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1
Low, Nigel Murray. "Mimicking antibody affinity maturation." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364567.
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Molari, Marco. "Modeling and Bayesian inference for antibody affinity maturation." Thesis, Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLE017.
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La Maturation d’Affinité (MA) est le processus biologique grâce auquel notre système immunitaire génère de puissants anticorps contre les nouveaux agents pathogènes rencontrés. Ce processus est également à la base de la vaccination, l’une des procédures médicales les plus efficaces jamais mises au point, qui permet de sauver des millions de vies chaque année. La MA présentent encore de nombreuses questions ouvertes, dont les réponses peuvent améliorer la manière dont nous vaccinons. Les mécanismes à la base de la MA sont extrêmement complexes, avec des interactions non linéaires entre nombreux cellules différentes. Dans ce contexte, les modèles théoriques et l’inférence Bayésienne sont des outils précieux pour relier les hypothèses qualitatives aux descriptions quantitatives et extraire informations des données expérimentales. Dans ce manuscrit, nous utilisons ces outils pour aborder certaines questions ouvertes, comme l’effet du dosage de l’antigène sur la qualité de la vaccinationAffinity Maturation (AM) is the biological process through which our Immune System generates potent Antibodies (Abs) against newly encountered pathogens. This process is also at the base of vaccination, one of the most successful and cost-effective medical procedures ever developed, responsible for saving millions of lives every year. AM still present many open questions, whose answers have the potential of improving the way we vaccinate. The mechanisms at the base of AM are extremely complex, involving non-linear interactions between many different cellular agents. In this context theoretical models and Bayesian Inference are invaluable tools, respectively to link qualitative hypothesis to quantitative descriptions and to extract information from experimental data. In this manuscript we make use of these tools to tackle some of the open questions, such as the non-trivial effect of Ag dosage on the outcome of vaccination
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Lang, Birthe Agnetha. "Nanofibrous affinity membranes containing non-antibody binding proteins." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15326/.
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The specific removal of molecules from various media is an area receiving increasing attention. Affnity membranes, i.e. membranes containing ligands, which can specifically capture target molecules, can meet this demand. One important area, in which the use of affinity membranes will be beneficial, is blood filtration, specifically haemodialysis treatments. The specific removal of toxins can reduce treatment time and/or frequency and therefore increase patients' quality of life as well as reduce costs for the health care sector. The presented research investigates the feasibility of combining a new class of nonantibody binding proteins (AdhironTM binders), which can be specifically designed to capture target molecules, with electrospun nanofibrous polysulphone (PSu) membranes to create an affinity membrane for the specific removal of target molecules. Adhiron binders against a model target protein (modified green fluorescent protein (mGFP)) were successfully produced and characterised. Suitable parameters for the electrospinning of PSu into smooth bead-free fibres were identified. Two different approaches for the functionalisation of PSu fibres were evaluated: incorporation of the Adhiron binders within the fibre and attachment of the binders to the functionalised PSu fibre surfaces. With the latter approach functionalisation was achieved by means of attaching Adhiron binders to surface functionalised fibres, on which the Adhiron binders were immobilised via a biotin-streptavidin bridge. The functionalised membrane specifically removed target molecules out of simple and complex solutions.
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Attiya, Said. "Antibody labeling methods for automated affinity electrophoresis on microchips." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0010/NQ59926.pdf.
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Hey, Carolyn McKenzie. "Antibody Purification from Tobacco by Protein A Affinity Chromatography." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/42645.
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Antibodies represent the largest group of biopharmaceuticals. Due to the nature of
their clinical applications, they often need to be produced in large quantities. Plants have
distinct advantages of producing large quantities of recombinant proteins, and tobacco is
arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high
biomass yields and robust transformation technology. However, to produce proteins using
transgenic tobacco for human applications, purification of the proteins is challenging. On
the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus
aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and
purification of antibodies. An affinity chromatography purification step utilizing Protein
A resin introduced early in the purification process can reduce successive unit operations,
thereby reducing the overall process cost. However, directly applying tobacco extract to
Protein A chromatography columns may be problematic due to the non-specific binding
of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA
High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were
studied to provide valuable information for future downstream processes for antibody
purification from transgenic tobacco. The efficiency of the post load wash buffer to
reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the
ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post
load wash preformed best at reducing the non-specific binding of NTP to the ProSep A
resins, while higher salt concentrations were more effective at reducing the amount of
NTP contaminants present during elution of the columns. Using a post load wash buffer
with an intermediate pH between the binding buffer and the elution buffer was more
efficient at eluting our model antibody, human IgG. However, lowering the ionic strength
and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely
eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced
the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract
samples were loaded onto the column. Nevertheless, cleaning the columns with
denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was
effective in regenerating the DBC of the resins and prolonging the life cycle of the resins.
This is important to evaluating the economic feasibility of directly using Protein A
chromatography to recover antibodies from tobacco extract. Of the three Protein A resins
studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a
PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of
5.Master of Science
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Sundberg, Mårten. "Protein microarrays for validation of affinity binders." Licentiate thesis, KTH, Proteomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-48256.
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Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.QC 20111117
Development and applications of protein microarrays
The Swedish Human Proteome Resource (HPR) program
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Qundos, Ulrika. "Antibody based plasma protein profiling." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-126270.
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This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.QC 20130821
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Ye, Jianmin. "The relationship between antibody redox structure and affinity in rainbow trout." W&M ScholarWorks, 2008. https://scholarworks.wm.edu/etd/1539616918.
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Teleost immunoglobulin M (IgM), an 800 kDa tetramer, possesses considerable structural diversity due to the non-uniform disulfide polymerization of its halfmeric or monomeric subunits. However, to date, no plausible functional role for this diversity has been demonstrated or proposed. This research was, therefore, designed to investigate the possible functional role(s) for this diversity using the trout model. The possible relationship between this structural diversity and affinity was specifically addressed. The relationship between high levels of disulfide polymerization and high affinity was demonstrated by selective immunoadsorption and analysis of antibodies isolated during the process of affinity maturation. A pivotal determinative role of antigen/BCR affinity in conferring graded levels of disulfide bonding was demonstrated by the induction of high and mixed affinity antibodies from a single lymphocyte source in vitro. Additionally, transfer of immunopurified antibodies and labeled non-immune immunoglobulins revealed a direct effect of polymerization on antibody half-life, with selective removal of less polymerized Igs and/or retention of more fully polymerized Igs. Thus, this differential effect on half-life also results in an increase of average affinity, accentuating the process of affinity maturation. The converse, modulation of affinity by disulfide variation; however, could not be demonstrated.
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Midelfort, Katarina Senn. "Biophysical characterization of high affinity engineered single chain Fv antibody fragments." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/30051.
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Thesis (Ph. D. in Molecular Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Vita.
Includes bibliographical references.
High affinity antibody binding interactions are important for both pharmaceutical and biotechnological uses. However, designing higher affinity interactions has remained difficult. Both high affinity interactions from nature and the results from directed evolution affinity maturation processes may yield clues about the important structural and energetic contributions to attain these tight associations. In this Thesis, we investigate affinity maturation of antibodies for very high affinity binding. Two single chain antigen-binding fragment (scFv) antibody systems that were engineered to obtain higher affinity interactions through directed evolution were probed using biophysical techniques to illuminate affinity modulation in proteins. First, anti-c-erbB-2 antibodies and their binding partner, the extracellular domain of the glycoprotein tumor antigen c-erbB-2, were examined. Thermodynamic studies were carried out on the originally identified human scFv and three higher affinity mutants. Although the first two steps included either entropic or enthalpic gains to affinity, the third improvement came from both types. This study demonstrates that a single energetic component is not generally responsible for the increased affinity within a given protein-protein affinity maturation pathway. Second, a family of anti-fluorescein antibodies and their binding to the small molecule fluorescein-biotin were explored. The femtomolar affinity matured anti-fluorescein antibody, 4M5.3, was compared to its wildtype high affinity precursor, 4-4-20. Affinity, thermodynamic, kinetic, and structural characterization of the binding identified 4M5.3 as one of the highest engineered affinity protein binding interactions known and
(cont.) illuminated how subtle structural changes can lead to large consequences for the kinetics and free energy of binding. The affinity mechanisms were further studied by the creation of a series of partial mutants. Context dependent and independent mutational effects on binding affinity indicated the extent of complexity in higher affinity mechanisms attained through directed evolution affinity maturation processes. These studies emphasize the importance of a large number of residues working in concert to create a very high affinity binding molecule. Based on these results, both rational design and directed evolution studies will need to allow for mutations in a spatially broad range around the binding site and involve many biophysical contributions to the binding free energy to reach very high antigen binding affinities.
by Katarina S. Midelfort.
Ph.D.in Molecular Systems Toxicology and Pharmacology
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Fernandes, Telma Godinho Barroso Maciel. "Functional monolithic platforms for antibody purification." Doctoral thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/11550.
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Dissertação para obtenção do Grau de Doutor em Química SustentávelFundação para a Ciência e Tecnologia - contracts PEst-C/EQB/LA0006/2011, MIT-Pt/BS-CTRM/0051/2008, PTDC/EBB-BIO/102163/2008, PTDC/EBBBIO/ 098961/2008, PTDC/EBB-BIO/118317/2010 and doctoral grant SFRH/ BD/62475/2009, and Fundação Calouste Gulbenkian
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Marillet, Simon. "Modélisation de la réponse des anticorps : de la structure des complexes immunoglobuline - antigène à la complexité clonale des répertoires de chaines lourdes d'immunoglobulines." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4120/document.
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Cette thèse étudie trois sujets relevant de la biologie structurale, de lagénétique et de l'immunologie.Premièrement, nous développons de nouveaux prédicteurs de l'affinité deliaison de complexes protéiques, produisant des résultats de niveau ``état del'art''. Nous calculons d'abord 12 variables modélisant diverses propriétésstructurales des complexes. Nous générons et évaluons des estimateursutilisant des sous ensembles de ces variables, de façon à identifier les plusperformants. Le logiciel associé est distribué dans la Structural BioinformaticsLibrary.Deuxièmement, nous proposons de nouvelles analyses de complexes Ig-Ag.D'une part nous concevons un classificateur distinguant les types de ligand desIg. D'autre part, nous montrons que le modèle précédent prédit fidèlementl'affinité de complexes Ig-Ag. Enfin, nous quantifions la contribution des CDR3de la chaine lourde à l'affinité de liaison, et montrons qu'il contribuesignificativement plus que les autres CDR.Enfin, nous nous intéressons à la modélisation de la diversité des répertoiresde chaîne lourde des Igs, à partir de données de séquençage de CDR3, dans unmodèle de vaccin chez le poisson. Nous analysons les répertoires dans troisconditions: naifs, vaccinés et vaccinés + infectés. Nous comparons lesrépertoires de deux individus en utilisant la « earth-mover distance », laquelleexploite la correspondance entre clonotypes de deux répertoires, révélant ainsides informations inaccessibles aux méthodes basées sur les indices dediversité.Dépôt de thèseDonnées complémentairesPour caractériser la notion de réponse immunitaire publique / privée, nousquantifions le chevauchement des clonotypes exprimés entre individus de lamême ou de différentes conditionsThis thesis investigates three topics at the cross-roads of structural biology,genetics and immunology.First, we develop a pipeline to design and select binding affinity predictors forprotein complexes, yielding state-of-the art results. The first step is the designand computation of 12 different variables accounting for geometric andphysico-chemical properties of the complexes. The second step is thegeneration and evaluation of models using subsets of these variables, followedby the selection of the best performing ones. The corresponding software isdistributed within the Structural Bioinformatics Library.Second, we provide an analysis of the interface properties of Ig-Ag complexes.In particular, we design a classifier using two descriptors, which is able todistinguish ligand types. We also apply the previous binding affinity predictionmodel to Ig-Ag complexes and obtain accurate predictions. We then develop aquantitative model for the contribution of VH CDR3 to the binding affinity andinteraction specificity, and show that it contributes significantly more thanother CDRs.Third, we model the diversity of VH CDR3 repertoires from Ig RNA sequencingdata in a fish vaccination model. We analyze repertoires from three conditions:naive, vaccinated and vaccinated + infected fish. Comparison of the repertoiresof two individuals uses the earth-mover distance (EMD). By exploiting amapping between the clonotypes of the repertoires, we show that EMD revealsinformation beyond classical methods based on diversity indexes. Tocharacterize the notion of public / private immune response, we quantify theoverlap of clonotypes between individuals of the same or different conditions
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Fong, Robin B. "Affinity bioseparations with smart polymer conjugates containing DNA, streptavidin, and antibody fragments /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/8071.
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Skamaki, Kalliopi. "In vitro evolution of antibody affinity using libraries with insertions and deletions." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/286439.
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In Nature, antibodies are capable of recognizing a huge variety of different molecular structures on the surface of antigens. The primary factor that defines the structural diversity of the antibody antigen combining site is the length variation of the complementarity determining region (CDR) loops. Following antigen stimulation, further diversification through the process called somatic hypermutation (SHM) leads to antibodies with improved affinity and specificity. Sequence diversification by SHM is mainly achieved by introduction of point substitutions and a small percentage of insertions/deletions (indels). Although the percentage of indels in affinity matured antibodies is low, probably due to the low rate incorporation of in-frame indels throughout the course of the SHM diversification process, it is likely that the antibody fold can accommodate higher diversity of affinity-enhancing indels. By in vitro evolution, other researchers have sampled either only restricted diversity of indels or extended diversity of insertions only in specific positions chosen based on structural information and natural length variation. The aim of this thesis was to study the impact of random and high diversity indels on antibody affinity by in vitro evolution. New approaches for construction of libraries with in-frame amino acid indels were applied to enable sampling of indels of different lengths across the entire antibody variable domains. I followed two different approaches for construction of indel libraries. Firstly, a recently developed random approach allowed the construction of libraries with random insertions and deletions. Secondly, a semi-random approach was developed to build libraries with different lengths of insertions that could be widely applied in future in vitro antibody affinity maturation campaigns. Libraries constructed by either of these approaches yielded variants with insertions with improved affinity. Overall, this thesis demonstrates that insertions besides offering alternative routes to affinity maturation can also be combined with point substitutions to take advantage of additive effects on function.
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Karlsson, Mikael. "Determination of antibody affinity and kinetic binding constants in Gyrolab Bioaffy microfluidic CD." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11616.
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Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants.
Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.
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Zhang, Haili. "Functional and Molecular Analysis of Antibody Affinity Maturation in Rainbow Trout (Oncorhynchus mykiss)." W&M ScholarWorks, 1999. https://scholarworks.wm.edu/etd/1539617980.
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陳磊碩 and Lui-sek Chan. "Chemical modification of immunoglobulins and the effects on antigen binding site affinity." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B29913378.
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Chan, Lui-sek. "Chemical modification of immunoglobulins and the effects on antigen binding site affinity /." [Hong Kong] : University of Hong Kong, 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13731506.
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Falk, Ronny. "Systems enabling antibody-mediated proteomics research." Doctoral thesis, Stockholm, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4025.
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Berglund, Lisa. "Selection of antigens for antibody-based proteomics." Doctoral thesis, Stockholm : School of Biotechnology, Royal Institute of Technology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4706.
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Sundberg, Mårten. "Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes." Doctoral thesis, Uppsala universitet, Analytisk kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-259623.
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Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.
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Lippow, Shaun Matthew. "Computational analysis, design, and experimental validation of antibody binding affinity improvements beyond in vivo maturation." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38886.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (leaves 98-110).
This thesis presents novel methods for the analysis and design of high-affinity protein interactions using a combination of high-resolution structural data and physics-based molecular models. First, computational analysis was used to investigate the molecular basis for the affinity improvement of over 1000-fold of the fluorescein-binding antibody variant 4M5.3, engineered previously from the antibody 4-4-20 using directed evolution. Electrostatic calculations revealed mechanistic hypotheses for the role of four mutations in a portion of the improvement, subsequently validated by separate biochemical experiments. Next, methods were developed to computationally redesign protein interactions in order to rationally improve binding affinity. In the anti-lysozyme model antibody D1.3, modest binding improvements were achieved, with the results indicating potentially increased sucesss using predictions that emphasize electrostatics, as well as the need to address the over-prediction of large amino acids. New methods, taking advantage of the computed electrostatics of binding, yielded robust and significant improvements for both model and therapeutic antibodies.
(cont.) The antibody D44.1 was improved 140-fold to 30 pM, and the FDA-approved antibody cetuximab (Erbitux) was improved 10-fold to 52 pM, with an experimental success rate of greater than 60% for single mutations designed to remove undersatisfied polar groups or improve misbalanced electrostatic interactions. Finally, a physics-based improvement to the calculation of the nonpolar component of solvation free energy was implemented and parameterized to address the over-prediction of large amino acids. These results demonstrate novel computational capabilities and indicate their applicability for enhancing and accelerating development of reagents and therapeutics.
by Shaun Matthew Lippow.
Ph.D.
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Thomas, Roula. "The effect of point mutations on the binding affinity of anti-blood group A antibody AC1001." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0021/MQ48186.pdf.
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Swers, Jeffrey Seth. "Isolation and engineering of a high affinity antibody against P-selectin glycoprotein ligand-1 (PSGL-1)." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/32323.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Vita.
Includes bibliographical references (leaves 87-91).
We aim to develop novel protein antagonists of P-selectin adhesion as anti- inflammatory therapeutics. Blocking P-selectin adhesion is particularly attractive because this adhesion mediates leukocyte rolling which occurs early in the inflammatory cascade before extensive tissue damage caused by the amplification of inflammation by proinflammatory cytokines. Currently, no subnanomolar antagonists of selectin adhesion are available. The low affinity of current antagonists results in the need for frequent administration and large doses in order to obtain inhibition. High affinity antagonists are desirable because they can be administered in smaller amounts thus reducing the risk of harmful side effects and reducing production costs. Our approach for developing high affinity antagonists is to combine error prone PCR and in vivo homologous recombination to mimic in yeast the broad spectrum of mutagenic strategies exploited by B cells such as somatic hypermutation, receptor revision (... CDR replacement), receptor editing (chain shuffling), and amino acid insertions and deletions. Together with yeast surface display and flow cytometric screening (FACS), this approach has been used to effect at least a five order of magnitude affinity improvement in a single chain antibody (scFv) directed against the N-terminal 19 amino acids of P-selectin glycoprotein ligand- 1 (PSGL- 1). Three rounds of engineering were performed after an initial pool of binders was isolated from a non-immune scFv library. Chain shuffling was found to be important for generating an improved mutant in the first round of engineering.
(Cont.) For the final round of engineering, four different libraries were generated: one with random mutations, one with preferential replacement of the ... CDR1, one with preferential replacement of the ... CDR1 and the ... CDR2, and one with preferential replacement of the light chain. All of these methods produced two order of magnitude affinity improvements except the light chain exchange library. However, the CDR exchange libraries gave equivalent affinity improvement despite the fact that they were 77 fold smaller than the random mutagenic library. In addition, an insertion in CDR2 of the VH was isolated in the best binder from both of the CDR exchange libraries and this mutation could not have been found through random mutagenesis. These results suggest that chain shuffling is best used when the affinity of the antibody to be matured is weak (> 1 [mu] M). In addition, receptor revision is an equally robust method as random mutagensis for the generation of ultra-high affinity binders. The best antibody from the library with preferential replacement of ... CDR1 and ... CDR2 was converted to an IgG and characterized. It was found to better inhibit P-selectin binding to PSGL-1 than the commercially available antibody KPLI in a static adhesion assay and an in vitro rolling assay. Our integrated approach, made possible by in vivo homologous recombination in yeast, decreases the likelihood of convergence upon a single high affinity solution and increases the probability of obtaining an antibody with desired secretory properties and therapeutic potential. This facile method for combining all the mutational strategies used in nature should prove as a valuable tool in the antibody engineering field.
by Jeffrey Seth Swers.
Ph.D.
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Conti, Sofia Alessandra. "Monoclonal antibodies purification via Protein G and protein A affinity chromatography." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2021.
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realization of a pilot scale affinity protein G chromatography for the purification of small volumes of supernatant from different harvests from the same antibody clone. The objective was to make a study on the productivity of each harvest, investigating for a dilution effect in case of an increasing number of harvests, and to have a rapid retention kit for the quality control of the purified antibody. Moreover, an identical column packed with protein A has been tested with the same products, in order to see if there was space for a yield improvement, and a further scale-up.
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Newman, Peter Michael Pathology UNSW. "Antibody and Antigen in Heparin-Induced Thrombocytopenia." Awarded by:University of New South Wales. Pathology, 2000. http://handle.unsw.edu.au/1959.4/17485.
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Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.
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Hjelm, Linnea. "Selection of affibody and domain antibody binders to the Binding Region (BR) domain of theadhesion protein PsrP of Streptococcus pneumoniae." Thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215246.
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Canelle, Quentin. "Real Time Surface Plasmon Resonance Biosensors, a Powerful Technology to Assess Polyclonal Antibody Avidity." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/216754.
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The present research focused on the development of a new methodology to assess the strength of the interaction between vaccine antigens and elicited polyclonal antibodies through SPR biosensors. Quantifying the binding strength of polyclonal antibodies is of first importance to evaluate the quality of the vaccine as well as to increase the scientific knowledge of immune protection mechanisms. To now the development of such tool has been complicated by the non-specific binding caused by high protein abundance in the blood and serum samples but also by the way of interpreting the data resulting from multi-interaction events measured at the same time. At first, we unsuccessfully tried to segregate the individual affinity contribution of each antibody population by measuring the signal as the sum of singular interactions. Differentiation of the singular contribution would have needed the fulfillment of the “additivity” hypothesis, meaning that each antibody bind identically alone or in mixture with other antibody. This hypothesis was not met and mathematical assessment by the sum of singular contribution led to fitting results that did not reflect the biological reality. It was therefore decided to switch the analysis method and to measure the end association binding level reached by the different samples injected at the same specific antibody content. The dissociation behavior was interpreted by the percentage of binding after long and fixed dissociation time. In a first application, we compared the antibodies elicited by two different commercially available vaccines and we showed that the binding interaction was not concentration dependent as, highly different levels were reached when injecting identical antibody concentration. No statistical significant difference was observed between both vaccines. Research firstly focused on the decrease of the non-specific binding and we found that ionic strength was a key parameter, increasing the buffer salt concentration reduced the non-specific binding without diminishing the binding strength. The sample composition was also a key parameter and purifying the IgG allowed to decrease dramatically the undesired binding events. A second application aimed at showing the equivalence between two different antigen constructions for two antibodies population. Even if identical antigen level immobilization is a challenge, the methodology is completely suitable to perform a 2-dimensional comparison (ligand and analyte). A last application was dedicated to the comparison between D and Q-pan Flu vaccines, and results showed that there was no statistical evidence of significant differences between both vaccines. End association level correlated well with haemagglutination inhibition assay at least when serum samples were not diluted at the same antibody content. This last application also showed that throughput may be extended to more than 50 samples per 80 hoursDoctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished
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Zielonka, Stefan [Verfasser], Harald [Akademischer Betreuer] Kolmar, Stefan [Akademischer Betreuer] Dübel, and Florian [Akademischer Betreuer] Rüker. "The shark strikes twice: Generation of Mono- and Bispecific High-Affinity vNAR Antibody Domains via Step-Wise Affinity Maturation / Stefan Zielonka. Betreuer: Harald Kolmar ; Stefan Dübel ; Florian Rüker." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2015. http://d-nb.info/1111112983/34.
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Cui, Yalun. "Design, Development, and Production of Therapeutic Immunoglobulins for Inhibition of Carboxyethylpyrrole-Induced Angiogenesis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1386090936.
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Byström, Sanna. "Affinity assays for profiling disease-associated proteins in human plasma." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-202616.
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Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer. The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided. Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD. In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies.QC 20170302
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Zuo, Ziwei. "Development of an Optical Fiber Biosensor with Nanoscale Self-Assembled Affinity Layer." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/54590.
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Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have been proven to be highly sensitive and reliable in many applications. With increasing public recognition of threats from bacteria-induced diseases and their potential outbreak among densely populated communities, an intrinsic, low-cost biosensor device that can perform quick and precise identification of the infection type is in high demand to respond to such challenging situations and control the damage those diseases could possibly cause.
This dissertation describes the development of a biosensor platform that utilizes polymer thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity- enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross- linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface of the ISAM-coated fiber, which form the core component of the biosensor.
By immersing such biosensor fiber into a sample suspension, the immobilized antibody molecules will bind the specific antigen and capture the target cells or cell fragments onto the surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and changing the refractive index of the cladding. This change occurring at the surface of the fiber results in a decrease of optical power emerging from the LPG section of the fiber. By comparing the transmitted optical power before and after applying the sample suspension, we are able to determine whether or not certain bacterial species have attached to the surface of the fiber, and as a consequence, we are able to determine whether or not the solution contains the targeted bacteria.
This platform has the potential for detection of a wide range of bacteria types. In our study, we have primarily investigated the sensitivity and specificity of the biosensor to methicillin- resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies have also been conducted on infected mouse tissues and clinical swab samples from human ears, noses, and skin, and in each case, the system was in full agreement with the results of standard culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA infections in clinical swab samples taken from infected patient wounds. It is proposed that nonspecific binding due to insufficient blocking methods is the key issue.
Other bacterial strains, such as Brucella and Francisella tularensis have also been studied using a similar biosensor platform with DNA probes and antibodies, respectively, and the outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3 Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml, while for negative control samples at the same concentration, it only produces a signal reduction of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide range of bacteria in a rapid and relatively inexpensive manner.Ph. D.
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Gibbs, Ebrima. "Evolution of the anti-interferon beta (IFNβ) antibody response in multiple sclerosis patients : IgG subclass distribution, affinity maturation and clinical correlates." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/5281.
Full textAbstract:
Multiple Sclerosis (MS), a chronic degenerative disease of the central nervous system, is
characterized by demyelination, axonal damage, and inflammatory lesions in the white
matter. Symptoms include neurological deficits, relapses and progressive disability. Three
recombinant interferon beta (IFNβ) products and glatiramer acetate are licensed for
treatment. They have been shown to reduce the frequency and severity of relapses and slow
disease progression in about 30% of treated patients. Long-term administration of IFNβ can
result in the development of anti- (IFNβ) antibodies. Binding antibodies (BAbs) bind (IFNβ) and
neutralizing antibodies (NAbs) prevent interaction with its receptor, reducing IFNβ
bioavailability and clinical efficacy.
The detection and characterization of anti-IFNβ antibodies does not adhere to any
internationally recommended standards. A comprehensive strategy is required to elucidate
the antibody properties that play a role in the immune response against IFNβ. To this extent,
our objectives were: first, to investigate the IgG subclass-specificities of BAbs over time;
second, to ascertain the affinity maturation pattern of BAbs and NAbs; and third, to
investigate the effects of NAbs on clinical efficacy.
We used an enzyme-linked-immunosorbent assay (ELISA) to measure relative distribution of
IgG subclass-specific BAbs and found that subclasses not only change over time, but their
distribution varies between subcutaneous (SC) IFNβ-la and SC IFNβ-lb. We also found that
NAb+ patients tend to have higher levels of IgG4 subclass-specific BAbs than NAb- patients.
To investigate the affinity maturation of anti-IFNβ antibodies, we utilized BiacoreTM, a
biosensor device based on the optical phenomenon of Surface Plasmon Resonance (SPR).
Our results indicate that relative antibody affinities, as reflected by antibody dissociation
rates, improve over time in NAb+ patients. Furthermore, we found a close parallel between
antibody affinity and NAb levels.
Our investigation showed that the effects of NAbs on clinical efficacy are delayed, with an
increase in relapse rates being more evident in NAb+ patients than in NAb- patients at year 3
(IFNβ-1b), and at year 3 and 4 (IFNβ-la).
We conclude that there is a need for a quantitative and qualitative framework for monitoring
anti-IFNβ antibodies that could prove valuable for better management of IFNβ-treated MS
patients.
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Poma, Alessandro. "Automatic solid-phase synthesis of molecularly imprinted nanoparticles (MIP NPs)." Thesis, Cranfield University, 2012. http://dspace.lib.cranfield.ac.uk/handle/1826/7911.
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Molecularly Imprinted Polymers (MIPs) are potential generic alternatives to antibodies in diagnostics and separations. To compete with biomolecules in these technological niches, MIPs need to share the characteristics of antibodies (solubility, size, specificity and affinity) whilst maintaining the advantages of MIPs (low cost, short development time and high stability). For this reason the interest in preparing MIPs as nanoparticles (MIP NPs) has increased exponentially in the last decade. Cont/d.
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Hu, Francis Jingxin. "Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-205410.
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Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine.QC 20170418
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Silva, Ivani Jose da. "Avidez de IgG na Toxoplasmose: padronização do pH como caotrópico para quantificação direta de anticorpos de baixa avidez." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-27092011-110151/.
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A toxoplasmose é uma protozoose altamente prevalente que atinge pelo menos um bilhão de indivíduos no mundo. A infecção causada pelo Toxoplasma gondii é benigna e assintomática, mas pode causar perdas visuais ou morte em fetos e pacientes imunossuprimidos. Isto pode ser controlado com diagnóstico e instituição de tratamento, mas depende da determinação de infecção ativa ou recente. O diagnóstico parasitológico é complexo, demorado e só executado em poucos centros, sendo a sorologia específica essencial no diagnóstico da doença. A avidez de anticorpos IgG tem sido utilizada para determinação da infecção recente, porém os testes convencionais de avidez só permitem uma estimativa indireta destes anticorpos a partir dos anticorpos totais e os de alta avidez. A quantificação destes anticorpos de baixa avidez seria interessante devido aos altos títulos na fase aguda da infecção ou como marcadores da atividade da doença. Padronizamos um ensaio imunoenzimático (ELISA), utilizando o pH como agente caotrópico, para permitir a determinação e quantificação dos anticorpos de baixa avidez. Na padronização utilizamos amostras de soro de coelhos experimentalmente infectados ou amostras do banco de material biológico do Laboratório de Protozoologia do IMTSP. Nossos resultados mostraram que pH 3,5 apresentou poder caotrópico semelhante a uréia 6M (r2= 0,9909), e que nos soros experimentais, os anticorpos de alta avidez foram resistentes aos dois caotrópicos associados. Os anticorpos recuperados na eluição com pH 3.5 ou Uréia eram semelhantes quanto a especificidade antigênica por imunomarcação ou Western Blot. A neutralização do anticorpo eluído por pH permitiu seu reensaio por ELISA após 1 hora de renaturação, com a quantificação direta dos anticorpos de baixa avidez.. A reprodutibilidade intra e inter teste foi superior a 95%, embora com resultados piores para o pH 3,5. Uma vez padronizada a reação, foram analisadas 150 amostras de soros humanos com sorologia e avidez conhecidas, composta por grande maioria de soros de alta avidez. As medidas de avidez por porcentagem mostraram um resultado errático, atribuído ao uso de grande maioria de anticorpos de alta avidez, embora a medida dos anticorpos recuperados mantivesse correlação com estimativa a partir da medida indireta (r2= 0.48). Esta abordagem permite a determinação direta dos anticorpos de baixa avidez, que são os anticorpos inicialmente produzidos em um desafio antigênico. Nosso ensaio é semelhante ao imunológico, já que a apresentação de antígenos por exossomos ácidos de células dendríticas foliculares no centro germinativo parece ser o sistema de seleção de clones produtores de anticorpos de alta avidez. As perspectivas futuras de uso da medida dos anticorpos de baixa avidez na toxoplasmose são imensas, desde a relação com a gravidade da doença, pela sua quantidade, ou da presença de infecção recente, principalmente em infecção congênita ou e em imunossuprimidos, ou a reatividade da doença crônica, como na toxoplasmose ocular.Toxoplasmosis is a highly prevalent protozoosis, affecting at least one billion people worldwide. The infection caused by Toxoplasma gondii is asymptomatic and benign but it can cause visual losses in addition to death in fetuses and immunocompromised patients. The agent can be controlled by early diagnosis and treatment, but this therapy depends on the determination of active or recent infection. The parasitological diagnosis is complex, time consuming and only performed in few centers, so specific serology is essential for diagnosis. The IgG avidity tests has been used to determine recent infection, but avidity conventional tests only provide an indirect estimate of low avidity antibodies from the total and high avidity antibodies. The quantification of low avidity antibodies would be interesting due to high titers in the acute phase of infection or as markers of disease activity. Using reversible chaotrope such as pH, we standardized an enzyme immunoassay (ELISA) to allow the determination and quantification of low avidity antibodies. For standardization we used serum samples from experimentally infected rabbits or samples of biological material bank of the Laboratory of Protozoology, IMTSP. Our results showed that pH 3.5 is a chaotrope similar to 6M urea (r2 = 0.9909) in avidity ELISA, and high avidity antibodies had similar resistance to two associated chaotrope in experimental sera. The antibodies recovered on elution with pH 3.5 or urea had similar antigen specificity by immunostaining or Western blot. The neutralizing antibody eluted by pH allowed retest by ELISA after 1 hour of refolding, with direct quantification of antibodies of low avidity. The reproducibility inter and intra test were above 95%, but with worse results for pH 3.5. After standardization, we analyzed 150 samples of human sera with known serology and avidity, composed by a large majority of high avidity samples. Avidity as percent of high avidity antibodies showed erratic results in chaotrope comparison, attributed to the majority of high avidity samples, although the direct measure of low avidity IgG kept correlation with the indirect estimate (r2 = 0.48). This approach allows the direct determination of low avidity antibodies that are early produced in an antigen challenge. Our test is similar to the biology of antibody selection, since antigen presentation by acid exosomes of follicular dendritic cells in germinal center seems to be the system of selection of clones that produce high avidity antibodies. The prospective use of the quantification of low avidity antibodies in toxoplasmosis are attractive, either by the quantitative relationship with the severity of the disease; or the increased presence in recent infections, especially in congenital infection and in immunosuppressed patients, or their relative increase in reactivated chronic disease, such as ocular toxoplasmosis.
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Otani, Yuki. "Time-dependent structural alteration of rituximab analyzed by LC/TOF-MS after a systemic administration to rats." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225506.
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Lundberg, Emma. "Bioimaging for analysis of protein expression in cells and tissues using affinity reagents." Doctoral thesis, Stockholm : School of biotechnology, Royal institute of technology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4862.
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Mohseni, Nodehi Sahar [Verfasser]. "Improved Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Affinity Maturated and Fc-Engineered Antibodies Directed Against the AML Stem Cell Antigen CD96 / Sahar Mohseni Nodehi." Kiel : Universitätsbibliothek Kiel, 2011. http://d-nb.info/1033824208/34.
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D'Souza, Vijay Kenneth. "Pharmacological and molecular characterisation of P2Y receptors in endothelial and epithelial cells." Thesis, University of Wolverhampton, 2007. http://hdl.handle.net/2436/20512.
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In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes.
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Neiman, Maja. "Bead based protein profiling in blood." Doctoral thesis, KTH, Proteomik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-117960.
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This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles. A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays.QC 20130208
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Silva, Sandriana dos Ramos. "Estudo comparativo da região Fc de anticorpos IgG1 murinos anafiláticos e não-anafiláticos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-19052010-134913/.
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Está estabelecido que o processo de glicosilação é essencial para a conformação estrutural e função efetora dos anticorpos. Entretanto, não está completamente claro como diferenças nos carboidratos ligados aos anticorpos podem interferir na sua atividade biológica. Foi previamente descrito que anticorpos IgG1 murinos podem ser divididos em anafiláticos ou não-anafiláticos, de acordo com a sua capacidade de induzir in vivo reação de anafilaxia. Somado a isso, foi verificado que a cadeia de oligossacarídeos N-ligada à molécula de IgG1 é fundamental para a manutenção da sua função efetora. O objetivo do presente trabalho é estudar diferenças estruturais entre os subtipos de IgG murinos que poderiam determinar a sua atividade biológica. O seqüenciamento dos nucleotídeos que codificam os domínios CH2 e CH3 dos dois subtipos de IgG1 permitiu constatar homologia de 100% dessas regiões nas duas moléculas estudadas. Entretanto, ao analisar o padrão de carboidratos N-ligados aos anticorpos IgG1 foi observado maior conteúdo de ácido siálico e fucose na cadeia N-ligada ao anticorpo anafilático em relação à do não-anafilático. Contudo, a remoção de resíduos de ácido siálico por tratamento enzimático do anticorpo IgG1 anafilático resultou na perda da capacidade desta molécula de induzir desgranulação celular in vitro e reação anafilática in vivo, semelhante ao anticorpo IgG1 deglicosilado. Em contraste, a remoção de fucose não afetou a sua função anafilática. A análise por PCR em tempo real da expressão dos genes das enzimas envolvidas no processo de glicosilação das proteínas revelou menor expressão gênica de algumas glicosidases, principalmente as sialiltransferases, no hibridoma e linfócitos B secretores do subtipo IgG1 não-anafilático em relação ao obtido no hibridoma e linfócitos B que secretam a IgG1 anafilática. Além disto, foi observada menor atividade enzimática das sialiltransferases obtidas do hibridoma produtor da IgG1 não-anafilática em relação à do hibridoma que produz a IgG1 anafilática. Em conjunto, estes resultados comprovam que a capacidade de anticorpos IgG1 murinos de induzir anafilaxia é diretamente dependente do conteúdo de ácido siálico presente na cadeia de oligossacarídeos ligada à região Fc do anticorpo, além disso sugerem fortemente que essa maior sialilação observada no tipo anafilático seja resultante da maior expressão gênica destas enzimas e assim da sua atividade enzimática no momento da síntese dos anticorpos.It is well established that the glycosylation process is essential for the structural conformation and effector function of the antibodies. However, it is quite clear how differences in the carbohydrates attached to the antibodies may interfere with their biological activities. It was previously reported that murine IgG1 antibodies can be divided into anaphylactic or nonanaphylactic according to their ability to induce anaphylaxis. Furthermore, it was demonstrated that the oligosaccharide chain N-linked to the IgG1 is essential for its conformation and biological activity. The objective of this work is to study structural differences between these subtypes of murine IgG1 that could determine their biological activity. The sequencing of the nucleotides encoding the CH2 and CH3 domains of these two subtypes of IgG1 showed 100% of homology in the Fc regions of these molecules. In contrast, the analysis of the carbohydrates N-linked to the IgG1 antibodies demonstrated higher sialic acid and fucose contents in the chain attached to the anaphylactic antibody than in the nonanaphylactic IgG1. However, the removal of sialic acid residues by enzymatic treatment of anaphylactic IgG1 antibody resulted in the abrogation of its ability to induce mast cells degranulation in vitro and anaphylactic reaction in vivo as observed to deglycosylated IgG1 antibody. On the other hand, the removal of fucose did not change the anaphylactic activity. The analysis by real time PCR of the gene expression of enzymes that are involved in the protein glycosylation showed lower gene expression of some glycosyltransferases, mainly sialyltransferases, in the hybridoma and B lymphocytes that produce the non-anaphylactic IgG1 compared to those verified in the hybridoma and B cells producer of the anaphylactic IgG1. Furthermore, it was verified lower enzymatic activity of sialyltransferases purified from the hybridoma producer of the non-anaphylactic IgG1 in relation to the hybridoma producer of the anaphylactic antibody. Together, these results prove that the ability of murine IgG1 to induce anaphylaxis is directly dependent of the sialic acid content in the carbohydrate core attached to the antibody Fc region. It is also strongly suggested that this higher sialylation observed in the anaphylactic IgG1 may be resultant of the higher gene expression and enzymatic activity of some sialyltransferases during the antibody synthesis.
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Su, Dan. "Rational design, characterization and in vivo studies of antibody mimics against HER2." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/133.
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Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.
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Brown, Elizabeth L. "Consequences of genital herpes simplex virus infection among vulnerable populations /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/10885.
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Drobin, Kimi. "Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum." Licentiate thesis, KTH, Proteinvetenskap, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-225980.
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Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.QC 20180412
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Troiano, Claudia. "Caratterizzazione di membrane per la purificazione di anticorpi." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amslaurea.unibo.it/12532/.
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Negli ultimi decenni l'utilizzo delle biotecnologie e delle tecniche di DNA ricombinante hanno portato allo sviluppo di farmaci a base di anticorpi monoclonali efficaci nella cura di varie malattie. Lo stadio principale del processo di purificazione impiegato a livello industriale è la cromatografia di affinità con proteina A, ma le operazioni di purificazione comprendono anche altri stadi cromatografici. Molti studi sono mirati alla ricerca di alternative più economiche al processo convenzionale con il ligando naturale per le IgG: è in questo contesto che si inserisce la tesi di laurea. Si cercano dei ligandi che possano sostituire la proteina A utilizzando membrane di affinità; si studiano inoltre processi cromatografici a scambio cationico. Il ligando in esame è l’HPTA e, per facilitare l’interazione ligando-anticorpo, viene utilizzato uno spaziatore, il 2LP, identificato come responsabile del fenomeno dell’adsorbimento non specifico. Sono state eseguite prove di adsorbimento in batch con alcuni contaminanti, legando alle membrane solo il 2LP e valutando varie strategie di reticolazione dei gruppi epossidici ed amminici. Inoltre sono state eseguite prove di adsorbimento in batch con miscele utilizzando il complesso HPTA-2LP, che ha dimostrato scarsa capacità in fase di eluizione ed una perdita di efficienza nel legare le IgG nei cicli cromatografici successivi al primo. Nell’ambito della cromatografia a scambio cationico, sono state studiate membrane non commerciali (PTA-OH) e commerciali (Sartobind S); sono stati eseguiti cicli cromatografici in condizioni dinamiche con soluzioni pure di lisozima: tra i due set di membrane, le Sartobind S hanno mostrato i risultati più promettenti. Le ricerche future dovranno completare la caratterizzazione di questi supporti e, nell’ambito dei processi di affinità, si potrebbe studiare il complesso TRZ-HPTA, in quanto lo spaziatore 1,2,3-triazolo ha già dato buoni risultati in combinazione con altri ligandi.
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Cedergren, Linda. "Expression of recombinant protein including an His-tag to facilitate purification for diagnosis of CCHF and Lassa Viruses." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7064.
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Abstract
Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.
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Gargouri, Dorra. "Synthèse de réactifs multifonctionnels et d'analogues de mycotoxine. Application à la détection de mycotoxines dans des solutions alimentaires." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR041.
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Les mycotoxines sont des métabolites secondaires développées par différentes espèces de champignons. Elles sont présentes dans l’environnement et peuvent entrer dans la chaîne alimentaire, et provoquer des intoxications graves. De ce fait, des doses tolérables pour chaque mycotoxine ont été fixées par l’Organisation Mondiale de Santé. Une quantité infime de ces toxines peut contaminer l’ensemble de la chaîne alimentaire, il est donc nécessaire de mettre au point des méthodes de détection fines et sensibles des toxines résiduelles. Il existe des techniques de laboratoire longues et coûteuses pour détecter la présence de ces composés mais ces techniques ne sont pas à la portée des petits producteurs et difficilement exportable hors des laboratoires de recherche. Afin de mieux satisfaire les besoins du marché agroalimentaire, ce projet de thèse a eu pour but de développer un immunocapteur microfluidique pour la détection des mycotoxines (Aflatoxine B1 et Patuline) basé sur le phénomène de transfert d’énergie par résonance de fluorescence (FRET). Ce dispositif « OFF-ON » repose sur la Méthode SPIT-FRI. L’outil se compose d’un réactif hétérotrifonctionnel « tripode » portant trois fonctions orthogonales entre elles (oxyamine, azoture et thiol). Cette molécule est localisée sur une surface d’or par sa fonction thiol par la méthode SAM. Les deux autres fonctions oxyamine et azoture ont servi respectivement pour le greffage d’un fluorophore jouant le rôle d’un donneur d’énergie et un analogue de mycotoxine. Ce dernier permet d’accrocher un anticorps spécifique anti-analyte marqué par un quencher (Accepteur d’énergie) adéquat au fluorophore. De ce fait, le phénomène de FRET entre ces deux partenaires s’opère. En présence de l’analyte introduit en flux, la forte affinité entre l’analyte par l’anticorps marqué permet une bioreconnaissance entre eux, ce qui a pour conséquence le déplacement de celui-ci. L’éloignement de l’anticorps et de son quencher permet le rétablissement du signal de fluorescence. D’où, une bioreconnaissance de l’analyte par l’anticorps marqué a lieu, ce qui a pour conséquence le déplacement de celui-ci. L’éloignement de l’anticorps et de son quencher permet le rétablissement du signal de fluorescence « ON »Mycotoxins are substances naturally produced by microscopic fungal species. Contamination of human food by such mycotoxins can lead to serious health problems. That's why, the World Health Organization recommended to control the mycotoxin concentration in food. Mycotoxins are detected by various conventional analytical methods. Even if these techniques are efficient, they are frequently expensive and time consuming. The purpose of our present work is to develop a novel immunofluorescence-based device to detect mycotoxins (Aflatoxin B1 and Patulin as proofs of concept). We aim to have a simple and accessible biosensor based as a competitive immunoassay for continuous flow detection involving a fluorescence resonance energy transfer (FRET). The procedure is based on SPIT-FRI method which can be used in different environments. Thus, my PhD work focused on the synthesis of heterotrifunctional crosslinkers « tripods », containing three different orthogonal groups (aminooxy, azido and thiol). This crosslinker is attached on a gold surface through the thiol functional group by self assembly monolayer adsorption (SAM). The aminooxy and azido functional groups are respectively used to graft a fluorophore as a donor energy D and a mycotoxin analog which structurally close to the target (respectively Aflatoxin B2 or Patulin ). The latter will be recognized by a specific antibodies equipped with the appropriate quencher (Acceptor) to enhance the FRET efficiency. Once the antibody bounds to the toxin analogue, the fluorescence in turned « OFF » through Fluorescence Resonance Emission Transfer (FRET). The detection of toxins on flow is based on a competetion immunoassay between the toxin and its analogue. The strong affinity of immobilized antibodies with toxins in food samples leads an interaction between them and will displace the antibody/quencher from the solid phase, and thus turn the fluorescence « ON »
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Boström, Tove. "High-throughput protein analysis using mass spectrometry-based methods." Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-154513.
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In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers. The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.QC 20141022
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Weiser, Armin. "Amino acid substitutions in protein binding." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15962.
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Die Modifizierung von Proteinsequenzen unter anderem durch den Austausch von Aminosäuren ist ein zentraler Aspekt in evolutionären Prozessen. Solche Prozesse ereignen sich nicht nur innerhalb großer Zeiträume und resultieren in der Vielfalt des Lebens, das uns umgibt, sondern sind auch täglich beobachtbar. Diese mikroevolutionären Prozesse bilden eine Grundlage zur Immunabwehr höherer Wirbeltiere und werden durch das humorale Immunsystem organisiert. Im Zuge einer Immunantwort werden Antikörper wiederholt der Diversifizierung durch somatische Hypermutation unterworfen. Ziele dieser Arbeit waren, neue Kenntnisse über die Mikroevolution von Antikörpern während der Immunantwort zu gewinnen und die Beziehung zwischen Aminosäureaustauschen und Affinitätsänderungen zu verstehen. Zu diesem Zweck wurde zunächst gezeigt, dass die SPOT Synthese eine präzise Methode ist, um Signalintensitäten drei verschiedenen Bindungsaffinitätsklassen zuzuordnen. Antikörper-Peptid Bindungsdaten, die aus SPOT Synthese Experimenten generiert wurden, bildeten die Grundlage zur Konstruktion der Substitutionsmatrix AFFI - der ersten Substitutionsmatrix, die ausschließlich auf Bindungsaffinitätsdaten beruht. Diese bildete die Grundlage für die Gewinnung eines reduzierten Aminosäuresatzes. Durch einen theoretischen Ansatz konnte gezeigt werden, dass der reduzierte Aminosäuresatz eine optimale Basis für die Epitopsuche darstellt. Für den Prozess der somatischen Hypermutation und Selektion wurde ein neuer Ansatz präsentiert, um für die Affinitätsreifung relevante Mutationen zu identifizieren. Die Analyse zeigte, dass das Spektrum der selektierten Mutationen viel umfangreicher ist als bisher angenommen wurde. Die Tatsache, dass auch einige stille Mutationen stark bevorzugt werden, deutet darauf hin, dass entweder die intrinsische Mutabilität stark unterschätzt wurde oder, dass Selektion nicht nur auf Affinitätsreifung von Antikörpern basiert sondern auch auf ihrer Expressionsrate.A central task of the evolutionary process is the alteration of amino acid sequences, such as the substitution of one amino acid by another. Not only do these amino acid changes occur gradually over large time scales and result in the variety of life surrounding us, but they also happen daily within an organism. Such alterations take place rapidly for the purposes of defense, which in higher vertebrates, is managed by the humoral immune system. For an effective immune response, antibodies are subjected to a micro-evolutionary process that includes multiple rounds of diversification by somatic hypermutation resulting in increased binding affinity to a particular pathogen. The goal of this work was to provide insights into the microevolution of antibodies during the immune response, including the relationship between amino acid substitutions and binding affinity changes. A preliminary step in this work was to determine the accuracy of the SPOT synthesis technique, which could be shown to be an accurate method for assigning measured signal intensities to three different binding affinity classes. A substitution matrix based on data produced with these binding experiments was constructed and named AFFI. AFFI is the first substitution matrix that is based solely on binding affinity. A theoretical approach has additionally revealed that an AFFI-derived reduced set of amino acids constitutes an optimal basis for epitope searching. For the process of somatic hypermutation and selection, a novel approach to identify mutations relevant to affinity maturation was presented. The analysis revealed that the spectrum of mutations favored by the selection process is much broader than previously thought. The fact that particular silent mutations are strongly favored indicates either that intrinsic mutability has been grossly underestimated, or that selection acts not only on antibody affinity but also on their expression rates.
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Kirchgatter, Karin. "Plasmodium vivax: Caracterização Molecular de Recaídas Utilizando um Segmento Polimórfico do Gene MSP1 como Marcador Genético." Universidade de São Paulo, 1997. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-01102004-140121/.
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Plasmodium vivax é a espécie de malária humana de maior distribuição geográfica, com 35 milhões de casos por ano. No Brasil, é a espécie mais prevalente, sendo responsável por cerca de 70% dos casos de malária. Diferentemente do P. falciparum, o P. vivax apresenta hipnozoítas, formas que se mantêm em estágio dormente no fígado e que, após um período de tempo variável, por mecanismos ainda desconhecidos, causam novo ataque malárico denominado recaída. Para contribuir para um melhor conhecimento acerca das recaídas causadas por P. vivax, neste trabalho foram analisadas amostras pareadas referentes ao ataque primário e à recaída de 10 pacientes que se infectaram na Amazônia Brasileira. Através da amplificação de um segmento polimórfico do gene que codifica a Proteína de Superfície do Merozoíta 1 (PvMSP1), foi encontrado um índice de 40% de infecções mistas, presentes inclusive durante a recaída, indicando que a ativação de hipnozoítas não é clonal. Em análise mais detalhada deste segmento polimórfico, utilizando técnicas de clonagem e sequenciamento, foi possível verificar que a população de parasitas obtida durante o ataque primário é idêntica àquela que surge nas recaídas. O estudo da resposta IgG específica naturalmente adquirida contra a região C-terminal da PvMSP1, a mais imunogênica da molécula, demonstrou, durante a recaída, um aumento nos títulos acompanhado por uma maturação na afinidade destes anticorpos além de um predomínio de IgG1.Plasmodium vivax is the most widely distributed human malarial parasite causing an estimated 35 million cases annually. In some parts of the world, including Brazil where it reaches almost 70% of malaria cases, this is the most prevalent species. Unlike P. falciparum, P. vivax has hypnozoites, hepatocyte dormant stages that cause clinical and parasitological relapses. Unfortunately, the molecular basis of relapses remain poorly understood. This work compared paired primary attack and relapse samples obtained from 10 infected patients from the brazilian Amazon Region using a polymorphic segment of the gene encoding the Merozoite Surface Protein 1 (PvMSP1) as a genetic marker. PCR, Southern blot and DNA sequence analysis demonstrated that the parasite population from the primary attack is identical to the one arising during relapses and that the activation of hypnozoites is not clonal; moreover, a large percentage (40%) of mixed infections, were detected. Studies on the naturally acquired human specific IgG response of these patients against the C-terminal region of the PvMSP1 molecule, the most immunogenic region, demonstrated an increase in the titers, affinity maturation and a predominance of the IgG1 subclass during the relapse.