Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.

Lymphocytes play a key role in the mammalian immune system and migrate around the body interacting with both soluble factors and tissues in their role of detecting and eliminating disease causing pathogens. These interactions are mediated by the molecules expressed at the cell surface of the leukocyte which have become a popular paradigm for the study of intercellular communication. Proteins which belong to the immunoglobulin superfamily (IgSF) are the most abundant of these cell surface molecules and one of these (OX-2) is the major focus of this thesis. The OX-2 glycoprotein is expressed in both the neuronal and lymphoid compartments of rats and has no known function. However, a highly avid OX-2 binding reagent was previously shown to specifically interact with a cell surface receptor expressed by macrophages. A monoclonal antibody, MRC OX-102, which bound rat macrophages and blocked the binding of OX-2 was raised and used to molecularly identify the receptor on the macrophage by a combination of protein purification and PCR-based strategies. The rat OX-2 receptor (OX-2R) was identified as a novel member of the IgSF and had a close evolutionary relationship to the OX-2 protein itself. The two glycoproteins interacted with an affinity of 2.5μM and K_off 0.8 sec^-1, values typical of interactions between cell surface proteins. A mouse form of the OX-2 receptor was also cloned. The cytoplasmic region of the OX-2R contained conserved tyrosine residues which were shown to be phosphorylated upon pervanadate treatment of macrophages. Preliminary distribution data suggest that the receptor is restricted to cells of myeloid origin and the functional consequences of the interaction are discussed. A monoclonal antibody, MRC OX-104, was raised to the human OX-2 protein to initiate the translation of this work from rodent models to humans. OX-2 showed highly conserved patterns of expression between the two species.

i) Human cardiac cDNA libraries were constructed and clones encoding human cardiac troponin I (cTnI) isolated. ii) Both the entire <i>cTNI</i> cDNA and a 5'-portion, were expressed in <i>Escherichia coli</i> as fusion products with β-galactosidase. The full-length cDNA was also expressed unfused. iii) The murine monoclonal antibody 29Mu is specific for human and baboon cTnI whereas the 31Mu antibody reacts with cTnI from a range of species. These antibodies might be useful in the imaging of necrotic cardiac tissue. In this study, 31Mu was found to bind to all prepared forms of cTnI antigen, in enzyme-linked immunosorbant assays (ELISAs) and, where tested, in Western blots. Thus, its epitope is localised towards the N-terminus of cTnI. 29Mu bound to the bacterially-produced unfused cTnI but not to the fusion polypeptides or crude bovine cTn. Its ability to bind to human cardiac extracts was related to the method of their preparation, indicating that the epitope of 29Mu shows greater conformational dependency than that of 31Mu. iv) cDNAs encoding the variable domains of 29Mu and 31Mu were cloned and chimaeric antibodies, comprising murine variable and human constant regions produced. Humanised antibodies, in which only the antigen-binding sites were of murine origin, were also produced. Such recombinant antibodies would be expected to exhibit reduced immunogenicity in man. v) Neither the chimaeric nor humanised antibody versions of 29Mu bound cTnI. Chimaerised 31Mu reacted with all forms of cTnI but did not show complete equivalence to 31Mu. An antibody containing the humanised 31 kappa chain and the chimaeric heavy chain was reactive to all forms cTnI in ELISAs but its efficiency of binding, relative to that of the chimaeric antibody, was dependent upon the antigen source. Humanised heavy chains were produced utilising two different human frameworks and the framework, showing closer homology to the 31Mu variable domain, supported antigen binding with fewer murine residue substitutions. However, both successful humanised 31 antibodies showed some cross-reactivity.

The cytoskeletal component of the muscle membrane, dystrophin and its associated proteins (DAPs), are essential for the maintenance of muscle integrity, since the absence of these molecules results in a variety of muscular dystrophies. The purpose of this work was to create and characterize monoclonal antibodies (mAbs) designed to recognize components of the DAP complex (DAPC), in order to provide tools for the study of its structure and function. / The first mAb generated, 1137, was raised against a 33 amino acid sequence of the core protein at the c-terminus of alpha-dystroglycan (alpha DG), a cell surface member of the DAPC linked to dystrophin via its co-transcript, the transmembrane protein, beta-dystroglycan. 1B7 was used to perform a comparative study in denervated rat muscle tissue in parallel with IIH6, a mAb which recognizes a different, more glycosylated form of alpha DG. The second and third mAbs were raised against a complex of proteins purified by succinylated Wheat Germ Agglutinin (sWGA) following extraction from rabbit skeletal muscle. (Abstract shortened by UMI.)

Includes bibliographical references (leaves 281-318) / xxv, 318 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Characterises the antigen recognized by mAb UA009 and investigates whether it is an endothelial adhesion marker. The identification of a CD36-like molecule led to targeted histological studies on the expression of the molecule in specific tissues of interest. / Thesis (Ph.D.)--Adelaide University, Dept. of Microbiology and Immunology, 2001

Thesis (Ph. D.)--Lehigh University, 1998. / Includes vita. Bibliography: leaves 51-64.

No description available.

The causative agent of the most deadly form of malaria, P. falciparum, was identified over 130 years ago, yet this disease still causes 430,000 deaths each year. Although naturally-acquired immunity exists, it requires a heavy and sustained exposure to the parasite, with most succumbing as young children, before this immunity has fully developed. Effective treatments exist but with small-molecule drug resistance on the rise and little in the way of affordable alternatives, the need for an efficacious malaria vaccine is as great as ever. A successful malaria vaccine is likely to necessitate targeting each stage of the parasite's lifecycle. Immunity directed to the blood-stage, the stage which causes all the symptoms of malaria, is unique in that it would allow for a concomitant development of naturally-acquired immunity along with a reduction in morbidity and mortality. To date, antibody-mediated immunity to the blood stage requires intractably high levels of antibody and this problem is compounded by a paucity of viable candidates with which to effectively target different strains. Other fields of vaccinology, over the past decade, have been employing various structure-based strategies to increase the specific activity of the immune response thus lowering the antibody levels required for protection. However, very few detailed investigations of this kind have been conducted on a P. falciparum vaccine candidate, and certainly none as promising as PfRH5. In a world's first, fully-human antibodies raised in response to PfRH5 vaccination were isolated and extensively characterised, both functionally and structurally with the intention of elucidating the important features necessary to inform the design of an improved PfRH5-based vaccine. Synergistic and antagonistic effects of antibody combinations were noted and highlight new complexities of the immune response to PfRH5, opening the door to unanticipated potential for rational vaccine design.

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:50:03Z : No. of bitstreams: 1 cardoso_ra_me_botfm.pdf: 896730 bytes, checksum: 97ac1a0ea8e45d9861c31c7feaac2ecb (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / A tecnologia de produção de anticorpos monoclonais revolucionou a investigação diagnóstica, especialmente a análise dos grupos sanguíneos em imuno-hematologia. O polimorfismo das hemácias humanas torna a produção de novos insumos extremamente necessária. Dentre os sistemas de grupos sanguíneos descritos, o sistema Rh tem se mostrado um dos mais complexos pela vasta composição de antígenos. Analisando aspectos estruturais do antígeno D associado às técnicas de biologia molecular, identificam-se numerosas variantes deste antígeno por trocas de DNA entre genes similares levando a alteração de epítopos e consequentemente de padrão de expressão fenotípica na superfície das hemácias. Desvendar e identificar estas variantes com insumos biotecnológicos desenvolvidos no país foi o desafio desta pesquisa, onde anticorpos monoclonais contra antígenos eritrocitários foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados com hemácias fenótipo D categoria IVa. Nove fusões foram realizadas e os sobrenadantes de cultura foram triados por teste de hemaglutinação em microplacas. Foram congelados e mantidos em nitrogêncio líquido 41 híbridos. Estes híbridos foram clonados e mantidos em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 12 clones IgG1, 5 IgG2a e 10 IgM. Os clones foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. Os anticorpos monoclonais produzidos, inicialmente não demonstraram a especificidade foco do presente trabalho que foi a produção de anticorpos anti-D, no entanto, os hibridomas podem ter secretado diferentes especificidades de anticorpos monoclonais, de acordo com os antígenos presentes na membrana das hemácias utilizadas para a imunização dos camundongos. Uma segunda fase da pesquisa está sendo... / The blood groups analyses in immunohematology have been highly benefited based on the advance of the monoclonal antibody technology improvement on diagnostics investigation. The human red blood cell polymorphism increases the need for new alternatives. The Rh system blood group is the most complex of all, due to the vast antigenic composition. When analyzing the structural aspects of the D antigen in combination with the molecular techniques, based upon DNA exchange between similar genes it is possible to identify a huge number of partial D variants, this situation leads to a change of epitopes and hence the red blood cell phenotypic expression pattern. Unveil and identify these variables through the use of local biotechnology was the challenge of this research. Monoclonal antibodies against human red blood cell were produced by cell fusion, using spleen cells from immunized BALB/c mice and DIVa red blood cells. Nine fusions had been made and the culture supernatants were selected to the hemaglutination test using microplates. Forty-one hybrids had been frozen and kept in liquid nitrogen. These hybrids had been cloned and kept in the culture to collect monoclonal antibodies, resulting in 12 IgG1, 5 IgG2a e 10 IgM. The clones had been selected for ascitic fluid production, by injection in BALB/c mice. The monoclonal antibodies produced initially had not produced the anti-D as expected by the research. However, the hibrydomas could have created different specificities of monoclonal antibodies, according to the antigen located in the donor red blood cells membrane used in the mice’s immunization. The second phase of the research is being done to confirm the monoclonal antibodies specificities produced through the deployment of new tests to immunoglobulin identification

Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.