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No description available.

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.

Hepatocellular carcinoma (HCC) is the most common malignant tumor in Taiwan with more than one million new cases annually in the world. Growth factors play important roles in liver carcinogenesis. Hepatoma-derived growth factor (HDGF), originally isolated from the cultured media of human hepatoma HuH-7 cells, stimulate the growth of fibroblast cells, endothelial cells, and hepatoma cells. Overexpression of HDGF is related to the transformation of human hepatoma, lung cancer and melanoma. Besides, HDGF also exerts strong influence on the prognosis of patients with hepatoma. In this study, recombinant HDGF was expressed and purified with higher purity than 90%. The recombinant protein was used to raise polyclonal HDGF antibodies in rabbits and to generate three lines of HDGF monoclonal antibodies in mice. After antibodies characterization, an in-house, sandwich HDGF ELISA system was established using the purified polyclonal anti-HDGF IgG as the capture antibodies and the monoclonal anti-HDGF IgG as the detection antibodies. By using recombinant HDGF as standard, this ELISA system accurately evaluate the changes of HDGF release in SK-Hep-1 cells after gene delivery. In addition, we also evaluated the effect of anti-HDGF on the growth of hepatoma cells. Application of either polyclonal or monoclonal HDGF antibodies, but not preimmune antibodies, inhibited the proliferation of SK-Hep-1 hepatoma cells in a dose-dependent manner. In summary, the present study generated HDGF monoclonal antibodies for development of HDGF ELISA and application on suppressing HCC progression. Future studies should be carried out to enhance the sensitivity of HDGF ELISA and to evaluate the therapeutic potential of HDGF antibodies for treatment of HCC.

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.