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Studies on the structure, mechanism and protein engineering of Bacillus subtilis pimeloyl-CoA synthetase (PCAS)Wang, Menglu January 2017 (has links)
Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14, UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic pathway and acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid (C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis describes the results of biochemical, structural and mechanistic studies of B. subtilis PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high purity and yield. Enzyme activity and kinetic constants were measured using reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at the University of St. Andrews various commercial and in-house screens were used to obtain diffraction-quality crystals suitable for X-ray crystallography. This also included the generation of seleno-methionine (SeMet) labelled PCAS, as well as heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate pimelic acid and the pimeloyl-adenylate intermediate and product PPi were determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily of adenylate forming enzymes. The enzyme is a homodimer composed of two domains, a short N-terminus and a large C-terminal domain and the ligand-bound structures revealed the residues potentially involved in substrate specificity and enzyme catalysis. The enzyme uses an internal ruler composed of a number of conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct dicarboxylic acid substrate. The X-ray structures guided the production of a number of site directed mutants to identify residues involved in the catalytic mechanism and stabilising the acyl-adenylate intermediate. This also allowed rational engineering of the PCAS active site to generate mutants with altered substrate specificity. Mutant PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the synthetic potential of PCAS. The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin pathway, a pyridoxal 5'phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase (AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give AON which is converted to biotin by the action of three other enzymes. We used genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion protein was expressed and purified from E. coli and initial studies suggest that it forms a functional, fused, dimeric enzyme.
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Development of Halomethyl-Triazole reagents for installation of protein post-translational modification mimicsBrewster, Richard Christian January 2018 (has links)
Triazoles have been widely used as amide bond isosteres in chemical biology as linkers and to enhance proteolytic stability. The use of triazoles has grown exponentially since the discovery of the copper (I) catalysed alkyne azide cycloaddition reaction in 2002 as the reaction is solvent and functional group tolerant, and usually high yielding. The reaction is also orthogonal to reactions used in nature, meaning it has become a powerful coupling tool. In post-translational modification (PTM), proteins are modified by covalent attachment of functional groups to amino acid side chains. These PTM processes are generally thought to be dynamic and highly regulated by cell machinery, controlling protein function in response to stimuli. The ability to control function post protein synthesis allows organisms to have a smaller genome, which is advantageous as it reduces the energy required for DNA replication and repair. Research into the function of PTMs has been limited by the difficulty in generating recombinant proteins that bear a single PTM in a specific location. Although many elegant methods have been proposed that solve this problem, to date cysteine alkylation is one of the most successful techniques. For lysine PTMs, thia-lysine II (sLys) derivatives have been shown to be excellent mimics of lysine, where the only perturbation between the native lysine-containing analogue is the switch of a CH2 for S in the side chain. Biotin is a well-known PTM in biotin dependent carboxylases, where biotin is involved in CO2 transfer. Recently biotinylation has also been shown to be a PTM on many other proteins, however the role of biotinylation is not well understood. Biotin triazole III has been shown to be a good mimic of the biotin amide bond and retains excellent affinity to Avidin (Av). In Chapter 1 the effects of modification to the valeryl side chain, and orientation of the biotin triazole bond affect affinity to Av using ITC are investigated. Compounds III, V and VI are shown to have a KD < 120 pM, but further information on the binding affinity of these compounds could not be assessed by ITC. Biotin triazoles III-VI were also shown to be resistant to hydrolysis in serum, unlike the native biotin amide bond, which is hydrolysed by the enzyme biotinidase (BTD). Generation of amide sLys derivatives has been shown to be synthetically challenging. In Chapter 2, the synthesis and applications of chloromethyl-triazole biotin as a sulfhydryl selective alkylation reagent are investigated. The electron withdrawing nature of the triazole was proposed to give a ‘pseudo-benzylic’ halide α to the triazole, thus increasing reactivity. The controlled alkylation of peptides and proteins has shown that chloromethyl-triazole biotin shows enhanced reactivity over many commercial alkylation reagents and also gives good selectivity for cysteine. Alkylation of histone H4K12C gave the singly alkylated product, accompanied by low amounts of double alkylation. Biotinylation was confirmed by Western blot with anti-biotin. Due to the wide range of readily available functional azides, it was envisaged that halomethyltriazoles could be incorporated into other PTM mimics. In Chapter 3, efforts to expand the range of PTMs accessible using halomethyl-triazoles and further enhance the reactivity of chloromethyl triazoles by preparation of bromo- and iodomethyl triazoles are detailed. Synthesis of reagents to mimic malonylation, succinylation and GlcNAcylation PTMs is described and the reactivity of these halomethyl-triazole reagents is assessed. An alternate approach to the development of PTM mimics through cysteine propargylation and subsequent CuAAC coupling is also described in chapter 3. In conclusion, a series of new reagents have been developed to mimic protein PTMs through alkylation of cysteine. The reagents, which include biotin, GlcNAc, succinyl and malonyl mimics, are based on a halomethyl-triazole scaffold and have been successfully reacted with cysteine containing peptides and proteins.
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Chitosan for biomedical applicationsAbbas, Aiman Omar Mahmoud 01 December 2010 (has links)
Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization.
In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization.
It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice.
Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly.
Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses.
Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation.
In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.
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Population pharmacokinetic/pharmacodynamic analysis of erythropoiesis kineticsSaleh, Mohammad Issa Mahmoud 01 May 2012 (has links)
In USA more than 12.5% of all infants are born preterm. Approximately 75% of all perinatal deaths occur among these preterm infants. Preterm infants are frequently very low in birth weight (VLBW) and receive multiple red blood cell (RBC) transfusions. These transfusions pose increased risk of infections and other complications. Since erythropoietin (EPO) stimulates RBC production, EPO treatment of VLBW infants has received attention as a modality for reducing transfusions in this group.
The overall hypothesis of this work is that treatment optimization of EPO of anemia in preterm infants requires a comprehensive knowledge of the behavior of RBC and the pharmacokinetic/pharmacodynamics (PK/PD) relationship between EPO and erythropoiesis. Under that overall hypothesis, the specific aims were: 1) To describe erythropoiesis dynamics in preterm infants, 2) To determine and explain the variability in the response to EPO in preterm infants, 3) To evaluate newborn sheep as an experimental model for erythropoiesis in preterm infants, 4) To test the hypothesis that RBC lifespan is shortened under acute hypoxic stress conditions, 5) To test the hypothesis that EPO receptor (EPOR) pool size increases under hypoxic stress conditions and the change in EPOR pool size can be predicted using EPO clearance measurements, 6) To describe the effect of EPOR pool size changes on erythropoiesis kinetics.
A model that describes erythropoiesis dynamics in preterm infants as a function of the plasma EPO concentration is presented in Chapter 2. In Chapter 3, several covariates are tested for their ability to identify infants with good EPO responsiveness. The lamb is also tested as an animal model for the erythropoiesis in preterm infants (Chapter 4). In Chapters 5-7, the effect of hypoxic stress conditions on RBC survival was explored defining the relation between the efficacy of EPO and survival of RBC produced as a result of EPO administration. RBC lifespan measurement methods are reviewed in Chapter 5. In Chapter 6, a new methodology for the measurement of RBC lifespan under stress conditions is developed. This new methodology is applied in Chapter 7 to explore the effect of hypoxic stress conditions on the survival of RBC. The study presented in Chapter 8 is undertaken to investigate changes in both EPOR pool size and EPO clearance under hypoxic conditions. An erythropoiesis model that accounts for change in the EPOR pool size under stress conditions is presented in Chapter 9.
Analysis of erythropoiesis dynamics in preterm infants demonstrated that a three fold increase in the amount of RBC produced by preterm infants is possible by EPO administration. This emphasizes the potential of using EPO for the management of anemia in preterm infants. Covariate screening identified gestational age as a potential marker for the responsiveness to EPO treatment. PD analysis results in lambs demonstrated similarities between lambs and preterm infants in different erythropoietic characteristics such as sensitivity to EPO in producing RBC, Hb production rate before birth and blood volume. Survival analysis demonstrated that RBC lifespan is not shortened under acute hypoxic conditions Analysis of EPOR mRNA level demonstrated an up regulation of EPOR level under stress conditions accompanied by a parallel increase in EPO clearance. EPOR up regulation under stress conditions level was incorporated in a PD model presented in Chapter 9. The developed model provides a framework for optimizing EPO dosing. Accordingly, an optimal dosing strategy should in general maximize the interaction between EPO and EPOR. Specifically, EPO should be administered when the number of EPOR are close to maximally up-regulated.
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Tumor cells surface-engineered with polymeric particles for use as cancer vaccinesAhmed, Kawther Khalid 15 December 2016 (has links)
Cancer is a group of diseases caused by aberrant continuously proliferating cells capable of metastasis. Despite significant advances in preventive, diagnostic and treatment measures, cancer is one of the major causes of death in the United States, second only to heart diseases. Main treatment approaches are surgery, radiotherapy, chemotherapy, and the recently expanding immunotherapeutic approaches. The main challenge in treating cancer is the ability of cancer cells to mutate and develop resistance to drug treatments therefore lowering the efficacy of chemotherapy in preventing metastatic tumors. Cancer vaccines are a treatment modality that employs the potential of the immune system to recognize and eliminate tumor cells by unmasking tumor cell antigens and generating an effective anti-tumor immune response with an immune memory capable of preventing metastases formation. This dissertation describes and evaluates an innovative cell-particle hybrid cancer vaccine construct involving irradiated tumor cell surface-engineered with polymeric particles using streptavidin-biotin cross-linking. The tumor cells were biotinylated indirectly using biotin-linked antibodies targeting a surface integrin and the particles were loaded with an immune adjuvant and coated with streptavidin. The tumor cells served as the source of tumor antigens and the anchored particles served to confine loaded immune adjuvant to the tumor cells. The vaccine construct was designed to co-deliver tumor antigens and the immune adjuvant to the same antigen presenting cell, a criteria that has been suggested recently to be important for optimal cancer vaccine potency.
The first report on this cell-particle construct was published in my master’s thesis defended in May 2013. In that report, the feasibility of assembling the cell-particle hybrid was demonstrated. However, loading of the immune adjuvant, CpG ODN (cytosine phosphate guanine oligonucleotide), into streptavidin-coated particles was not optimal. In the current studies, this problem was addressed and the cancer vaccine potential of the cell-particle construct was assessed.
We first evaluated a new TLR4 (toll like receptor 4) agonist, PET lipid A (pentaeryhtritol lipid A), for its potential use in cancer vaccines with the intention to incorporate it in the cell-particle hybrid. PET lipid A is a fully synthetic lipid A analog that has been demonstrated to have immunostimulatory properties. We evaluated the potential use of PET lipid A in cancer vaccine applications and the effect of particulate formulations on its adjuvant properties. Results showed improved in vitro immunostimulatory properties for particle based formulations. Upon testing the immunostimulatory properties of PET lipid A in vivo, moderate enhancement in antigen specific cytotoxic T cells stimulation was observed when PET lipid A was delivered in particles, which then translated into a corresponding trend toward increased survival in a prophylactic tumor study. PET lipid A was concluded to be a weak potential cancer vaccine adjuvant and was not chosen as the immune adjuvant to use in the cell-particle hybrid assembly. Instead, CpG ODN (TLR9 agonist) was chosen due to its strong record of efficacy as a cancer vaccine adjuvant.
The second part of this research project aimed at addressing the challenges we encountered previously in achieving acceptable CpG ODN loading of the final streptavidin-coated PLGA (Polylactic-co-glycolic acid) particles. The approach taken was to modify the method used earlier to make the particles in order to circumvent CpG ODN loss. In the modified method the number of steps required to make streptavidin-coated CpG ODN-loaded PLGA particles was reduced and the fabrication media was altered to allow simultaneous particle fabrication and activation of surface carboxyl groups. The modified method resulted in 5-fold higher loading in the final streptavidin-coated particles compared to the original method.
Subsequent to establishing the feasibility of constructing the cell-particle hybrid and characterizing the assembled hybrid in vitro, the in vivo cancer vaccine potential of the designed construct was examined. Two independent murine tumor models were chosen for this purpose, namely prostate cancer and melanoma. The proposed cell-particle hybrid vaccine construct had significant therapeutic outcomes in the prostate cancer tumor model where mice vaccinated with cell-particle hybrids were the only group to show significant improvement in survival compared to untreated controls whereas no other vaccine formulation had such an effect. Unfortunately, no prophylactic benefit was observed from any of the vaccine formulations used in the melanoma tumor model involving irradiated GM-CSF (granulocyte macrophage colony stimulating factor)-secreting B16.F10 cells. In vitro examination of the immunostimulatory properties of all cell lines used in these studies revealed that transfected and parent B16.F10 cells (representing murine melanoma) were possibly immunoinhibitory whereas RM11 (representing murine prostate cancer) cells lacked such immunosuppressive effect in vitro.
Our objective was to design and evaluate a new cancer vaccine construct that improved the immunostimulatory properties of irradiated tumor cell based vaccines. The approach taken was to surface engineer tumor cells with immune adjuvant loaded polymeric particles. We reported a simple method for fabricating streptavidin-coated PLGA particles and a versatile method of tumor cell surface engineering. We found that the efficacy of tumor cell-based vaccines can be inconsistent across tumor models and the in vitro immunosuppressive effect of tumor cells might be a contributing factor.
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PLP-Dependent α-Oxoamine Synthases: Phylogenetic Analysis, Structural Plasticity, and Structure-Function Studies on 5-Aminolevulinate SynthaseTurbeville, Tracy D 29 June 2009 (has links)
5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are two of four homodimeric members of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. The evolutionary relationships among α-oxoamine synthases representing a broad taxonomic and phylogenetic spectrum have been examined to help identify residues that may regulate substrate specificity.
The structural plasticity of ALAS has been documented in studies of functional circularly permuted ALAS variants and the single polypeptide chain ALAS dimer (ALAS/ALAS) exhibiting a greater turnover number than wild-type ALAS. An examination of the contribution of each ALAS/ALAS active site to the enzymatic activity shows that each active site makes distinct contributions to the steady-state activity of the enzyme. Chimeric ALAS/AONS proteins exhibited an oligomeric structure with two sites having ALAS activity and two sites having AONS activity. Remarkably, the steady-state rates for both the ALAS and AONS activities were lower than that observed in the parent enzymes, while the reactivity of the ALAS sites in ALAS/AONS was similar to that of wild-type ALAS. We propose that the different contribution of each active site to the steady-state activity of ALAS/ALAS and the reduced steady-state activities of the ALAS/AONS chimera, compared to the parent enzymes, relate to different extents of conformational changes associated with product release due to the strain caused with the linking the two ALAS (or ALAS and AONS) subunits. Thus, the extensive plasticity seen in ALAS extends to another member of the α-oxoamine family, AONS.
In the α-oxoamine synthase family a conserved histidine hydrogen bonds with the phenolic oxygen of PLP and may be significant for substrate-binding, PLP-positioning, and maintaining the pKa of the imine nitrogen. The replacement of this conserved histidine, H282, with alanine in murine erythroid ALAS has multiple effects on the spectral, binding, and kinetic properties of the enzyme and supports the conclusion that H282 plays multiple roles in the enzymology of ALAS. Altogether, these results imply that amino acid H282 coordinates the movement of the pyridine ring with the reorganization of the active-site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.
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Rational and combinatorial protein engineering for vaccine delivery and drug targetingWikman, Maria January 2005 (has links)
This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting. In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines. In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.
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A Novel Approach for Detection of Several Tuberculosis Markers Using Diffractive OpticsKim, Nari 30 May 2011 (has links)
Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.
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A Novel Approach for Detection of Several Tuberculosis Markers Using Diffractive OpticsKim, Nari 30 May 2011 (has links)
Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.
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A generic capture assay for immunogenicity, using BiacoreEngqvist, Martin January 2013 (has links)
The purpose of this investigation was to create and optimise a capture assay for the detectionof anti-drug antibodies (ADA) in human plasma, using Biacore. We also dealt with the nonspecificplasma binding to mouse-derived anti-biotin which may occur in the capture assay.By paying attention to these things we aimed at reaching as high sensitivity as possible for theADA detection. The capture assay also benefited and gained flexibility from using the same regenerationsolution irrespective of drug and from having a composition that minimises the risk ofdamaging drug epitopes.
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