Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Screening and characterization of human anti-GPC3 single chain Fv antibody fragment selected by phage display / 药学学报

The aim of the experiments is to screen human single-chain variable fragment (scFv) targeting glypican 3 from phage display library, and analyze its biological activity. After several rounds of panning, the binders with high affinity were obtained through phage ELISA and IMGT analysis. The desired scFv gene was then ligated with pET-22b vector yielding recombinant plasmids, which was then introduced into E. coli Rosetta (DE3). Soluble scFv protein was expressed and further purified using Ni2+ affinity chromatography. The purified proteins were identified by SDS-PAGE and Western blot. Subsequently, the affinity and cell based binding activity were measured using Surface Plasmon Resonance (SPR) and flow cytometry assay, separately. Four enriched sequences with relatively high binding affinity were found (1F7, 1D7, 1D4 and 1B10). We also found that 1F7 scFv showed better targeting ability and higher affinity. The scFv could pave the way for new immunotherapies, such as bispecific anitbody, antibody-drug conjugate and chimeric antigen receptor T-cell immunotherapy cell, etc.

Combination of scFv-AFP with doxorubicin inhibits ceII proIiferation in hepato-ceIIuIar carcinoma ceII Huh7 / 中国药科大学学报

This study was to investigate the inhibitory effects of single-chain variable fragment of alpha fetopro-tein (scFv-AFP)in combination with doxorubicin on the proliferation of human hepatocellular carcinoma cell lines Huh7.Huh7 cells were treated with different concentration of scFv-AFP or doxorubcin alone or their combi-nation.The inhibitory effects were detected by MTT assay,and cycle arrest and apoptosis of Huh7 cells were ana-lyzed by flow cytometry in different groups using PI and Annexin V /PI-staining respectively.Results showed that scFv-AFP,doxorubicin alone or in combinations dose-dependently inhibited the proliferation of Huh7,and a syn-ergistic effect was observed in their combined action.The combination treatment resulted in significantly higher apoptosis than those in other groups (P <0.05).scFv-AFP (40 μg/mL)markedly blocked the Huh7 cell pro-gression by arresting the cells in the G0 /G1 phase,and the percentage of cells in S phase decreased dramatically (P <0.05);and scFv-AFP combined with doxorubicin blocked the Huh7 cell progression by arresting the cells in G2 /Mphase (P <0.01).

Screening scFv Specific to Vcam-1 by Phage Display Library and Its Activity Evaluation / 华中科技大学学报(医学版)

Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones were transferred into E.coli ,and the clone with the highest expression was regarded as the final requisite one.Competent cells were infected by the final requisite clone and scFv was expressed.After purification ,the activity of scFv was tested by ELISA and its affinity was evaluated.Results Molecular weight of Vcam‐1 antigen protein was 85-90 kD.Positive clones were screened out by taking Vcam‐1 protein as the antigen ,and 9 high titer clones were obtained by single phage ELISA.Gene sequencing of these clones was carried out and 3 sequences were obtained ,1 of which got the highest expression.Molecular weight of the expressed scFv was about 30 kD.The scFv got high affinity to Vcam‐1 antigen according to ELISA ,in spite of its lower activity than full‐length monoclonal antibody.Conclusion scFv antibody specific to Vcam‐1 was successfully obtained from phage display librar‐y ,which laid the foundation of subsequent in vivo diagnosis and therapy.

Construction of a Single Chain Variable Fragment Antibody Against Sulfonamides and Its Interaction with Sulfathiazole / 分析化学

The interaction between the antibody and the corresponding target molecule determines the characteristics of immunoassay. In this study, a single chain variable fragment antibody (scFv4C7) derived from the hybridoma strain 4C7 were prepared via genetic engineering technique. The recognition properties of scFv4 C7 was determined and compared to those of the parent monoclonal antibody by indirect competitive enzyme-linked immunosorbent assay(ic-ELISA). Three dimensional structure of the scFv4C7 was presented by Swiss-Model, and sulfathiazole ( STZ) was docked to the scFv4C7 model to obtain the structure of the binding complex. The results from the ic-ELISA showed that the binding properties of scFv4C7 were comparable with the parent monoclonal antibody and STZ was almost completely buried in a deep binding pocket formed by the heavy chain and light chain of the antibody. The interaction between STZ and scFv4C7 was more closely related to the heavy chain and the complementarity-determining region ( CDR ) H3 loop played more important role than other CDR loops. The study preliminary provided the necessary structural information for the preparation of antibody with broader specificity and higher affinity.

Screening and primary identification of anti-CCR7 scFv from large phage library / 中国药理学通报

Aim To screen and identify anti-CCR7 single chain fragments variable(scFv)from lager phage library and to detect the scFv efficiency.Methods The insert ratio of ScFv antibodies library was identified by PCR.The products digested by Sfi I/Not I double enzyme.Panning against breast cancer cell and CCR7 were performed four and three rounds respectively.Positive clones were transformed to E.coli HB2151, and their dissolvability was assayed.The soluble scFv was purified by affinity chromatography, and its relative molecular mass was determined by Western blot.The ELISA assay was used to identify the immunocompetence of the antibody.Immunocytochemical staining and radioimmunoimaging were employed to determine the affinities of scFv binding with CCR7 in cell line and in nude mice.Results The insert ratio of ScFv gene was 90%(18/20), enzyme digest reaction showed the aim products on 1% agarose gel.ScFvs were obviously enriched after 7 round panning.Western blot result showed soluble scFv's molecular mass was about 34 KD.ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435 s cells.Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435 s cells which expressed CCR7.Conclusions ScFvs against CCR7 are successfully acquired by screening the phage antibody library.The soluble ScFvs have high affinity and specifical binding to human breast cancer cells.

Screening of human phage single chain antibody against the recombinant surface protein of Streptococcus mutans and identification of its biological activities / 中国人兽共患病学报

To isolate human phage single chain antibody against surface protein of Streptococcus mutans,the recombinant surface protein of S.mutans(rAP) was used to coat the immune tubes and the phage single chain antibody was prepared through pDAN5 phage antibody library after 5 rounds of panning.The eluted phage was enriched nearly 30 times.In these ways,13 positive clones were obtained and found to be able to bind with rAP in ELISA assay.Then one of the 13 positive clone phage plasmid was used to infect E.coli HB2151 to induce the expression of the non-fusion single chain antibody (ScFv) with IPTG induction.As demonstrated by SDS-PAGE,the molecular mass of this single chain antibody was proved to be 30 kDa and the amount of expression constituted to 30% of the total bacterial proteins.Apparently,the human phage single chain antibody against surface protein of S.mutans with biological activity was successfully screened.

Construction and screening of human phage display antibody library against lung adenocarcinoma / 第二军医大学学报

Objective:To construct human single-chain variable fragment (ScFv) antibody library, and screen out antibodies against lung adenocarcinoma from the library. Methods: The total RNA was isolated from tumor adjacent lymph nodes of the lung adenocarcinoma patients and was used to amplify VH and VL genes by RT-PCR. VH and VL genes was joined with a DNA linker by SOE-PCR to form the ScFv. The gel purified ScFv gene repertoires were cloned into the phage vector pCANTAB5E to construct the primary phage library. Panning against lung adenocarcinoma cell line A549 was performed for four rounds and the phage library was identified. Results: A recombinant phage antibody library was successfully constructed. The fourth phage harvest yielded 115 times as much as that of the first one. During affinity screening, the antibody was enriched with the increase of panning rounds. Positive reactions to A549 were detected in 7 of 10 randomly selected clones, with a positive rate of 70%. Conclusion: A human phage-display antibody library has been successfully constructed. The selected ScFv fragment can specifically bind to human lung adenocarcinoma cell line A549.

Construction of human phage display antibody ScFv library and identification of antibody ScFv against lung adenocarcinoma / 基础医学与临床

Objective To construct a human phage single chain-antibody library, and to sieve out the antibody ScFv against lung cancer from the library. Methods Total RNA was abstracted from lymph node tissue of the lung cancer, and was used to amplify V_H and V_L gene by RT-PCR. V_H and V_L were joined by a DNA linker by SOE-PCR to form the single chain variable fragment ( ScFv) gene. ScFv gene was coloned into the phage vector pCANT-AB5E. Panning against lung cancer cell line A549 was performed and positive clones were chosen for soluble expression. Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning, the number of eluted phages increased by 115 times. Positive reactions to A549 were detected in 7 of 10 random clones. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis. Conclusion ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has specificall avidity to human lung cancer cells.

Construction of gene library of human-originated single-chain variable fragment antibody associated with lung adenocarcinoma / 重庆医科大学学报

Objective:To construct human single-chain variable fragment (scFv) antibodies gene library associated with lung adenocarcinoma. Methods:Total RNA was extracted from the lymphatic tissue near the lung adenocarcinoma.VH and VL fragments were amplified with RT-PCR,the purified VH and VL fragments were used to produce scFv fragment by splicing-overlap-extension PCR. The scFv gene was cloned in pCANTAB-5E Phasmid,then transferred to Ecoli TG1.The insertion ratio of antibody gene was identified with RT-PCR,the positive enzyme-cutting products were identified with 1.5% agarose gel electrophoresis. Results:Clear 28 S and 18 S strips were found in total RNA;The size of VH is about 370 bp,VL is about 350 bp.The size of scFv is about 750 bp.After transformation into Ecoli TG1,2.2?107 CFU/?g ampicillin resistant bacteria colonies grow after overnight culture,and the positive insert ratio was 83.3%. Conclusion:Human single-chain variable fragment(scFv) antibodies gene associated with lung adenocarcinoma was constructed successfully. It could provide bases to further screen the antibody library construction.

Construction of human phage display antibody ScFv library and identification of antibody ScFv against lung adenocarcinoma / 基础医学与临床

Objective To construct a human phage single chain-antibody library,and to sieve out the antibody ScFv against lung cancer from the library.Methods Total RNA was abstracted from lymph node tissue of the lung cancer,and was used to amplify VH and VL gene by RT-PCR. VH and VL were joined by a DNA linker by SOE-PCR to form the single chain variable fragment (ScFv) gene. ScFv gene was coloned into the phage vector pCANTAB5E. Panning against lung cancer cell line A549 was performed and positive clones were chosen for soluble expression.Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning,the number of eluted phages increased by 115 times. Positive reactions to A549 were detected in 7 of 10 random clones. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis.Conclusion ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has specificall avidity to human lung cancer cells.

Expression of Anti-Tumor-Associated Glycoprotein-72 Single Chain Variable Fragment in Human Breast Cancer Cells / 中国普外基础与临床杂志

Objective To investigate the expression of tumor associated glycoprotein-72 (TAG-72) in human breast cancer cells. Methods Two breast cancer cell lines MCF-7 and Bcap-37 were cultured and prepared. TAG-72 expressions in MCF-7 and Bcap-37 were detected immunochemically with anti-TAG-72 single chain variable fragment (scFv). Results TAG-72 was positively expressed in MCF-7 cells but negatively expressed in Bcap-37 cells. Conclusion Tumor associated antigen TAG-72 is expressed in certain human breast cancer cells, which indicate TAG-72 may be used as a tumor maker and anti-TAG-72 scFv may play a role in the diagnosis and treatment of breast cancer.

Screening and identification of human anti-HIF-1? scFv of lung adenocarcinoma / 第三军医大学学报

Objective To screen human anti-hypoxia-inducible factor (HIF)-1? scFv of lung adenocarcinoma from large phage antibody library and identify the positive clones. Methods Panning of large phage antibody library against lung adenocarcinoma cell line A549 and HIF-1? was conducted respectively to select specific antibodies. E. coli HB2151 was infected to induce the expression of soluble scFv. The binding activity and specificity were tested by ELISA and immunocytochemical assay. The expression and relative molecular weight of the soluble scFv was detected by SDS-PAGE and Western blot analysis. Results After panning,the target scFv was enriched,and ELISA results showed that positive reactions to HIF-1? were detected in 5 of 10 random clones with a positive ratio of 50%. Immunocytochemical analysis showed the specific affinity of the antibodies to A549 cells. The soluble human anti-HIF-1? scFv fragments of lung adenocarcinoma were expressed in E. coli HB2151 and then confirmed by SDS-PAGE. The result of Western blotting showed that the relative molecular weight of the soluble scFv was about 30?103. The binding activity and specificity were confirmed by ELISA. Conclusion Human anti-HIF-1? scFv of lung adenocarcinoma is successfully obtained with large phage antibody library technique.

Construction of and screening for phage antibody library of human lung adenocarcinoma / 第三军医大学学报

Objective To construct human single-chain antibody gene library associated with lung adenocarcinoma, and screen lung adenocarcinoma cell A549-specific antibody. Methods The lymphatic tissue near lung adenocarcinoma was used to construct human single-chain antibody gene library. The specific antibody to lung adenocarcinoma cell A549 was screened from the antibody library. Positive clone bacteria were transformed to E. coli HB2151, and their dissolvability was observed. The soluble scFv was purified by affinity chromatography and its relative molecular mass was determined by SDS-PAGE and Western blotting. The specificity of scFv to human lung adenocarcinoma cells was identified with ELISA. Results The phage antibody library of 4.6?107 was constructed successfully. Single-chain antibody of human lung adenocarcinoma was enriched. The ratio of yield was increased gradually, 181 times higher after 5 rounds of panning than after the first round of panning. SDS-PAGE and Western blotting results showed soluble scFv' molecular mass was about 30 000. ELISA showed dissolved antibody had high specificity to bind A549 cells, not MDA-MB-435. Conclusion The single-chain antibody gene library of human lung adenocarcinoma was constructed successfully, and specific antibodies to lung adenocarcinoma were screened, providing the basis for single-chain antibody radionuclide imaging and treatment.

Constructing a fusion molecular with T cell receptor for activation of carcinoembryonic antigen specific cytotoxic T lymphocytes to gastrointestinal tumors / 中华消化杂志

Objective To generate specific targeting T cells against gastrointestinal cancer, a eukaryotic expression vector encoding carcinoembryonic antigen(CEA) specific single chain variable fragment(scFv) fused to the transmembrane and intracellular domains of the signal transducing chain of CD3? was constructed. Methods Anti CEA scFv cDNA was obtained by RT PCR. The transmembrane and intracellular domains of CD3?cDNA was amplified from human T lymphocyte using RT PCR to clone into a eukaryotic expression vector, and anti CEA scFv cDNA was inserted upstream of CD3?. Finally, the eukaryotic expression vector containing scFv CD3? pcDNA3.0 was constructed and sequenced. Results A 810 base pair of anti CEA scFv was in accordance with sequence concerned, a 438 base pair of cDNA of the transmembrane and intracellular domains of CD3? was confirmed as sequence concerning of GenBank. Further identification was carried out through agarose electrophoresis after enzyme digestion. Conclusion We constructed a fusion molecular with both CEA targeting and CD3? signal transduction perporties, which lay a good foundation for generation of modified cytotoxic T lymphocytes against gastrointestinal tumors.

Tumor vessel-specific antibody ScFvH1: Targeting efficiency and anti-tumor ability / 中国肿瘤生物治疗杂志

Objective: To investigate the targeting and anti-tumor ability of the tumor vessel-specific antibody ScFvH1 selected from phage-ScFv library, and to discuss the application of the antibody in clinical diagnosis and therapy of cancer. Methods: The ScFvH1 gene was inserted into pET-28a(+)/EGFP vector containing green fluorescent protein(GFP) gene and pTIG-Trx vector containing thioredoxin gene; the products were then expressed in E.coli and purified by using Ni-NTA. Tumor-bearing mice model was established by subcutanuous injection of cervical cancer cell line HeLa. The mice were injected with purified ScFv-EGFP fusion protein through vena caudalis and the GFP signals were observed by fluorescent microscope to evaluate the targeting ability of the antibody. Meanwhile, the mice model also received intratumoral injection of purified ScFv-EGFP fusion protein to evaluate the anti-tumor effect of the antibody. Results: Soluble ScFvH1 gene and ScFvH1-EGFP protein were successfully expressed in E.coli; a single band was showed in SDS-PAGE after the purification by Ni-NTA. We found that ScFvH1-EGFP fusion protein was enriched to tumor tissues, but there was only weak fluorescent signal when EGFP protein was injected. No EGFP signal was observed in the lung of tumor-bearing mice. Tumor inhibition experiment showed that the tumor growth in the antibody treatment group was similar to that of the PBS control group. Conclusion: The tumor vessel-specific antibody ScFvH1 selected from phage-ScFv library can specifically target tumor vessels, but it has no obvious inhibitory effect on tumor growth. Our findings pave a way for antibody in cancer diagnosis and treatment.

Construction of tumor targeting ScFv library and screening of tumor vessel-specific antibody by phage display in vivo / 中国肿瘤生物治疗杂志

Objective:To obtain phage-displayed ScFv library targeting tumor tissues and to screen for antibodies specifically binding to tumor vessels using in vivo phage display,so as to lay a foundation for diagnosis and treatment of cancer.Methods:The membrane proteins were extracted from the specimens of esophageal carcinoma,stomach carcinoma,brain cancer,lung cancer,and spinal cord tumor.The recombinant phage-antibody system was used to construct a single-chain Fv fragment(ScFv)cDNA library from the total RNA of the BALB/c mice immunized with purified membrane protein.The specific primers of VH and VL were used to amplify the cDNA of VH and VL,respectively,which were then assembled into ScFv gene with a specially constructed linker DNA.The ScFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated samples were transformed into competent E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phage.Using the animal model of human cervical carcinoma(HeLa cells),sepecific phage-ScFvs were selected by phage displaying and panning in vivo.After four rounds,24 phage-ScFvs,which were identified by PCR,were analyzed immunohistochemically.The ScFvs expressed in the tumor tissue slices and negative in control kidney tissue slices were sequenced.Results:Tomors-bearing animal models were established with 7 different kinds of carcinoma cell lines in BALB/c nude mice.It was found that inoculation with HeLa cells resulted in most satisfactory tumorigenesis in nude mice.A ScFv library of 1.6?106 was obtained and a tumor vessel specific phage-ScFv named ScFvH1(VH-linker-VL)was selected from the library.Conclusion:A tumor targeting ScFv library has been successfully constructed and a tumor vessel-specifrc antibody has been identified from the library,which provides a new way for the early diagnosis and therapy of cancer.

Construction and screening of human-originated phage single-chain antibody library associated with esophageal cancer / 中国药理学通报

Aim To construct human phage single-chain antibody library associated with esophageal cancer and to screen the specific scFv against Eca109 cells from the liberary. Methods Metastatic periesophageal lymph nodes of esophageal cancer patients were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. First, we screened graticulely two pairs of primers of the heavy and light regions separately, then the V_H and V_L fragments were first amplified from the cDNA by the polymerase chain reaction (PCR). Second, the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last, the V_H-linker and V_L-linker were assembled into scFv gene fragments by SOE-PCR,and then Sfi I and Not I restriction site were inlet in it. ScFv gene was cloned into the pCANTAB-5E phagemid. Phagemids were introduced into E.coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Recombinant scFv phage library was constracted and PCR was used to identify the insert ratio of scFv antibodies library. Results of SfiI/Not I double digestion reaction positive insert clone were identified by 1.5% agarose gel electrophoresis. The phage library was panned with NHEEC and Eca109 cancer cells in suspension for four rounds. Strongly positive recombinant phage clones were used to infect E.coli HB2151. Expression of soluble scFv was induced by IPTG. Soluble scFv from periplasm were purified by affinity chromatography and identified by SDS-PAGE and Western blot. Cell ELISA , immunohistochemical staining and immunocytochemical staining were used to identify the activity of the soluble scFv. Results The result of agarose gel electrophoresis showed that total RNA of these B cells had two bands of 28 S and 18 S. The size of V_H fragment is about 450 bp,V_L fragment is about 350 bp and scFv is about 850 bp. The competence is 108 cfu??g-1 pUC18 DNA. Randomly digestive reac-tion showed that the positive insert ratio was 91.7% (22/24). After four rounds of panning, the fourth phage yield is 141 times as much as that of the first one. SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30 ku and the brand of 30 ku was stained. Immunohistochemical staining showed strong stainning of the tissue of esophageal cancer, but not the liver and gastric cancer tissue. Immunocytochemical staining showed significant staining of the esophageal cancer line Eca109. The result of cell ELISA assay revealed that soluble scFv had highly specific and could combined with Eca109 cells, but not with BGC-823 and NHEEC. Conclusion A human scFv phage display library associated with esophageal cancer has been constructed successfully and the specific scFv antibody against Eca109 has been identified from the liberary.