviation of average NN intervals,SDANN)偏高[SDNN:143.00(122.00,166.00)与110.00(95.00,130.50),Z=5.780,P<0.05;SDANN:112.00(100.00,136.00)与96.00(76.00,118.50),Z=4.013,P<0.05]。多元Logistic回归分析HRV不同指标显示,HRV时域指标SDNN、SDANN诊断陈旧性心肌梗死后碎裂QRS波均有统计学意义(SDNN:OR=0.949,95%CI:0.922~0.977,P<0.001;SDANN:OR=1.036,95%CI:1.005~1.068,P=0.022),HRV时域指标SDNN、SDANN的ROC曲线下面积显示,SDNN、SDANN对于陈旧性心肌梗死后碎裂QRS波具有一定诊断准确性(SDNN:AUC:0.737,95%CI:0.666~0.807,灵敏度:0.818,特异度:0.634;SDANN:AUC:0.664,95%CI:0.587~0.741,灵敏度:0.737,特异度:0.673。0.5<AUC<1)。结论碎裂QRS波群与陈旧性心肌梗死患者室性心律失常发生率、室性心律失常发生严重程度呈正相关,与陈旧性心肌梗死患者心率变异性时域指标SDNN、SDANN呈正相关。展开更多
Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not c...Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs select ed were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×10 6 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically an d had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construct ion also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.展开更多
Monoclonal antibodies(MAbs) against natural products with low-molecular weights have become an important tool when combined with other analytical systems. Eastern blotting involves a typical staining system wherein, f...Monoclonal antibodies(MAbs) against natural products with low-molecular weights have become an important tool when combined with other analytical systems. Eastern blotting involves a typical staining system wherein, for example, glycosides can be blotted to a membrane and cross-linked and stained using MAbs. An immunoaffinity column combined with a monoclonal antibody allows a one-step purification of hapten compounds or preparation of a knockout extract that removes only the target hapten molecules from a crude extract. Here, we discuss the application of these extracts. Single-chain variable fragment(scFv) proteins have led to novel assay systems such as fluobodies or antibodies coupled with green fluorescent protein for natural products. A typical novel application of a scFv gene would be in the context of plant breeding; this is designated "missile-type" molecular breeding, intended to increase hapten molecule concentrations in transgenic plants.展开更多
基金supported by the National Natural Science Foundation of China,No.30360100,30760234,30860260,81160373,81360458
文摘Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
基金This study was supported by grants from the National Natural Science Foundation of China (No. 30572213)and Student Innovation Program of Shanxi Medical University (No.200404).
文摘BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.
基金Supported by the National Natural Science Foundation of China(No.30970608)the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045)+1 种基金the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948)the Innovation Method Fund of China (No.2008IM040800)
文摘In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.
基金the National Natural Science Foundation of China, No. 30500573
文摘BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.
基金supported by the National Basic Research Program of China (2009CB118701,2009CB118704)the National Natural Science Foundation of China (31072233,31172434)
文摘Grass carp （Ctenopharyngodon idella） is an important species of freshwater aquaculture fish in China. However, grass carp reovirus （GCRV） can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic ca- pacity of the virus＇s structural proteins for preparing an effective vaccine has not been available. In this study, some sin- gle-chain variable fragment antibodies （scFv）, which could specifically recognize grass carp IgM, were selected from a con- structed mouse naive antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from vi- rus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.
基金supported by National Natural Science Foundation of China (81070622, 81370939)Provincial Six Talent Foundation of Jiangsu (2010-022)+1 种基金Municipal Science and Technology Foundation of Nanjing (009010684)A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801)
文摘Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment （scFv） phage display library consists of approximately 1 ~ l0s clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Topl0F＇ and then purified by affin- ity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.
基金National Natural Science Foundation of China(No.31570937 and No.81871391)Natural Science Foundation of Hubei Province of China(No.2017CFB707)+1 种基金the Fundamental Research Funds for the Central Universities of China(No.HUST:2018KFYYXJJ086)Graduates'Innovation Foundation of Huazhong University of Science and Technology(No.5003510001).
文摘Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.
文摘目的探讨碎裂QRS波群与陈旧性心肌梗死患者心率变异性分析(heart rate variability,HRV)及室性心律失常发生情况的关系。方法2018年8月至2019年10月首次就诊于河北北方学院附属第一医院心脏功能检查科的陈旧性心肌梗死患者200例,依据患者首次就诊于我院的病例库资料及常规十二导联心电图诊断分为陈旧性心肌梗死碎裂QRS波群组99例与陈旧性心肌梗死无碎裂QRS波群组101例,之后回顾性分析患者出院后1年内复查的24 h动态心电图,采用χ^(2)检验比较两组室性心律失常的发生情况,秩和检验比较两组心率变异性的差异,采用多元Logistic回归分析心率变异性不同指标的对陈旧性心肌梗死碎裂QRS波群的评估价值,绘制受试者工作特性曲线(receiver operating characteristic,ROC),通过曲线下面积(area under the curve,AUC)分析心率变异性不同指标的对陈旧性心肌梗死碎裂QRS波群的诊断准确性。结果依据室性期前收缩Lown分级,Lown分级室性期前收缩Ⅰ级、室性期前收缩Ⅲ-Ⅴ级患者室性心律失常发生率陈旧性心肌梗死碎裂QRS组均高于陈旧性心肌梗死无碎裂QRS组[室性期前收缩Ⅰ级:54.5%(54/99)与39.6%(40/101)(χ^(2)=4.484,P<0.05;室性期前收缩Ⅲ-Ⅴ级:34.3%(34/99)与9.9%(10/101)(χ^(2)=17.406,P<0.05)]。室性期前收缩0级陈旧性心肌梗死碎裂QRS组发生率为8.1%(8/99),低于陈旧性心肌梗死无碎裂QRS组48.5%(49/101)(χ^(2)=37.995,P<0.05)。室性心律失常陈旧性心肌梗死碎裂QRS波群组发生率为91.9%(91/99)高于陈旧性心肌梗死无碎裂QRS波群组51.5%(52/101)(χ^(2)=57.146,P<0.05)。室性期前收缩Ⅱ级陈旧性心肌梗死碎裂QRS组室性心律失常阳性发生例数与陈旧性心肌梗死无碎裂QRS组之间比较差异无统计学意义(P>0.05)。陈旧性心肌梗死碎裂QRS波群组较陈旧性心肌梗死无碎裂QRS波群组HRV时域指标全部窦性心搏间期的标准差(standard diviation of NN intervals,SDNN)、窦性心搏间期均值的标准差(standard diviation of average NN intervals,SDANN)偏高[SDNN:143.00(122.00,166.00)与110.00(95.00,130.50),Z=5.780,P<0.05;SDANN:112.00(100.00,136.00)与96.00(76.00,118.50),Z=4.013,P<0.05]。多元Logistic回归分析HRV不同指标显示,HRV时域指标SDNN、SDANN诊断陈旧性心肌梗死后碎裂QRS波均有统计学意义(SDNN:OR=0.949,95%CI:0.922~0.977,P<0.001;SDANN:OR=1.036,95%CI:1.005~1.068,P=0.022),HRV时域指标SDNN、SDANN的ROC曲线下面积显示,SDNN、SDANN对于陈旧性心肌梗死后碎裂QRS波具有一定诊断准确性(SDNN:AUC:0.737,95%CI:0.666~0.807,灵敏度:0.818,特异度:0.634;SDANN:AUC:0.664,95%CI:0.587~0.741,灵敏度:0.737,特异度:0.673。0.5<AUC<1)。结论碎裂QRS波群与陈旧性心肌梗死患者室性心律失常发生率、室性心律失常发生严重程度呈正相关,与陈旧性心肌梗死患者心率变异性时域指标SDNN、SDANN呈正相关。
基金ThisworkwassupportedbytheWHOSpecialProgramforResearchandTraininginTropicalDiseases (No 970 978) ,theScienceFoundationfromtheMinistryofHealthofChina (No 98 1 85 ),andtheGuangdongProvincialScienceFoundationfor"2 11"Engineering
文摘Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs select ed were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×10 6 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically an d had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construct ion also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.
文摘Monoclonal antibodies(MAbs) against natural products with low-molecular weights have become an important tool when combined with other analytical systems. Eastern blotting involves a typical staining system wherein, for example, glycosides can be blotted to a membrane and cross-linked and stained using MAbs. An immunoaffinity column combined with a monoclonal antibody allows a one-step purification of hapten compounds or preparation of a knockout extract that removes only the target hapten molecules from a crude extract. Here, we discuss the application of these extracts. Single-chain variable fragment(scFv) proteins have led to novel assay systems such as fluobodies or antibodies coupled with green fluorescent protein for natural products. A typical novel application of a scFv gene would be in the context of plant breeding; this is designated "missile-type" molecular breeding, intended to increase hapten molecule concentrations in transgenic plants.
