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Johansson, Daniel X. "Expression and interaction studies of recombinant human monoclonal antibodies /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-137-1/.
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Sheikholvaezin, Ali. "Recombinant antibodies and tumor targeting." Doctoral thesis, Umeå : Univ, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-875.
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López, Cerro Maria Teresa. "Strategies to improve Chlamydomonas reinhardtii as a recombinant protein host: from a small growth factor to a complex monoclonal antibody production." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586310.
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Chlamydomonas reinhardtii has emerged as a promising alternative host for recombinant protein expression. Despite its advantageous characteristics and low-cost production, its use is hampered by low expression levels of nuclear transgenes. In this thesis we test several strategies designed to reduce or overcome this limitation. As a result, on the base of a secreted fusion protein comprising a small growth factor and a reporter, the use of regulatory and stabilizing regions resulted in expression levels ranging from 1 to 100 µg /L of culture. We report the expression of a fully-assembled monoclonal antibody in Chlamydomonas nucleus, therefore, validating Chlamydomonas as a host for complex protein production. The cassettes and high throughput screenings developed emerge as innovative tools expanding the molecular toolbox available for Chlamydomonas nucleus. In addition, a scalable purification method to recover the target protein from culture medium has been developed and validated indicating a simple downstream processing for secreted recombinant protein production. Finally, we report that Chlamydomonas secreted components induce proliferation of murine fibroblasts and have a synergistic effect with supplied hEGF, unveiling the potential of extracellular components of Chlamydomonas for a variety of applications.Las proteínas recombinantes ofrecen un gran potencial para diversas aplicaciones impactando procesos industriales, investigación, el mercado cosmético y el mercado terapéutico. Chlamydomonas reinhardtii es un huésped prometedor para expresión de proteínas recombinantes. A pesar de ofrecer características ventajosas y bajos costes de producción, su uso se ve limitado por bajos niveles de expresión de transgenes nucleares. En la presente tesis se testan diversas estrategias con el objetivo de superar esta limitación. Como resultado, en base a la secreción de una proteína de fusión formada por un factor de crecimiento y un reportero, el uso de regiones reguladoras y estabilizadoras ha resultado en niveles de expresión entre 1 y 100 µg /L de cultivo. Además, en la presente tesis se recoge la expresión nuclear de un anticuerpo monoclonal en Chlamydomonas, así como su secreción y acumulación en el medio. Este anticuerpo está correctamente plegado y reconoce su antígeno. Esto representa un punto clave para Chlamydomonas ya que significa su validación como huésped para la expresión de proteínas complejas. Los vectores y cribados desarrollados emergen como recursos innovadores que expanden la batería de herramientas disponible para la modificación genética del núcleo de Chlamydomonas. Además, se ha desarrollado y validado un método de purificación de proteína recombinante des de medio. La simplicidad de este método de purificación indica la potencia de Chlamydomonas como huésped industrial para la expresión de proteínas recombinantes. Finalmente, en la presente tesis se reporta la proliferación de fibroblastos murinos inducida por componentes secretados por Chlamydomonas y su efecto sinérgico cuando se aplican con el factor de crecimiento humano, revelando así el potencial de los componentes extracelulares de Chlamydomonas.
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Fiddes, Jane L. Sutton Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Development of recombinant human monoclonal antibodies suitable for blood grouping using antibody engineering techniques." Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences, 2007. http://handle.unsw.edu.au/1959.4/40503.
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Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens, monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they perform as well as or better than the human polyclonal antibody preparations which they have replaced. However, the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines in the form ofRNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and cell based RBC antigens. In addition a large naIve library was biopanned against the same antigens in an attempt to isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with respect to size (> 107 clones) and diversity were generated. Biopanning of recombinant libraries resulted in enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human polyclonal sera in the identification of Duffy a RBC antigen phenotyping.
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Cromwell, Mary Ellen Miley. "Self-association, crystallization, and phase separation : understanding intermolecular interactions for a monoclonal antibody /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
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Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 209-236). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Bailey, Laura. "Investigating the influence of long-term culture and feed additions on recombinant antibody production in Chinese hamster ovary cells." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-influence-of-longterm-culture-and-feed-additions-on-recombinant-antibody-production-in-chinese-hamster-ovary-cells(1ccfdb8f-c0a6-49c8-a7a7-5e79b84e2862).html.
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Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.
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Thirumangalathu, Renuka. "Understanding physical and chemical stability of proteins in solution : relevance to therapeutic protein and monoclonal antibody formulations /." Connect to abstract via ProQuest. Full text is not available online, 2007.
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Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 133-143). Online version available via ProQuest Digital Dissertations.
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Maiocchi, Rebecca. "Recovery of rare cells and single cells analysis: different opportunities and challenging applications." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1128669.
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Single-cell biology is a new discipline which aims to address and solve the problem of cellular
heterogeneity. Single-cell omic, which allows the molecular investigation of different cell types in a
high throughput manner, is driving the Precision and Personalized Medicine approaches.
Single B cell isolation strategies, the starting points of the single-cell omics, have become essential
research procedures for efficiently sampling the natural repertoire of immunized animals and
humans (Tiller et al, 2008). The goal of the first study is to demonstrate the feasibility of a novel
approach to rapidly generate recombinant mAbs recovering rare antigen-specific plasma cells
from complex samples derived from immunized mice. TLS Foundation, where this work has been
carried out, has recently invested in Precision Medicine activities and, in particular, in the
development of an alternative method to hybridoma technology to isolate rare antigen-specific B
cells from blood of immunized or infected individuals. In Study 1 we have set-up a FACS sorter
free method for a fast identification and isolation of antigen-specific plasma cells producing IgG
with unique and desirable features and, moreover, we have obtained high yield TAP-PCR products.
This could simplify the actual hybridoma technology procedure for the identification and
molecular cloning of antigen-specific antibodies from single-B cells.
Single-cell biology is also used to address and solve problems related to tumour heterogeneity.
Cancer is one of the research areas that has greatly benefited from single-cell analysis. The
complexity of immune responses to cancer has hampered the development of novel therapeutical
approaches with the exception of monoclonal antibody based-therapies that target specific
immunomodulators. In the last 10 years PD-1 blockade monoclonal therapy has indeed
revolutionized cancer treatments but a substantial population of patients is still unresponsive. To
rescue unresponsive patients, the mechanism of unresponsiveness and phenotype must be
elucidated. The second part of this work (Study 2) deals with the possibility to reveal one of the
mechanism responsible for T cells exhaustion in patients that are non-responsive to monoclonal
anti-PD1 therapy. We demonstrated that there is a correlation between the overexpression of TFX
(Transcriptional Factor X, the real name of the factor has been hidden for confidentiality) and the
onset of the characteristic features of exhaustion. Indeed the KO of this gene restores in T cells
their functionality through a more active metabolism. Further in-depth study of the modulation of
this transcription factor may elucidate the pathways and the genes responsible for the exhaustion
phenotype in T cells.
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Caucheteur, Déborah. "Nouveau format de banques d’anticorps recombinants humains pour un criblage fonctionnel à grande échelle." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT012.
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Les anticorps monoclonaux (mAb) sont depuis les années 2000 devenus des médicaments incontournables et de routine en thérapie et notamment en cancérologie. Le domaine continue à croître très rapidement et devant l’abondance des molécules disponibles, il est de plus en plus important d’apporter des molécules innovantes à haute valeur ajoutée pour la thérapie. Deux grandes approches sont utilisées pour sélectionner ces mAbs : l’hybridation lymphocytaire à partir de souris normales ou humanisées ; Les systèmes de display comme le phage-display. Les intérêts majeurs du phage display sont la rapidité de développement des mAbs, la facilité de manipulation chez E. coli, l’accès aux techniques de "protein engineering". Classiquement, les anticorps sont d’abord sélectionnés sur leur capacité de liaison à l’antigène puis ensuite testés pour leur efficacité fonctionnelle dans des modèles cellulaires. Cependant, seulement une partie de l'activité des anticorps est expliquée par leur liaison à l’antigène et l’activité thérapeutique dépend aussi fortement de leur capacité à recruter le système immunitaire (ADCC) et à activer la cascade du complément (CDC).Ce projet de thèse consiste à développer un nouveau format de banque d'anticorps recombinants combinant la puissance de la sélection par phage display à un criblage fonctionnel au format IgG entières produites en cellules eucaryotes. Ce nouveau système est basé sur des régions initiatrices hybrides contenant à la fois des promoteurs et des séquences signal procaryotes et eucaryotes permettant l’expression dans ces deux systèmes cellulaires, et des évènements de recombinaisons sites-spécifiques transférant le fragment Fab du vecteur de display vers le chromosome d’une lignée cellulaire de mammifère spécialement développée pour aboutir à la sécrétion d’un anticorps humain monoclonal par la cellule. L’approche habituelle de reclonage un par un du vecteur E. Coli au format IgG n’est plus nécessaire puisqu’il se fait directement par transfection. Ce nouveau système rend possible le couplage d’une sélection par phage display à un criblage fonctionnel direct sur une large population de clones monoclonaux humainsSince 2000, monoclonal antibodies (mAb) have become essential and routine drugs in therapy and particularly in oncology. The field continues to grow very quickly and given the abundance of molecules available, it is increasingly important to bring innovative molecules with a high added value for therapy.Two main approaches are used to select these mAbs: hybridoma technology using normal or humanized mice; display systems such as phage-display. The major interests of phage display are the speed of mAb development, the facilities offered by E. coli and the easy access to protein engineering techniques. Typically, antibodies are first selected on their ability to bind to the antigen, and then tested for their functional efficiency in cellular models. However, only a part of the activity of antibodies is explained by their binding to the antigen, and the therapeutic activity also depends strongly on their ability to recruit the immune system (ADCC) and activate the complement cascade (CDC).This thesis project consists in the development of a new recombinant antibody library format combining the power of phage display selection with functional screening in a whole IgG format produced in eukaryotic cells. This new system is based on hybrid promoter and signal peptide regions allowing expression both in prokaryotic and eukaryotic cells, and a site-specific recombination event that exchanges the Fab between the display vector and the chromosome of an especially developed mammalian cell line resulting in the secretion of a monoclonal human antibody by the cell. The usual approach of recloning one by one from E.Coli vector to an IgG format is no more needed since it is done directly by transfection. This new system makes possible to couple selection by phage display with a direct functional screening of a large population of human monoclonal clones
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Lien, Ting-Ya, and 連亭雅. "Selection of Monoclonal Antibody Against Recombinant Grb2-SH2 Domain." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/bzuekd.
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碩士國立中興大學
生命科學院碩士在職專班
102
Cancer was the leading cause of death in Taiwan. Breast cancer was the most prevalent cancer in female. Clinical breast cancer therapies include surgery, chemical and radial therapy. Several drugs were used for chemical therapy, however, these drug treatments have many side effects, even leading to some other cancers development. The drug with low side effect is needed to avoid these side effects. Antibody, from the immune system?has high specificity to a candidate specific cell or a molecul and becomes a potential drug for targeted therapy. Cancer development comes from abnormal growth mechanism from normal cells. According to previous study, Grb2 protein (Growth factor receptor-bound protein 2) with SH2 domain (Src-homology 2 domain) was connected to cancer cell proliferation. In this study, we tried to acquire a SH2 domin specific antibody to prevent cancer cell abnormal growth. MCF-7 cell was used and its genome was served as template to amplified Grb2 gene SH2 domain fragment to further insert this DNA fragment into expression plasmid. SH2 domain fragment of Grb2 was expressed in E.coli expression system. Expressed protein was gathered as antigen to immune the mouse. After elevated immune response, mice were screened and sacrifice to obtain antibody in sera. The B Cells were fused with NS1 myeloma cells to produce hybridoma. We got 2 limes of SH2 antibody producing hybridoma after the monoclonal selection. The 6H3 antibody was highly specific to SH2 protein and will be use to enginerring for scFv drug development in the future.
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Chang, Jui-Shin, and 張瑞昕. "Development and in vitro expression of the recombinant monoclonal antibody against grouper nervous necrosis virus." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/35673236655608790211.
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碩士國立臺灣大學
動物學研究所
97
The aquaculture production of marine fish is an important economic industry in Taiwan. One of the highly value marine fish is groupers. However, the occurrence of viral nervous necrosis disease persists as a stumbling block that besets sustainable production in grouper larvae. Nervous necrosis virus (NNV) is the causative agent of mass mortality of cultured grouper at larval stage. It belongs the betanodavirus of Nodaviridae, with 2 segments of single strand positive sense RNA. The immune system of grouper larvae are not mature enough to be immunized until 20-30 days post hatchery (d.p.h.). Therefore, the larvae are very sensitive to NNV infection within 30 d.p.h. Passive immunization was therefore an alternative prophylaxis strategy against NNV infection at this stage. Five NNV neutralization monoclonal antibodies (mAb) are developed in our previous study. The mAb 9D exhibits high neutralizing activity and recognizes linear epitope of capsid protein. In the present study, the heavy and light chain genes of hybridoma cells of mAb 9D were cloned. The full length of these two genes were determined by 5’ and 3’ RACE PCR, and then inserted into the expression vector, respectively. The constructed vectors were transfected into GF-1 cells. The mRNA of heavy chain and light chain genes were detected by RT-PCR, and the antibody proteins expressed in the transfected cells were detected by western blot. Moreover, the expressed mAb 9D in the culture supernatant of transfected cells was confirmed to recognize NNV capsid protein by western blot. In the future, the constructed mAb may be applied in the passive immunity of grouper larvae in order to decrease threaten of VNN disease.
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郭自晏. "Expression of recombinant classical swine fever virus(CSFV) glycoprotein E2 and production of a monoclonal antibody." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/82815983283740967787.
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Lin, Wei-Fang, and 林為方. "The production of monoclonal antibody with broad spectrum reactivity against calla lily-infecting potyviruses using recombinant capsid protein." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/08634104774290660909.
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碩士國立臺灣大學
植物病理與微生物學研究所
95
Calla lily originated from southern Africa belongs to the genus Zantedeschia, the family Araceae. Recently calla lily has become one of economically important flowering plants and continues to increase in popularity because of its unique shape, spathe coloration and long-term lifespan. During the cultivation of calla lily, viral disease is one of the limiting factors in Taiwan as well as in other countries. Potyviruses are the major viruses infecting calla lily and often cause symptoms of stunt, mosaic, growth decline and flower deformation. The yield and quality of cut flowers are seriously affected. At present, detection methods for potyviruses in calla lily such as reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) are commonly employed. Interestingly, ELISA is considered by the industry to be more suitable than RT-PCR as routine indexing technique of large amount of samples. To save the materials, labors and time in detection, therefore in this study, we tried to generate a monoclonal antibody which can detect as many potyviruses as possible. The highly conserved region of capsid protein (CP) gene of calla lily-infecting potyviruses reported in Taiwan including Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV) was selected after amino acid sequences aligned. The conserved region of 121 amino acids in length was PCR amplified by specific primers of each virus. The PCR fragments were ligated to construct two kinds of expression vectors, recombinant proteins were expressed in E. coli expression system and then purified as antigens for immunization. After cell fusion, we used the expressed proteins and potyvirus-infected plant samples as antigens to screen the hybridoma cell lines by indirect-ELISA. One stable cell line secreting potyvirus-specific antibodies named as MAb C12-C4 with best reactivity and was used for ascites production. The mouse ascites from MAb C12-C4 gave well detectable reactions to antigens at dilution up to 105 times. In the spectrum test, this MAb could detect at least ten other potyviruses by indirect-ELISA. From our experimental results, MAb C12-C4 has the potential to be used for further researches and applications.
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Lin, Wei-Fang. "The production of monoclonal antibody with broad spectrum reactivity against calla lily-infecting potyviruses using recombinant capsid protein." 2007. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-1007200717185100.
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Barros, Georgia. "Downstream Processing of Recombinant Proteins from Transgenic Plant Systems: Phenolic Compounds Removal from Monoclonal Antibody Expressing Lemna minor and Purification of Recombinant Bovine Lysozyme from Sugarcane." Thesis, 2012. http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10774.
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Transgenic plant systems have been proposed as bioreactors in the production of pharmaceutical and industrial proteins. The economic benefits of inexpensive plant production systems could be erased if the downstream processing ends up being expensive.
To avoid monoclonal antibody (mAb) modification or fouling of chromatography resins, removal of phenolics from plant extracts is desirable. Removal of major phenolics in Lemna extracts was evaluated by adsorption to PVPP, XAD-4, IRA-402 and Q-Sepharose resins. Analysis of phenolics adsorption to XAD-4, IRA-402 and Q-Sepharose showed superior dynamic binding capacities at pH 4.5 than at 7.5. The economic analysis using SuperPro Designer 7.0 indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The overall mAb processing cost can be reduced by implementing a phenolics removal step.
To understand phenolics-resin interactions, adsorption isotherms of phenolic compounds (chlorogenic acid, ferulic acid, rutin, syringic acid and vitexin-2-O-rhamnoside) from different phenolic classes on three resins (IRA-402, PVPP, XAD-4) at pH 4.5 and 7.5 were determined. Differences in adsorption with the type of phenolics were observed, and PVPP was not efficient for phenolics removal.
Screening of sugarcane lines for bovine lysozyme (BvLz) accumulation indicated that expression levels are still inadequate for commercial development. To maximize BvLz extraction, pH and ionic strength were evaluated; five conditions resulted in equivalent BvLz/TSP ratio. Membrane filtration process using BvLz extracts attained partial removal of native proteins by the 100 kDa membrane step, but also BvLz loss (21-29%). Regardless of the extraction condition, at least 47% of the starting BvLz was lost during the membrane processing. None of the evaluated extraction conditions caused a substantial recovery of BvLz in the concentrate.
Alternative purification options for the IEX+HIC process, which achieved 95% BvLz purity, were tested. Direct loading of sugarcane extract concentrate on HIC and XAD-4 pretreatment of juice did not recovered BvLz as effectively as the IEX chromatography. Pure BvLz was obtained by the XAD+HIC process, but higher purification fold and HIC yield were achieved by the IEX+HIC process, due to the complete separation of BvLz and 18-kDa protein.
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Van, Blarcom Thomas John. "Antibody discovery and engineering using the anchored periplasmic expression (APEx) Escherichia coli display system with flow cytometric selection." 2009. http://hdl.handle.net/2152/6903.
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The development of recombinant proteins for therapeutic applications has revolutionized the pharmaceutical industry. In particular, monoclonal antibodies are the safest class of all therapeutic molecules and account for the majority of recombinant proteins currently undergoing clinical trials. A variety of technologies exist to engineer antibodies with a desired binding specificity and affinity, both of which are a prerequisite for therapeutic applications. This dissertation describes the implementation of a novel combinatorial library screening technology for the discovery and engineering of antibodies with unique binding properties. Combinatorial library screening technologies are used for the in vitro isolation of antibodies from large ensembles of proteins (libraries) typically produced by microorganisms using molecular biology techniques. Our lab has developed a powerful antibody discovery technology that relies on E. coli display by anchored periplasmic expression, otherwise known as APEx. First, I compared the effects of using combinatorial libraries comprising either smaller, monovalent single-chain antibody fragments (scFv), or the much larger, bifunctional full-length IgG antibodies. These technologies were used to isolate a small panel of antigen specific antibodies from the same library of antibody variable domains amplified from a mouse immunized with the Protective Antigen (PA) component from Bacillus anthracis, the causative agent of anthrax. Overall, IgG display resulted in the isolation of a broader panel of variable domain sequences. Most of these variable domains exhibited substantially reduced affinity when expressed as scFvs, which is consistent with the finding that none of these could be isolated from the equivalent scFv library. These results indicate that the antibody format used during in vitro selection affects which antibody variable domains will be discovered. Second, I developed several modifications of the APEx methodology to allow for more efficient recovery of antibodies with desired properties. Specifically, the system was reengineered to simultaneously account for antibody binding and expression levels in order to isolate the highest affinity antibodies with favorable expression characteristics. Third, the new approach, coupled with optimized fluorescence activated cell sorting (FACS) settings, was used to increase the affinity of an antibody by 35-fold resulting in a K[subscript D] of 100 pM. It was demonstrated that genetic transfer of this high affinity antibody specific for the V antigen of Yersinia pestis, the etiologic agent of the plague, conferred increased protection against intranasal challenge with a 363 LD₅₀ of Y. pestis in mice.text
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Liu, Yang-Wei, and 劉楊威. "Expression of the Major Capsid Protein of Cobia (Rachycentron canadum) Lymphocystis Disease Virus by recombinant E. coli and the production of its monoclonal antibody." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/01089572118826163198.
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碩士國立臺灣大學
微生物與生化學研究所
93
The material of this study is the Cobia(Rachycentron canadum) which were infected by iridovirus and had lymphocystis syndrome. We isolated the genomic DNA of several organs and tissues and designed specific primers to detect and amplify the major capsid protein gene mcp by polymerase chain reaction (PCR). After sequencing, the total length of mcp is 1295 base pairs. Then, we used the software Bioedit to analyze the restriction map and simulate its translation product, MCP. The number of amino acid composing MCP is 431, which suggested that the molecular weight should be between 45 to 50 kD. We isolated viral particles from infected organs and tissues by blending method and emulsified viral particles to immune Balb/c female mouse in order to produce monoclonal antibody (mAb). Immunization methods are traditional method and RIMMS. Then the spleen cells of immuned Balb/c mouse were collected and fused with NS-1 myeloma cells by PEG-inducing method. The fused cells were then selected in HAT medium. The hybridomas, which could secrete anti-MCP antibodies, were screened with enzyme-linked immunosorbant assay (ELISA). Then the hybridomas were subcloned by limit dilution. Two hybridomas producing anti-MCP mAb were obtained and designated as 3C6 and 2A10. The isotypes of 3C6 and 2A10 were IgG1 and IgG2a as heavy chain and same κ as light chain. 3C6 hybridoma secreted higher titer of anti-MCP antibodies and had better growth conditions than 2A10 hybridoma, thus we chose 3C6 hybridoma to produce anti-MCP mAb. Then we purified antibodies by following methods: ammonium sulfate participation, ion-exchange (DEAE) chromatography, and Protein-G affinity chromatography. SDS-PAGE and ELISA were used to analyzed purified products. Finally, we cloned mcp gene into pQE31 expression vectors and expressed MCP by E. coli DH5α. The recombinant MCP protein was purified easily because it carried 6x His-tag on N-terminal. After Ni-NTA affinity chromatography, pure MCP protein was collected, and its molecular weight was about 48 kD. Western blotting showed that recombinant MCP would interact with 3C6 mAb specifically. With indirect ELISA, purified 3C6 antibodies were diluted to 104 times to interact with different concentrations of MCP protein. The MCP concentration ranged form 0.5 to 5 μg/mL showed a better linear relationship, and the detection limit was 0.5 μg/mL. In summary, we produced antibodies which interacted with major capsid protein of iridovirus that were isolated from cobia with lymphocystis syndrome. We hope that the mAb produced in this study can apply in virus detection. Otherwise, the recombinant MCP also has a potential to develop as vaccines.
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Daffis, Stephane [Verfasser]. "Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display / vorgelegt von Stephane Daffis." 2006. http://d-nb.info/980613906/34.
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Chen, Wen-Hung, and 陳文鴻. "Using recombinant protein to develop monoclonal antibody specific to coat proteins of hepatopancreatic parvovirus (HPV)、infectious hypodermal and hematopoietic necrosis virus (IHHNV) and taura syndrome virus (TSV)." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/53642380548667407452.
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碩士國立交通大學
生物科技系所
94
As the FAO (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS) reported, the aquaculture of shrimp is a very important industry. It can support high nutritional food and provide the income for the developing nation. Because of the viral disease, this industry suffers huge loss recently. Therefore, if the detection of virus can be effective as soon as possible, we may prevent the diseases from the shrimp. In this study, we refer to the NCBI database to design primers which are used for amplification of the gene fragment of hepatopancreatic parvovirus (HPV)、infectious hypodermal and hematopoietic necrosis virus (IHHNV) and taura syndrome virus (TSV). These gene-encoding structural coat proteins were cloned into expression vector and transformed into E. coli. The objective was to produce recombinant coat protein with a 6-histidine tag. After induction, the recombinant proteins were produced, purified by Nickel column and used for immunization of BABL/c mice for polyclonal antibody production. The mouse antiserum showed specific immunoreactivity to the recombinant protein as verified by ELISA and Western blot. In HPV, the western blot data indicated that two monoclonal antibodies against the HPV recombinant protein were constructed. The dectection of shrimps with monoclonal antibody 3-24 strain exhibited parallel result as compared with that of PCR diagnosis. In IHHNV, 3-62 strain showed immunoreactivity against the IHHNV recombinant protein or coat protein purified from the IHHNV infected shrimp.