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by Julia Luen-wah Woo. / Thesis (M.Ph.) -- Chinese University of Hong Kong, 1988. / Bibliography: leaves 90-94.

by Yu-Wai Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 122-129.

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.

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