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Chen, Desheng, and chen desheng@deakin edu au. "Development of monoclonal antibodies against Vibrio pathogens." Deakin University. Department of Biological Science, 1991. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20080626.140825.
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Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria.
Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios.
In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as
V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals.
Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates.
All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios.
Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS.
Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.
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Austin, Eric B. "Human monoclonal antibodies." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276187.
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Plumpton, Christopher. "Monoclonal antibodies against phytochrome." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358677.
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Ribeiro, Maricy Alves. ""Contribuição ao imunodiagnóstico da leptospirose humana: ênfase ao uso de anticorpos monoclonais"." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-15032004-161427/.
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A prova sorológica de referência na leptospirose ainda é a soroaglutinação microscópica (SAM). Devido à complexidade desta prova avaliamos alguns testes rápidos para triagem dos anticorpos anti-leptospiras na fase aguda da infecção. Na década de 80, uma hemaglutinação passiva, utilizando frações polissacarídicas de leptospiras, foi considerada apropriada ao diagnóstico precoce, porém esta preparação antigênica incluía muitos antígenos comuns reconhecidos por anticorpos de 4% dos indivíduos normais. Um novo ELISA (enzyme-linked immunosorbent assay) utilizando uma suspensão de antígenos imunodominantes, resistentes à proteinase K, foi padronizado e avaliado quanto ao seu valor diagnóstico. Com 89,9% de sensibilidade e 97,4% de especificidade, esta técnica, referida como PK-ELISA, satisfaz os requisitos necessários para as provas de triagem da leptospirose humana. No entanto, em virtude de alguns reagentes usados nesta preparação antigênica serem importados e muito instáveis, foi proposta a introdução de novos métodos empregando-se anticorpos monoclonais. Em um Acordo de Pesquisa Cooperativa entre o Instituto Adolfo Lutz e o Laboratório Fleury foram produzidos hibridomas contra leptospiras. Dois deles foram selecionados para dar continuidade ao estudo: um, secretando anticorpos monoclonais (AcM) para um epítopo detectado em 16 de 23 sorovares do gênero Leptospira mais freqüentes em nosso meio (clone A12P4), e outro específico a somente um sorogrupo patogênico, icterohaemorragiae (clone H7P1). O AcM A12P4, uma imunoglobulina G2B (IgG2B), reagiu com epítopo presente nos componentes de pesos moleculares (PM) de 16-18 kDa dos lisados de leptospiras das cepas RGA e M-20, quando separados na eletroforese em gel de poliacrilamida, e com componentes de PM de 75-84kDa dos sorovares copenhageni e canicola. Por sua vez, o AcM H7P1, uma imunoglobulina G, reagiu com um epítopo comum a várias frações de PM acima de 21 kDa da cepa RGA e com componentes de PM de 21-22 kDa e de 75-82 kDa da cepa M-20. Os monoclonais foram empregados em provas imunoenzimáticas para a detecção de anticorpos específicos em amostras séricas pareadas coletadas de 52 pacientes com leptospirose, e do grupo controle que incluiu amostras séricas de 57 pacientes com outras doenças consideradas no diagnóstico diferencial, e de 68 indivíduos normais. Estas provas, no entanto, não foram satisfatórias. Finalmente, um novo ELISA foi desenvolvido no presente estudo que utiliza a suspensão de antígenos AgMc, purificados por cromatografia de afinidade utilizando a Sepharose 4B ativada com CNBr acoplada aos anticorpos monoclonais descritos acima. Os resultados obtidos com esta prova foram comparados aos obtidos com outros testes disponíveis em nosso meio, como a SAM e o ELISA clássico (ELISA c). Este novo método, o ELISA AgMc, com 80,70 % e 83,33 % de sensibilidade e especifidade, respectivamente, em relação à SAM; valores preditivos positivo e negativo de 69,70% e 90,10% respectivamente e índice de concordância geral de 82,49%, não parece ser um protocolo promissor para o diagnóstico rápido na leptospirose humana. Além disso, tomando-se a SAM como diagnóstico verdadeiro, os resultados obtidos no novo teste, após a conclusão diagnóstica do grupo de pacientes com a leptospirose, mostrou uma discordância significativa. São discutidas as possíveis explicações para os resultados encontrados.The best serological test for leptospirosis laboratory diagnosis remains the microscopic agglutination test (MAT). Because of the complexity of MAT, we have been developed some rapid screening tests for leptospiral antibodies detection in the acute phase of infection. In the decade of 80, a passive hemagglutination test employing polysaccharide fractions of leptospires was considered appropriate for early diagnosis, but its antigen preparation included common antigens recognized by antibodies from 4% of healthy individuals. A new ELISA (enzyme-linked immunosorbent assay) employing proteinase K resistant immunodominant antigens was developed and its potential diagnosis evaluated. This technique, the PK-ELISA, presented 89.9% sensitivity and 97.4% specificity, and satisfied the requeriments needed for serological screening tests of human leptospirosis. However, some of the reagents used in its antigen preparation are imported and very unstable. So, it was proposed, in a Cooperative Research Accordance between Instituto Adolfo Lutz and Laboratório Fleury, to try new approaches with monoclonal antibodies. Two hibridomas secreting specific monoclonal antibodies (MAb) were selected: one, against an epitope detected in 16 of 23 members of the genus Leptospira (clone A12P4) and the other, specific to the icterohaemorragiae serogroup (clone H7P1). The MAb A12P4, a G2 (IgG2B) immunoglobulin, reacted with an epitope present in the 16-18 kDa components of icterohaemorragiae serogroup and with the 75-84 kDa components of serovars copenhageni and canicola, after whole-cell lysates of the leptospires were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The MAb H7P1, which is an IgG, reacted with an epitope common to several fractions of molecular weight above 21 kDa of strain RGA and with the 21-22 kDa and the 75-82 kDa components of strain M-20. Both monoclonal antibodies were employed in enzyme immunoassays for detecting specific antibodies in serum samples serially colleted from 52 patients with leptospirosis, and from the control group, which consisted of sera from 57 patients with other diseases included in the differential diagnosis, and from 68 healthy individuals. These tests, however, were not satisfactory. A new ELISA was developed in the present study employing an antigen suspension AgMc, purified by affinity chromatography with CNBr-activated Sepharose 4B coupled to the monoclonal antibodies described above. The results obtained with this test were compared to the MAT and to the classical IgM ELISA (ELISA c). The new method, AgMc ELISA, presented serological indices, relatively to reference test MAT, of 80.70 % and 83.33 % of sensitivity and specificity, respectively; positive and negative predictive values of 69.70 % and 90.10 %, respectively, and general agreement index of 82.49 %. So, this test was not considered a promising approach to rapid diagnosis of human leptospirosis. Moreover, the proportion of patients diagnosed as having leptospirosis by the AgMc ELISA and the MAT differ significantly. The possible explanations for the results obtained are discussed.
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Qin, Shi-Xin. "Transplantation tolerance with monoclonal antibodies." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305697.
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Heron, Andrew David. "The stability of monoclonal antibodies." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252169.
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Isaacs, John Dudley. "Improving serotherapy with monoclonal antibodies." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386115.
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Benjamin, Richard John. "Tolerance induction with monoclonal antibodies." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253988.
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Paudel, Subhash. "Shear thinning in monoclonal antibodies." Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/32833.
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Master of ScienceDepartment of Physics
Jeremy D. Schmit
Antibodies are large Y-shaped proteins which are used by immune system to identify and neutralize pathogens. Monoclonal antibody therapy is used to treat different patient conditions. There are problems associated with the manufacturability and deliverability of mAb solutions due to the viscous nature of the protein. The viscosity of antibody solutions increases with the increase in concentration and decreases with applied shear. We want to know why these behaviours are seen and to address this problem we have developed a theory describing the rapid viscosity increase with increasing concentration. We use the polymer theory to explain this behaviour. Here antibodies are treated as polymers. The length of the polymer depend on the aggregation. The reptation time increases approximately as the cubic power of size of aggregate (N³ ). We see the shear thinning behaviour is dependent on the Ab-Ab binding energy and find the relationship between the size of the aggregate and the binding energy. We find aggregate size and morphology using several models for Ab-Ab interaction sites. We use the head to head binding (fAb-fAb binding) model to describe aggregation state in our viscosity theory. The size of the aggregate and hence the reptation time is captured by the binding energy. When the binding energy increases the zero shear viscosity increases and the reptation time decreases. Likewise when the binding energy decreases the zero shear viscosity decreases and the reptation time increases. We have yet to find the correct exponents for the shear thinning behaviour of different mAbs which would be our future work.
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Ueda, Yasuji. "MONOCLONAL ANTIBODIES TO CHICK CRYSTALLINS." 京都大学 (Kyoto University), 1989. http://hdl.handle.net/2433/86412.
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Ohlin, Mats. "Human monoclonal antibody technology a tool to investigate human antibody repertoires /." Lund : Dept. of Immunotechnology, Lund University, 1992. http://catalog.hathitrust.org/api/volumes/oclc/39693827.html.
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Yeung, Douglas Edward. "Characterization of six monoclonal antibodies against the Minute Virus of Mice NS-1 protein, and the use of one in the immunoaffinity purification of NS-1 expressed in insect cells." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/29405.
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Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells.
The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies.
Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Mirza, Myriam. "Characterization of new CFTR monoclonal antibodies." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66882.
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The available antibodies against CFTR are not sensitive enough to detect CFTR at endogenous or near endogenous levels making detection at native levels difficult. We raised two monoclonal antibodies, 22E8 and 23C5, against the R domain of human CFTR with the goal of identifying an antibody sensitive enough to detect CFTR in native airway cells. These antibodies were characterized for their ability to detect over-expressed as well as endogenous levels of CFTR in immunoblotting, immunoprecipitation and immunofluorescence. Their ability to detect CFTR was also compared with commercial antibodies M3A7 and 24-1. We show that 23C5 and 22E8 are more sensitive than the commercial antibodies and are able to detect CFTR in over-expressed and endogenous cells by immunoblotting. However, only 23C5 is able to immunoprecipitate CFTR and neither is able to detect CFTR in native airway cells by immunoblotting or are suitable for immunofluorescence. These antibodies will enable studies of CFTR biogenesis in endogenous cells.A l'heure actuelle les anticorps dirigés contre la protéine CFTR ne sont pas suffisamment sensibles pour détecter cette protéine de facon endogène rendant ainsi l'étude de cette protéine difficile dans les tissus. Notre laboratoire a fabriqué deux anticorps monoclonaux , nommés 22E8 et 23C5, dirigés contre le domaine R de la protéine CFTR. L'abilité de ces anticorps à détecter l'expression de CFTR que ce soit de façon endogène ou lorsque la protéine est surexprimée a été testée à l'aide des techniques d'immunoblotting, d'immunoprécipitation et d'immunofluorescence. Afin de verifier leur sensibilité et leur capacité à détecter la protéine CFTR, ces anticorps ont été comparés aux anticorps M3A7 et 24-1 qui sont disponibles dans le commerce et connus pour détecter de facon optimale la protéine CFTR. Les resultats obtenus dans les lignées cellulaires à l'aide de la technique d'immunoblotting ont permis de montrer que les anticorps 23C5 et 22E8 sont plus sensibles que les anticorps commerciaux, de plus ils sont capables de détecter à la fois les protéines endogènes et sur-exprimées. Bien que l'anticorps 23C5 soit capable d'immunoprécipiter la protéine CFTR, aucun des deux anticorps n'a permis la detection de la protéine CFTR par immunoblotting dans les cellules de culture primaire. De plus, ces anticorps n'ont pas permis la detection de la protéine CFTR par immunofluorescence. Ainsi l'utilisation de ces anticorps nous donnera l'opportunité d'étudier la protéine CFTR dans les cellules l'exprimant de facon endogène afin de mieux comprendre sa regulation et son traffic.
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Giorno, Caterina [Verfasser]. "Glycoengineering of Monoclonal Antibodies / Caterina Giorno." Konstanz : Bibliothek der Universität Konstanz, 2010. http://d-nb.info/1020366117/34.
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Noble, Philip W. "Characterisation of anti-glycan monoclonal antibodies." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12071/.
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The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-associated glycolipids with the intention of producing therapeutically valuable mAbs and to determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer. The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study aimed to determine their fine specificity using a glycan array. 692/29 displayed binding predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens. This was compared to other anti-Lewis mAbs, with differences in specificity being observed. Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful. This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-immune mediated cell death as well as to determine whether non-immune-mediated cell death would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer cells and mAbs stimulating apoptosis may not be very effective. Alternatively, cancer cells are driven to initiate apoptosis by genomic and other aberrations thus if pro-apoptotic pathways are stimulated these cells may be more susceptible to death than normal cells. To investigate the significance of apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was associated with good prognosis in colorectal cancer. This suggested that some tumours were still susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-associated stroma was also an independent marker of good prognosis in colorectal cancer. This may be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals originating from tumour-associated immune cells and stromal cells. As the tumour microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to induce direct cell death without the need for effector cells or complement would be an advantage. Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant colorectal cancers. Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response, which can yield IgG mAbs capable of binding a high proportion of ovarian cancers. In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis provides the basis for the production of new anti-glycolipid antibodies that may also be able to induce direct cell death.
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Holdsworth, Mary Louise. "Characterisation of phytochrome using monoclonal antibodies." Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35466.
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Characterisation of phytochrome using monoclonal antibodies Mary L. Holdsworth Native oat phytochrome has been purified to homogeneity and used to produce a panel of monoclonal antibodies (mAbs). Selection of mAbs followed early screening against native phytochrome by ELISA, and SDS-denatured phytochrome by "mini" western blotting. Six mAbs which recognised SDS-denatured phytochrome were mapped using proteolytically derived fragments of phytochrome and subsequent immunoblotting. LAS 31 and 33 map to the 6 kDa NH2-terminus and LAS 35 and 41 map to the adjacent 4 kDa sub-NH2- terminal domain. LAS 11 maps to the 64 kDa- chromophore-bearing domain and LAS 32 maps to between 74 and 88 kDa from the NH2-terminus on the COOH- terminal-half of the molecule. A novel protocol for the mapping of conformation-specific mAbs has been developed and used to assign LAS 21, 34 and 42 to the 64 kDa-chromophore-bearing domain. Determination of differential affinities towards Pr and Pfr demonstrated that LAS 42 exhibited a higher affinity for Pfr, LAS 31, 33, 34 and 35 exhibited a higher affinity for Pr. LAS 41 discriminates absolutely against Pfr. LAS 41 has therefore facilitated:- (i) the purification of PfrP, i.e. Pfr which is free of contaminating Pr, (ii) the development of an ELISA for phytochrome photoequilibrium, (iii) the first direct experimental evidence that phytochrome can exist as a stable heterodimer in vitro and (iv) an appraisal of ELISA protocols for determining differential affinities of mAbs towards Pr and Pfr. Spectral analyses of phytochrome in the presence of mAbs have underlined the importance of the 6 kDa NH2-terminus in the maintenance of the spectral integrity of the molecule but have also indicated that the 4 kDa sub-NH2-terminal domain also interacts with the chromophore. Cross reactivity studies amongst phytochrome from monocots and dicots demonstrate that the epitopes recognised by LAS 11, 31, 33, 35 and 41 are not highly conserved. However, LAS 32 recognises phytochrome from every plant species tested, and is therefore recognising a highly conserved region on the molecule.
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Thanh, Le Thiet. "Exon-specific monoclonal antibodies against dystrophin." Thesis, University of Salford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261661.
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Watson, Nigel. "Monoclonal antibodies to human immunoglobulin allotypes." Thesis, University of Strathclyde, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304897.
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Ortlepp, Susan. "Leucocyte integrin activation by monoclonal antibodies." Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359976.
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Alexandrovich, Susan K. "Characterization of monoclonal antibodies against digoxin /." Online version of thesis, 1987. http://hdl.handle.net/1850/10681.
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Machado, Marta Santos Serafim. "Estudo da imunogenicidade de antígenos de Neisseria lactamica: utilização de anticorpos monoclonais." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-14012009-102548/.
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Evidências epidemiológica e imunológica sugerem que o desenvolvimento da imunidade natural contra doença meningocócica pode está associado com a reação cruzada de antígenos em comuns com Neisseria meningitidis e outras bactérias comensais, como Neisseria lactamica. O Objetivo deste trabalho foi de investigar a imunogenicidade de antígenos de vesículas de membrana externa (OMV) de N. lactamica, com ou sem a presença de Bordetella pertussis (BP), utilizada como adjuvante. Grupos de camundongos neonatos da linhagem BALB/c foram imunizados com antígenos de N. lactamica. Os resultados de nossos estudos mostraram o predomínio de altos títulos de anticorpos dos isótipos IgG e IgM com alta e intermediária avidez, depois das imunizações pela via (i.n) com N. lactamica. A análise do soro por immunoblot mostrou proteínas com reatividade cruzada entre as espécies do gênero Neisseria e os anticorpos monoclonais utilizados neste trabalho. Estes resultados sugerem que antígenos de N. lactamica e N. meningititdis em comum, possam ser importantes na imunidade natural contra doença meningocócica, e no desenvolvimento de vacina.Immunological and epidemiological evidences suggest that the development of natural immunity to meningogoccal disease may be associated with crossreactive antigens together with Neisseria meningitidis and other commensal bacteria, like Neisseria lactamica. The present study aimed to investigate the immunogenicity of antigens of outer membrane vesicles (OMV) of N. lactamica with or without the presence of Bordetella pertussis (BP) used as an adjuvant. Groups of neonate BALB/c mice were immunized intranasally antigens of N. lactamica. The results of our studies showed the predominance of high titers of antibodies of IgG and IgM isotipes with high and intermediate avidity after intranasal immunization with N. lactamica. Immunoblot analysis of serum showed cross-reactivity proteins between the species of the gender Neisseria and the monoclonal antibodies used in this study. These results suggest that antigens of N. lactamica and N. meningitidis in common may be important in natural immunity against meningogoccal disease and in the development of vaccine.
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Telles, Andréia Elisa Rodrigues. "Clonagem e expressão de fragmentos de anticorpo de cadeia única (scFv) anti-LDL eletronegativa em Pichia pastoris." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-29092017-110802/.
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As modificações das lipoproteínas de baixa densidade (LDL) são uma etapa essencial na aterogênese pois acarretam a geração de LDL eletronegativa [LDL(-)] que apresenta propriedades quimiotática, citotóxica, imunogênica e pró-inflamatória. O objetivo deste trabalho foi a produção de hibridomas secretores de anticorpos monoclonais anti-LDL(-), a clonagem dos genes que codificam para as cadeias variáveis destes anticorpos, e sua expressão como fragmentos de anticorpo de cadeia única (scFv). A LDL(-) isolada de plasma humano foi utilizada como antígeno para imunização de camundongos BALB/c. Após triagem dos clones, dois anticorpos monoclonais foram obtidos baseados em sua reatividade pela LDL( -) e não pela LDL nativa: 1A3H2 (1A3) e 2C7D5F10 (2C7). Os cDNAs codificante para a cadeia pesada (VH) e cadeia leve (VL), de ambos os anticorpos, foram obtidos por meio de RT-PCR utilizando bibliotecas de oligonucleotídeos que reconhecem todas os genes de domínios variáveis das famílias de VH e VL murinas. Os genes da VH e VL obtidos foram clonados no vetor pGEM-T Easy (Promega®) e suas seqüências determinadas. A VH do anti-LDL(-) 1A3 pertence família J558.84 e fragmento gênico JH2, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A VH do anti¬-LDL(-) 2C7 pertence a família Vmu 3.2 (J558) e fragmento gênico JH4, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A partir disso, oligonucleotídeos sintéticos foram sintetizados a fim de clonar estes segmentos gênicos no vetor pPlgLE de expressão em Pichia pastoris. Foram realizadas três construções: o scFv 1A3, scFV 2C7 e um scFv híbrido (VH do 1A3 e VL do 2C7). Das três construções obtidas, conseguimos expressar o scFv do anti-LDL 2C7D5F10 que demonstrou ser capaz de reconhecer o antígeno. A proteína recombinante expressa tem grande potencial de ser usada no diagnóstico clínico incluindo imunoensaios in vitro e como reagentes para exames que envolvam a obtenção e análise de imagens.Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating electronegative LDL [LDL(-)], which has chemotactic cytotoxic, immunogenic and proinflammatory properties. The aim of this study was the generation of anti-LDL(-) mAbs, the cloning of the genes that code for their variable domains and their expression as single-chain Fv (scFv). LDL(-) was isolated from human blood plasma and used as an antigen for immunization of Balb/c mice. Upon screening, two different mAbs were selected based on their ability to recognize LDL(-) and not native LDL: 1A3H2 (1A3) e 2C705F10 (2C7). The cDNAs that code for VH and VL were obtained by RT-PCR using specific immunoglobulin primer libraries wich recognize all VH and VL murine families. The VH and VL genes were cloned in pGEM-T Easy (Promega®) and sequenced. The anti-LDL(-) 1A3 uses a VH segment from J558.84 and a JH2 segment, while VL uses a 8.24/Jk5 segments. The anti-LDL(-) 2C7 uses a VH segment from Vmu 3.2 (J558) and a JH4 segment, while VL uses a 8.24/Jk5 segments. Oligonucleotides were synthetized and those gene segments were cloned in pPIGLE a Pichia pastoris immunoglobulin expression vector. We obtained three scFv constructions: scFV 1A3, scFv 2C7 and a husk hybrid, harboring 1A3 VH and 2C7 VL. Among those, we expressed the scFv anti¬-LDL(-) 2C7 that are able to recognize the antigen. The recombinant protein has a great potential for clinicai diagnostic applications, including in vitro immunoassays and as imaging reagents.
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Scuro, Loren Semionatto. "Obtenção e estudo das propriedades de hibridomas produtores de anticorpos monoclonais anti-IL6 humana." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317407.
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Orientador: Wirla Maria da Silva Cunha TamashiroDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O objetivo do presente trabalho foi a obtenção e caracterização de anticorpos monoclonais contra a IL6 humana recombinante (hr) para serem empregados em ensaios imunoenzimáticos do tipo ELISA (Enzyme linked immunosorbent assay) de detecção da IL6 humana nativa, presente em fluídos biológicos de pacientes portadores de quadros onde os níveis de IL6 encontram-se elevados, ou então produzida por monócitos humanos e murinos ativados in vitro. Dois grupos de hibridomas (1A6 e 3B1) secretores de anticorpos monoclonais anti-IL6 foram obtidos pela fusão de células de mieloma da linhagem SP2 Ag14/0 com esplenócitos de camundongos BALB/c, previamente imunizados com a IL6 humana recombinante. Esses hibridomas foram selecionados com base em sua reatividade com a hrIL6, através de ensaios do tipo ELISA indireto. As imunoglobulinas (Igs) monoclonais produzidas pelos hibridomas dos dois grupos são do isotipo IgG1 Kappa e foram purificadas de líquidos ascíticos e dos sobrenadantes de cultura por cromatografia de afinidade. As proteínas purificadas foram conjugadas com biotina para uso em ensaios de ELISA de captura da hrIL6, de modo a se identificar um ou mais pares de anticorpos adequados a esse tipo de teste, bem como para definir a sensibilidade de detecção da citocina. O par de anticorpos monoclonais 3B1E4 x 1A6F10-biotinilado se mostrou mais promissor nos ensaios de ELISA, detectando a citocina recombinante entre 8 e 512 ng/mL, liberando densidades óticas mais elevadas. Todos os anticorpos monoclonais (AcMos) anti IL6 estudados foram capazes de neutralizar a atividade biológica da citocina em ensaios empregando o hibridoma B13.9, uma célula dependente de IL-6 para seu crescimento. Finalmente, o par de hibridomas anti IL6 3B1E4 e 1A6F10 foi estudado quanto às suas principais características de cultivo: crescimento, produção in vitro dos anticorpos, consumo de glicose e produção de lactato e amônia. O seqüênciamento da porção N-terminal do par 3B1E4 e 1A6F10 revelou que as cadeias leves dos dois anticorpos apresentam seqüência idêntica de aminoácidos. Porém, a análise dos dez resíduos de aminoácidos presentes na região variável das cadeias pesadas resultou em seqüências completamente distintas nos dois monoclonais, sendo um forte indício de diferença nos sítios de ligação ao antígeno dos anticorpos estudados
Abstract: In the present study we have developed monoclonal antibodies against human recombinant (hr) IL6, for the use in ELISA assays to detect the human native IL6 present in biological fluid of patients or in supernatant of activated human and rodent monocytes. Two families of hibridomas (1A6 and 3B1) secreting MAb against-IL6 were obtained from the fusion of mieloma cells from SP2 Ag14/0 lineage with spleen cells from BALB/c mice, previously immunized with human recombinant IL6. Those hibridomas were selected on the basis of their reactivity with the hrIL6, through an ELISA indirect assay. The monoclonal immunoglobulins (Igs) produced by these hibridomas are from IgG1 Kappa isotype and were purified from ascitic fluid and culture supernatant by affinity chromatography. The purified proteins from the ascitic fluid were conjugated with biotin for the use in ELISA hrIL6 capture assay, to identify one or more pairs of antibodies appropriated to this kind of test, as well to define the sensibility of cytokine detection. The pair of MAbs 3B1E4 x 1A6F10-biotinilates was shown promising in ELISA assays, detecting the recombinant cytokine between 8 and 512 ng/mL, liberating elevated optical densities. All of the a-IL6 MAbs studied were capable to neutralize the biological activity of the cytokine in attempt employing the hibridoma B13.9 IL-6 dependent. Finally, the pair of hibridomas a-IL6 3B1E4 and 1A6F10 was studied as regards his main cultivation characteristics: growth, MAb production in vitro, lactate and ammonia production and glucose consumption. The N-Terminal portion sequence of 3B1E4 and 1A6F10 revealed that both light-chains present identical amino acid sequence. However, the analysis of the ten amino acid residues present in variable region of heavy-chains resulted in completely distinct sequences in both antibodies, being a strong indication of difference in their ability to recognize the antigen
Mestrado
Imunologia
Mestre em Genética e Biologia Molecular
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Chen, Jianqing. "Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96 potential use for extracorporeal immunoadsorption with enhanced tumor radioactivity retention of iodine, indium and rhenium /." Lund : Lund University, the Jubileum Institute, Dept. of Radiation Physics, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39725797.html.
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Evans, Rachael Yvonne. "The production of anti-idiotopic antibodies to monoclonal anti-RhD antibodies." Thesis, Lancaster University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274194.
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Marques, Carlos Humberto. "Aspectos fundamentais à implantação da tecnologia de produção de anticorpos monoclonais humanizados com potencial aplicação terapêutica." Instituto de Tecnologia em Imunobiológicos, 2005. https://www.arca.fiocruz.br/handle/icict/5781.
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Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
Os anticorpos monoclonais possuem diversasaplicações em transplantes, na composição de conjuntosde reativos para diagnóstico, grande variedade de doenças auto-imunes e, principalmente, na terapia do câncer. A tecnologia de produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas, possibilitando a obtenção de anticorpos humanizados dirigidos a uma grande variedade de antígenos específicos. A baixa seletividade das metodologias atuais para diagnóstico e terapia de neoplasiasconstitui um dos principais empecilhospara a prática oncológica. Nesse particular, a utilização de imunoglobulinas submetidas à engenharia genética já é uma realidade e significa um avanço estratégico, abrangendo cerca de 25% do mercado biofarmacêutico global de proteínas terapêuticas. Este trabalho aponta os aspectos fundamentais à concretização da metodologia de humanização de anticorpos por transplante das regiões determinantes de complementaridade – CDR, com ênfase em uma proposta de produção do anticorpo anti – CD20 contrao Linfoma Não-Hodgkin. A introdução do Instituto de Tecnologia emImunobiológicos - Bio-Manguinhos nesse promissor e importante mercado de biofármacos através da implantação da metodologia de humanização do anticorpo monoclonal murino anti-CD20 é objeto desta dissertação. Viabilizar sua produção torna-se extremamente importante, tanto para a identificação precisa e precoce da enfermidade, quanto para atender um segmento do mercado brasileiro ainda desprovido de tratamento abrangente e eficaz. A apresentação do estudo dos anticorpos, sua estrutura e características, o estudo dosdiferentes sistemas de expressão, cultivo, purificação,bem como a proposta de reestruturação e redimensionamento do Laboratório de Tecnologia de Anticorpos Monoclonais, parcerias, colaborações, recursos humanos necessários e aspectos de mercado, são aqui considerados.
Monoclonal antibodies (Mabs) have several applications in transplants, reagents for diagnosis, a great variety ofauto-immune diseases and mainly, in cancer therapy. Mabs production employing recombinant echnologymade a revolution in immunoglobulinsgeneration, enabling the production of humanized antibodiesthat recognize specific antigens. The low selectivity of the current techniquesfor neoplasm diagnosis and therapy is one of the major impediments for oncology practice. In this regard, the use of eneticallyengineered immunoglobulins has become a reality and meansa strategic development comprising around 25% of the global biopharmaceutical market. This work shows the most important aspects in Mabs humanization through complementary determining regions (CDR) graft methodology, emphasizing a proposal ofanti-CD20 Mab production against non-Hodgkin lymphoma. Introducing the Instituto de Tecnologia emImunobiológicos – Bio-Manguinhos in this important and promising biopharmaceutical market through the establishment of humanization methodology is the main object of this dissertation. Making humanized Mabs production feasible is veryimportant not only for the earlyand precise identification of illnesses, but also to meet a demand of the Brazilian market that still lacks comprehensive and efficient treatment. The study of Mabs, their structureand properties, expression systems, cultivation, purification, new dimensions and structure of the laboratory, partnerships, cooperation,human resources and market analysis are considered herein.
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Storey, E. "Monoclonal antibodies to merozoites of Plasmodium falciparum." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371577.
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Banbury, David N. "New monoclonal antibodies to visualise vesicular compartments." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259782.
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Klutz, Stephan [Verfasser]. "Continuous processing of monoclonal antibodies / Stephan Klutz." München : Verlag Dr. Hut, 2016. http://d-nb.info/1115549731/34.
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Honey, C. R. "Immunosuppression with monoclonal antibodies in neural transplantation." Thesis, University of Oxford, 1990. http://ora.ox.ac.uk/objects/uuid:ea39dc7a-4ada-4c21-8cef-4649cb322646.
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Huang, Ling. "Investigation of soya globulins using monoclonal antibodies." Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302022.
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Anderson, J. "Investigations on bluetongue virus using monoclonal antibodies." Thesis, Open University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374892.
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The main aim of the thesis was the production and characterisation of monoclonal antibodies (Mabs) against bluetongue virus (BTV) and the application of such antibodies to the improvement of BTV diagnosis. The suitability of the indirect ELISA for the detection of antibodies to BTV was examined along with the published protocols for the purification of BTV ELISA antigen. When sera were examined from animals which had experienced either BTV vaccine or any other tissue-culture derived vaccine, host-cell protein contaminants in the BTV ELISA antigen reacted with anti-BHK cell antibodies to give false positive results. None of the published protocols completely removed host-cell protein contaminants. As a solution to these problems monoclonal antibodies were produced to BTV and characterised as to their reactivity against a panel of BTV antigen preparations. They were further characterised by competitive binding assays, and SDS-PAGE analysis of the various BTV preparations with which they reacted optimally. One Mab (3-17-A3), directed against BTV p7, proved suitable for use in a blocking ELISA for the detection of antibodies to all 22 serotypes of BTV. The blocking ELISA proved both more sensitive and more specific than the currently used agar gel precipitin test, and has been adopted as the screening test before importation of semen and embryos into the United Kingdom. A further group of antibodies (characterised by Mab F58) directed against BTV polypeptide NS1, used in a blocking assay detected anti-BTV tubule antibodies. This allows detection of BTV replication in infected animals, and may be used to discriminate between BTV infected animals and animals vaccinated with inactivated BTV vaccine. This work demonstrated the previously unreported BTV-specific nature of BTV NS1. Serum-free suspension culture of the hybridomas and purification of the Mab from tissue-culture supernatant was also developed.
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Bell, Ian Martin. "Monoclonal antibodies as catalysts for cationic cyclisations." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387041.
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Jones, Christine Ann. "Monoclonal antibodies in the study of neuropeptides." Thesis, Imperial College London, 1991. http://hdl.handle.net/10044/1/46848.
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Coswig, Lia Treptow. "Metapneumovirus aviario : suscetibilidade em diferentes sistemas celulares e produção de anticorpos monoclonais." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316628.
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Orientadores: Clarice Weis Arns, Dagmar Ruth Stach-MachadoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: o Metapneumovírus Aviário (AMPV), também denominado vírus da rinotraqueíte dos perus (TRT), é um vírus que acomete e causa infecção no trato aéreo superior das galinhas e perus. Além da infecção respiratória, em poedeiras e matrizes está associado com uma queda significativa na produção de ovos. Em galinhas o vírus está relacionado com a Síndrome da Cabeça Inchada (SCI), uma enfermidade multifatorial, e por este motivo é importante o diagnóstico diferencial. Testes realizados com anticorpos monoclonais (Mabs) e técnicas moleculares são capazes de detectar diferenças entre os subtipos do vírus. Os métodos de diagnóstico incluem isolamento ou detecção da partícula viral ou testes sorológicos. O isolamento das amostras virais SHS-BR-121 (subtipo A) e STG-SHS-1439 (subtipo B) foi realizado em cultura de anel de traquéia, em fibroblasto de embrião de galinha (FEG) e em células chicken embryo related (CER). A comparação das médias dos títulos obtidos para as duas amostras virais, em célula CER, apresentou diferença estatisticamente significativa (P=0,014) com p< 0,05. Neste projeto foi avaliada a suscetibilidades de seis sistemas celulares (CER, Vero, BHK-21, HEp-2, MDBK e ED) para a multiplicação das duas amostras virais (subtipos A e B). Destes sistemas as células CER, Vero e BHK-21 demonstraram ser apropriadas para a replicação vira!. Os títulos nestas células variaram de 105.5 a 107,o/mL DICC50 , para o vírus SHS-BR-121, e 105,5 a 106.0/mL DICC50 para o vírus STG-SHS-1439. As diferenças entre as médias dos títulos nos diferentes sistemas celulares foi estatisticamente significativa para a amostra SHS-BR-121 inoculada em CER em relação as células Vero e BHK-21 (P= 0,01 e P=0,004, respectivamente) com p< 0,05. Para a amostra STG-SHS-1439 não houve diferença estatística significativa, com p<0,05. A curva da cinética viral foi realizada para as duas amostras virais, em três sistemas celulares, CER, Vero e BHK-21, demonstrando estas diferenças. Foram produzidos anticorpos monoclonais contra o AMPV isolado no Brasil, sendo obtidos cinco anticorpos monoclonais para o antígeno viral através da fusão celular que apresentaram os isotipos IgG1, IgG2a e IgG2b quando da isotipagem. Dos cinco anticorpos monoclonais, três possuíam atividade neutralizante e quatro deles inibiram a fusão invitro. No teste de soroneutralização cruzada foram utilizadas três amostras virais para a análise, sendo elas SHS-Br-121, STG 854/88 e TRT-SHS-1439. Todos os anticorpos monoclonais apresentaram resultado positivo em relação à amostra homóloga, sendo que três apresentaram resultados positivos também para as amostras heterólogas. Os resultados confirmam que os dois anticorpos monoclonais descritos podem ser utilizados com importante ferramenta nos estudos epizootiológicos e para o diagnóstico específico dos subtipos na infecção pelo Metapneumovírus Aviário
Abstract: Avian Metapneumovirus (AM PV) , also denominated virus of the rhinotracheitis of the turkeys, it is a virus that attacks and it causes infection in the upper respirator) tract of chickens and turkeys. Besides the respiratory infection, in breeders and layers is associated with a significant fall in the production of eggs. In chickens the virus is related with the Swollen Head Syndrome (SHS), an illness multifactorial and for this reason it is very important the differential diagnosis. Tests accomplished with monoclonal antibodies (Mabs) and molecular techniques are capable to detect differences among the subtypes of the virus. Methods for the diagnosis of AMPV infections include detection or isolation of the virus itself, demonstration of a specific antibody response to the virus. For the primary isolation of the two samples of AMPV SHS-BR-121 (subtype A) and STG-SHS-1439 (subtype B) was it accomplished ir chicken embryo tracheal" organ culture (TOC), in chicken embryo fibroblast cell culture (CEF) and was it accomplished in cell line chicken embryo related (CER). Done the comparison of the averages of the titers obtain for the two strains, in cellline CER, did present statistically significant difference (P=0,014) with p< 0,05. The growth of SHS121-BR and STG-SHS-1439 was evaluated in six different celllines (CER, Vero, BHK21, HEp-2, MDBK and ED). CER, Vero and BHK-21 showed to be the most appropriate for virus multiplication. The titers in these cells varied from 105.5 to 107,o/mL !CID50, for the virus SHS-BR-121, and 105,5 to 106,o/mL TCID50 for the virus STG-SHS71439. The differences among the averages of the titers in the different cell lines was statistically significant differences (P=0,01) with p<0,05 for the strain SHS-BR-121 in CER cellline. One-step growth curves of the strains SHS-BR-121 and STG-SHS-1439 in CER, Vero and BHK-21 showed that there was not statistically significant difference in the infectious virus titers from 0 to 60 hours after infection. Five monoclonal antibodies were obtained for the viral antigen through the cell fusion that showed the isotypes IgG1, IgG2a and IgG2b, when of the characterization of Mabs. Three of them showed neutralizing activity and four inhibited the fusion in vitro. These MAbs were used to investigate antigenic relationship among three strains (SHS-BR-121, STG 854/8o/and TRT1439/91) of a MPV subtypes A and B using cross-neutralization test. When the five hybridomas were analyzed showed result positive for the homologus virus. In relation t4 two samples of heterologus AMPV three Mabs were positive to heterologus AMPV. Th4 results confirm that the two monoclonal antibodies described can be used as a valuable tool in the epizootiological and serological studies, and also for the specific diagnosis o the subtypes in the infection for Avian Metapneumovirus
Doutorado
Microbiologia
Doutor em Genetica e Biologia Molecular
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Hills, Anna E. "Control of monoclonal antibody N-glycosylation." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344101.
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Eastwood, David Geoffrey Douglas. "Immunotoxicology of the therapeutic monoclonal antibody TGN1412." Thesis, St George's, University of London, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.676898.
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Having passed all pre-clinical safety testing, the superagonistic anti -CD28 therapeutic monoclonal antibody (mAb ) TGN 1412, intended for the treatment of rheumatoid arthritis and B-cell chronic lymphocytic leukaemia, was approved by German and UK regulatory authorities for first-in-man Phase One clinical trial. Shortly after infusion, all six healthy trial volunteers suffered unexpected and profound systemic pro-inflammatory cytokine release, later termed a 'cytokine storm,' causing multi-organ failure. This unexpected and near fatal cytokine release syndrome (CRS) publically highlighted the failure of current pre-clinical safety testing procedures, emphasising an urgent need for novel cytokine release assays (CRAs) capable of predicting adverse properties of therapeutic mAbs. A wet coat mAb immobilisation approach, developed here, has proven predictive of clinical outcome and would have anticipated TGN1412 immunotoxicity in man. This approach IS now being widely applied by the pharmaceutical industry and contract research organisations (CROs). Comparative studies, testing TGN 1412 against a panel , of therapeutic mAbs, identified a unique mechanism of TGNl412-driven cytokine release. Substantial concentrations of the cytokine lL-2 were subsequently found to be a hallmark for the cytokine storm observed in-vivo, signifYing IL-2 as a TGN1412-like response biomarker. Multiple pro-inflammatory cytokine release was also shown to be principally effector memory T cell (T EM) derived in man. Human and macaque comparative immunophenotyping crucially identified macaque T EM cells as lacking CD28 expression, explaining pre-clinical animal model testing failures. A more physiologically relevant aqueous phase co-culture assay using monocyte-derived dendritic cells is also shown capable of eliciting a TGN1412-like cytokine release profile equivalent to that detected in-vivo and in-vitro using wet coat immobilisation; implying presentation in-vivo likely involved dendritic cells. This thesis describes the most likely mechanisms of action responsible for TGN1412 immunotoxicology in man and provides a plausible explanation for the pre-clinical safety testing failures, findings vital to the fields of immunomodulatory therapeutic mAb development and immunotoxicology.
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Watkins, I. D. "Monoclonal antibodies to Legionella pneumophila and related organisms." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354879.
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Tellides, George. "Immunosupression monoclonal antibodies to rat lymphocyte activation antigens." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236271.
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Chia, T. W. H. "The use of monoclonal antibodies for glycoside hydrolysis." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597597.
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Since 1986, a wide range of reactions have been catalysed by monoclonal antibodies, many with good rate enhancements, high regio- and stereo-selectivities and high substrate specificities. As antibodies can be elicited against practically any molecule of interest, this new class of antibody molecules, commonly known as abzymes, offers a unique way of generating tailor-made, enzyme-like catalysts. We propose to investigate the possibility of using monoclonal antibodies to catalyse the hydrolysis of glycosides. Glycosides are important components of many biological processes especially in glycoproteins which make up the cell walls of bacteria and the outer coats of viruses. Catalytic antibodies raised may serve as therapeutic agents to selectively hydrolyse such carbohydrate coats. In addition, they can be used to probe the mechanisms of glycoside hydrolysis. In our initial attempts, we synthesised nojirimycin-like transition state analogues (these compounds have an extra methylene group at the C2 position) which when protonated, copies a similar charge in the transition state. Although we were successful in synthesising the benzyl-protected compound and incorporating the uv-detectable nitrophenyl molecule at C2 (to mimic the transition state of analogous nitrophenyl glucosides), the deprotection step, unfortunately, removed the nitrophenyl group too. We next attempted to synthesise an amidine analogue which would mimic both the charge and conformation of the transition state. However, the synthesis of the key intermediate, D-gluconolactam, was unsuccessful. Another chemical was then synthesised. Hydroximo-lactones are good inhibitors of β-glucosidases and could also serve as transition state analogues. Incorporation of the dinitrophenyl group followed by deprotection of the acetyl-protecting groups provided the desired product without affecting the uv-group. Attempts to attach a linker molecule (this is to minimise the response directed to the protein when raising monoclonal antibodies) directly to this hapten were unsuccessful and the linker was ultimately introduced in a protected form although this esterification step was not regioselective. Deprotection of the tert-butyl group of one isomer provided the hapten.
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Mason, Caroline Margaret. "Characterisation of intestinal M cells using monoclonal antibodies." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260952.
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Perera, W. Shermal. "Binding and molecular characterisation of monoclonal RhD antibodies." Thesis, University of Aberdeen, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342706.
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Monoclonal RhD antibodies (anti-RhD) may potentially be used in preventing Rh alloimmunisation of women delivering RhD-positive babies, and thereby preventing haemolytic disease of the newborn (HDN). Binding and molecular analyses were performed on a range of monoclonal anti-RhDs to understand the interaction between the antibody and antigen. A flow cytometry (FCM) assay system was developed to analyse the binding of the anti-RhD to human group O R1R2 red blood cells (RBC). A panel of 30 anti-RhD preparations were studied using this assay and Vmax (maximum binding) and KD (equilibrium constant) were determined for each antibody. The antibodies were categorised into 3 groups (low, medium and high binders) according to their Vmax. Furthermore, the Vmax was converted to the number of antibody molecules bound per RBC (NMBR) by using a correlation curve generated from running parallel FCM and radioiodination assays (RIA). Scatchard analysis of the RIA data indicated that the total NMBR for O R1R2 RBC was 27,300 antigen sites/cell. Molecular analysis involved cloning and sequencing of 11 anti-RhD. Heavy chains (HC) preferentially used gene segments from the VH3 and VH4 families, and kappa chain (κ LC) usage was restricted to DPK9. Four sets of antibodies, showed restricted D gene segments (encode part of the HCDR3) which indicated possible importance for epitope specificity. Analysis of V gene sequence indicated that common VH and VL pairings were found used by the medium binders. The high and low binders had unique VH and VL pairings, although the high binders also showed greater somatic mutations from their respective germline genes. It was concluded that the fit of the antibody to the RhD antigen is dependent on both the VH and VL usage and pairing, and that the precise epitope specificity of these antibodies may require HCDR3 interaction.
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Duguid, I. G. M. "Prevention of corneal graft rejection with monoclonal antibodies." Thesis, University of Aberdeen, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387460.
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This thesis aims to place corneal allograft rejection in the context of general transplantation immunology, examine the role of lymphocyte subsets in the rejection process and consider the potential application of monoclonal antibody therapy in clinical corneal graft rejection. The literature relating to the current clinical practice of corneal grafting, with particular reference to corneal allograft rejection, is reviewed in chapter 1 to present the extent of the problem. Chapter 2 then reviews the mechanisms of allograft rejection from the literature of transplantation immunology, much of which has arisen from studies of kidney, heart, pancreatic islets and liver in animal models. The materials and methods are described in detail in chapter 3, and only the relevant experimental design is detailed in the Materials and Methods sections of the succeeding chapters. The experimental mouse model of transplanting corneal tissue into the renal subcapsular is evaluated in chapter 4, demonstrating that isografts survive indefinitely whereas allografts are rejected typically by 30 days. Pretransplant sensitisation decreased allograft survival time to 10 days. Immunohistochemistry demonstrated the presence of CD4+ and CD8+ lymphocytes and macrophages at the rejection site. Heterotopic corneal graft recipients were then treated with various monoclonal antibody regimes. Chapter 5 demonstrates that allograft survival can be increased by either anti-CD4 or anti-CD8 therapy, providing near total depletion of the respective lymphocyte subset is achieved. Xenograft rejection is shown to depend on mainly CD4+ lymphocytes in chapter 6, with no benefit being found of depleting the CD8+ subset in addition. A mild immunosuppressive effect of anti-Vβ8 monoclonal antibody is demonstrated and discussed in chapter 7. The final chapter discusses these results in the light of recent, related work in other transplant systems, and presents a case for a trial of intracameral pan-T-cell monoclonal antibody treatment.
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De, Silva M. G. "Human monoclonal antibodies in the study of diabetes." Thesis, University of Surrey, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233146.
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Tsim, Karl Wah-Keung. "Chick acetylcholinesterase : purification, molecular properties and monoclonal antibodies." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236054.
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Acetylcholinesterase (AChE, E.C. 3.1.1.7) from 1-day chick brain was enriched over 2,000-fold by N-methylacridinium affinity column. Using this preparation as immunogen, two monoclonal antibodies (mAbs), 1E2 and 3D10, were isolated. Both mAbs react with all molecular forms of AChE from chick but not with butyrylcholinesterases (BuChE, E.C. 3.1.1.8). The mAb, 1E2, was used to immunopurify chick brain globular and muscle asymmetric AChE to homogeneity. The purified brain AChE showed a specific activity of 2,200 U/mg of protein, and it appeared to be a hydrophobic tetramer with a subunit mass of 105 kDa. The purified muscle asymmetric AChE has a specific activity of 1,100 U/mg of protein, it exhibits catalytic and inhibition properties characteristic of both AChE and BuChE and contains three distinct subunits with an apparent size of 110 kDa, 72 kDa and 58 kDa in the ratio 2:2:1. The discovery of an AChE/BuChE hybrid asymmetric form has been further supported by: (1) the identification of active site properties of AChE in the 110kDa subunit and of BuChE in the 72-kDa subunit, (2) the purification and precipitation of both activities by a BuChE-specific mAb (7D11), and (3) the evidence that all subunits are bound in the asymmetric form by disulphide bonds. The 58-kDa subunit is the only one that is sensitive to digestion with purified collagenase; it carries the collagenous 'tail'.
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Gilliland, Lisa Kim. "The development of bispecific monoclonal antibodies for therapy." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278214.
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Buffetto, Fanny. "Generation of new monoclonal antibodies to rhamnogalacturonan fragments." Nantes, 2014. https://archive.bu.univ-nantes.fr/pollux/show/show?id=85b86558-be8b-4c38-ae82-702e14160fa0.
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Les pectines sont largement présentes dans la lamelle moyenne et la paroi primaire des dicotylédones. Elles sont composées de différents domaines structuraux liés de façon covalente. Les rhamnogalacturonanes II et les rhamnogalacturonanes I sont les domaines pectiques les plus complexes. Le rhamnogalacturonane II est formé d'une chaîne principale d'homogalacturonane sur laquelle sont branchées 5 chaînes latérales contenant de nombreux oses rares. Le rhamnoglaladuronane·I possède un squelette·hétérogène de rhamnoses et d'acides galacturoniques. Ce domaine contient des ramifications constituées d' arabinoses et de galactoses. Pour localiser ces structures in planta, la microscopie en immunofluorescence est une technique·sensible. . . Les immu nomarquages sont réalisés par le biais d' anticorps reconnaissant des structures très spécifiques. Certains anticorps spécifiques des pectines sont déjà disponibles, cependant la productiorn de nouvelles sondes est nécessaire. Pour générer de nouveaux anticorps, des oligosaccharides ,issus de rhamnogalacturonane II et rhamnogalacturonane I ont été isolés. La préparation de ces oligosaccharides a révélé une grande diversité de motifs structuraux composant ces domaines. Les immunisations menées avec les différents oligosaccharides ont permis la génération d'un nouvel amticorps contre·les chaînes latérales des rhamnogalacturonane I. Cet anticorps reconnaît un motif structural, jusqu'alors très peu décrit dans la littérature:, mais présent dans les parois cellulaires riches en galactanes. Cette structure in planta a été localisée dans le tubercule de pomme de terre ainsi que dans les jeunes racines d’Arabidopsis. La fonction de cette structure reste encore à détermi nerPectins are widely present in middle lamellae and primary cell walls of dicots. They are composed of different covalently linked structural domains. Rhamnogalacturonan II and rhamnogalacturonan I are the most complex pectic domains. Rhamnogalacturonan II is composed of a homogalacturonan backbone, which carries five different side chains containing rare sugars. Rhamnogalacturonan I has a heterobackbone of rhamnoses and galacturonic acids. This domain contains linear or branched arabinose ‐ and galactose ‐ rich chains. To localize these structures in planta, immunofluorescence microscopy is a sensitive technique. Immunolabelling is performed with antibodies, recognizing specific structures. Some antibodies, which label pectins are already available. However, the production of new probes is required. To generate new antibodies, oligosaccharides from rhamnogalacturonan II and rhamnogalacturonan I were purified. Structural analyses revealed the high diversity of motifs present in rhamnogalacturonan I. Immunizations carried out with the different oligosaccharides have enabled the generation of a new monoclonal antibody binding to rhamnogalacturonan I side chains. This antibody recognizes a molecular motif present in galactan rich sources, which has been poorly described in literature. This structure was localized in planta, in potato tuber and in roots of seedling Arabidopsis. However, function of this structure remains to be determined
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Bell, Graham Thomas. "Studies on the production of human monoclonal antibodies." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/19241.
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Sun, Ping. "Studies of sperm antigens using specific monoclonal antibodies." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/28405.
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This thesis work is focused on studies of sperm antigen: its purification and antifertility effect, its "internal image" by use of anti-idiotypic antibodies, and its capacity to induce antibodies of IgA class.
Mouse sperm acrosomal antigens that react with a monoclonal antibody generated against mouse spermatozoa, MS 207, were purified by immunoaffinity chromatography from soluble fraction of mouse testis homogenates. Analyzed by HPLC and SDS gel electrophoresis in the presence of reducing agent, the purified sperm antigens were shown to have a molecular weight of 600 to 700 KDa. It was shown to be glycoproteins with carbohydrate content of 23%. The purified mouse sperm antigen was used to isoimmunize female mice. After successive immunizations, antisera of high titers were raised and were shown to react specifically with mouse and human sperm, but not with any mouse somatic tissues as judged from results of immunofluorescent assay and Ouchterlony's double immunodiffusion. The antisera were shown to crossreact with human sperm at the acrosomal region. By using mouse in vitro fertilization experiments and human sperm penetration assay with zona-free hamster ova, the antisera were shown to exert strong inhibitory effects on fertilization. Another part of this thesis work was directed to generating anti-idiotypic antibody against a monoclonal sperm antibody MS 204. MS 204 was previously shown to react with a conformational epitope of sperm antigens. Anti-MS 204 antiserum was raised in a rabbit. A series of affinity chromatographic columns were applied to purify anti-Fab fragment of MS 204 (AId 204). The obtained Aid 204 possessed weak antigenicity in the mouse. After successive immunizations with Aid 204, the isoimmune sera against AId 204 were shown to crossreact with acrosomal region of mouse sperm, a pattern similar to that of MS 204. A partial inhibitory effect on in vitro fertilization was observed. The results indicate that the "internal image" of the specific sperm antigen reactive with MS 204 exists in AId 204. This could be a new strategy to obtain conformational epitope of a given antigen and may serve as the alternative of contraceptive vaccines.Medicine, Faculty of
Obstetrics and Gynaecology, Department of
Graduate
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Antonialli, Renan. "Efeito de ligantes de receptores semelhantes a Toll na resposta imune induzidas por antígenos direcionados ao DEC205 e DCIR2." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-03062014-160754/.
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As células dendríticas (DCs) expressam receptores que reconhecem padrões moleculares associados a patógenos (PAMPs), sendo capazes de capturar, processar e apresentar antígenos para os linfócitos. O contato com PAMPs induz a maturação das DCs e, consequentemente, a ativação dos linfócitos. Nossa estratégia para desenvolver vacinas é empregar anticorpos monoclonais contra os receptores endocíticos DEC205 e DCIR2, presentes nas subpopulações de DCs, em fusão com o antígeno de interesse. E estimular a maturação delas utilizando os adjuvantes poly I:C, CpG ODN 1826 e flagelina de S. Typhimurium . Imunizamos camundongos selvagens ou knockouts para os receptores TLR 3, 5 e 9 com adjuvantes e anticorpos quiméricos anti-DEC205, anti-DCIR2 ou isotipo controle (Iso) fundidos a proteína MSP119 de Plasmodium vivax. Avaliamos as repostas humoral e celular no grupos imunizados. Os resultados indicam que, seja qual for o adjuvante usado, a maioria dos parâmetros analisados aponta para a superioridade do direcionamento do antígeno para o receptor DEC205.Dendritic cells (DCs) express receptors that recognize pathogen-associated molecular patterns (PAMPs), and are able to capture, process and present antigens to lymphocytes. In recent years, a immunization strategy that directs antigens to DCs in vivo has been developing. This consists in the use of monoclonal antibodies fused with the antigen of interest. However, effective immune activation occurs mainly when the chimeric antibodies anti-DEC or anti-DCIR2 are administered in the presence of a DC maturation stimulus. We immunized wild type and knockout mice for TLR3, 5 and 9 with adjuvants (poly I:C, CpG ODN 1826 and flagellin from S. typhimurium) and chimeric anti-DEC205, anti-DCIR2 or isotype control fused to the Plasmodium vivax MSP119 protein. After the administration of two doses, we evaluated the humoral and cellular immune responses in the different groups. Our results indicate that, irrespectively of the adjuvant, the majority of parameters analyzed points out to the superiority of antigen targeting to the DEC205 receptor.