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.

An antibody library against Eimeria tenella sporozoites was constructed by phage display. Total RNA was isolated from the spleen, bone marrow, and ceca of immune chickens, and was used to reverse-transcribe cDNA. Heavy and light antibody variable genes were amplified from cDNA by the Polymerase Chain Reaction (PCR), using primer pairs that contain complementary sequences encoding a short linker sequence. The single-chain antibody fragment (scFv) was obtained by a secondary overlap PCR with primers that incorporate SfiI restriction sites, thus allowing for subsequent cloning into the phagemid vector pComb3X. Vector and scFv insert were digested with SfiI, ligated, and transformed into competent XL1-Blue Escherichia coli cells by electroporation, yielding a library with 7.4 x 107 total transformants. The culture was grown under carbenicillin selective pressure, rescued with helper phage, and the antibody-displaying phage was precipitated by PEG/NaCl, and subsequently used for panning. Five panning rounds were performed using cryopreserved E. tenella sporozoites, with a gradual increase of washing stringency to select for specific, highaffinity binders. A 1000-fold increase in phage output was obtained after 3 rounds of panning. There was clear enrichment of the positive clones over the panning rounds, with the 3rd round resulting in a 3,000-fold enrichment over the first one, as the binding clones became the dominant population in the library. Selected antibodies from the last round of panning were sequenced and characterized by immunoblotting. Soluble antibody fragments were produced in a non-suppressor E. coli strain, and recognized a 66-KDa sporozoite antigen on a Western blot. Primary cultures of chicken enterocytes were prepared in the hope of serving for invasion assays with E. tenella sporozoites. The isolation procedure, however, proved to be cumbersome and time-consuming. Future investigations will focus on purification and further characterization of antibodies selected from the constructed library. Such antibodies can be tested, alone or in combination, for their ability to block in vitro the invasion mechanism of E. tenella.

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.

No description available.

Η παρακολούθηση σε πραγματικό χρόνο των βιομοριακών αλληλεπιδράσεων παρουσιάζει ιδιαίτερο ενδιαφέρον διότι παρέχει άμεσα πληροφορία σχετικά με την κινητική πρόσδεσης και τις σταθερές ισορροπίας. Σε αυτή την αναφορά, παρουσιάζεται μια μεθοδολογία η οποία βασίζεται στη Φασματοσκοπία Ανάκλασης Λευκού Φωτός και ενδείκνυται για την παρακολούθηση σε πραγματικό χρόνο των βιομοριακών αντιδράσεων που λαμβάνουν χώρα σε στερεά υποστρώματα. Η οπτική διάταξη αποτελείται από μια VIS–NIR οπτική πηγή η οποία επικοινωνεί μέσω οπτικής ίνας με φασματοφωτόμετρο συνδεδεμένο σε Η/Υ. Το εξωτερικό τμήμα της οπτικής ίνας κατευθύνει το φως κάθετα πάνω στην επιφάνεια όπου συμβαίνουν οι αλληλεπιδράσεις μεταξύ των βιιομορίων. Το ανακλώμενο φως συγκεντρώνεται από το εσωτερικό τμήμα της οπτικής ίνας και κατευθύνεται στο φασματοφωτόμετρο. Οι αντιδράσεις λαμβάνουν χώρα σε επιφάνεια διοξειδίου του πυριτίου επικαλυμμένη με πολυμερικό υμένιο. Μια αντλία χρησιμοποιείται για την παροχή των αντιδραστηρίων με ελεγχόμενο ρυθμό σε ένα μικρορρευστομηχανικό κανάλι. Το φάσμα της ανάκλασης καταγράφεται σε πραγματικό χρόνο. Οι αντιδράσεις μεταξύ των πρωτεϊνικών μορίων γίνονται αντιληπτές σαν μετατοπίσεις του μήκους κύματος εκεί που παρατηρείται το φαινόμενο της ενισχυτικής συμβολής. Οι μετατοπίσεις αυτές στο μήκος κύματος συμβαίνουν λόγω του σχηματισμού πρωτεϊνικού στρώματος είτε κατά τη διάρκεια της προσρόφησης των αντισωμάτων στο στερεό υπόστρωμα ή λόγω αλλαγής στο πάχος του πρωτεϊνικού στρώματος όταν τα συμπληρωματικά αντιγόνα δεσμεύονται από τα ήδη ακινητοποιημένα αντισώματα. Η προτεινόμενη μεθοδολογία εφαρμόστηκε για την παρακολούθηση σε πραγματικό χρόνο, χωρίς τη χρήση ιχνηθέτη της αντίδρασης μεταξύ των συμπληρωματικών μορίων βιοτίνης-στρεπταβιδίνης όπως επίσης και για την ανίχνευση της πρόσδεσης των αντιγόνων mouse IgG από ήδη ακινητοποιημένα αντισώματα anti-mouse IgG. Mouse IgG σε συγκεντρώσεις μικρότερες των 150 pM ανιχνεύτηκαν σε πολύ μικρούς χρόνους αντίδρασης (10 min). Ο οπτικός αισθητήρας είναι μια απλή, γρήγορη, χαμηλού κόστους διάταξη για την παρακολούθηση σε πραγματικό χρόνο των βιομοριακών αλληλεπιδράσεων που τον καθιστά κατάλληλο για διάφορες αναλυτικές εφαρμογές. / Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide direct results concerning binding kinetics and equilibrium constants. In this report, a method based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable for real-time monitoring of biomolecular reactions taking place on a solid surface. The optical setup consists of a VIS–NIR light source connected through a bifurcated optical fiber to a PC-driven spectrophotometer. The outer part of the optical fibre guides the light vertically onto the surface where the biomolecular reactions occur. The reflected light is collected from the central part of the optical fibre and is directed to the spectrophotometer. The reactions take place on the top of a polymer covered silicon dioxide surface. A microfluidic module in combination with a micropump is used to supply the reagents in an adjustable rate. The reflectance signal is recorded in real-time. The biomolecular interactions are monitored as shifts of the wavelength where constructive interference is observed. These wavelength shifts correspond to biomolecular film thickness changes during either the adsorption of biomolecules onto the substrate or during biomolecule’s reaction with the immobilized counterpart molecules. The proposed methodology has been applied for real-time and label-free monitoring of streptavidin binding to surface-immobilised biotinylated protein as well as of mouse IgG onto immobilized anti-mouse IgG antibody. Mouse IgG at concentrations less than 150 pM were detected in short reaction times (10 min). The proposed reflectance spectroscopy sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions thus making the developed sensor suitable for various analytical applications.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

Some previous work has suggested that fetal rejection may be a cause of spontaneous abortion in humans. The aim of the work presented, therefore, was to determine the influence of maternal alloantibody formation against paternal B lymphocytes, detected by the erythrocyte antibody rosette inhibition (EAI) assay, on the outcome of semi-allogeneic pregnancies. Preliminary studies indicated that EA rosette inhibition was a suitable assay for these investigations, because it detected alloantibodies directed to any major histocompatibility complex (MHC) antigen, irrespective of the ability of the antibody to fix complement; this was an important consideration because alloantibodies induced by pregnancy are often only weakly lytic. In humans it was found that antibodies to paternal B lymphocytes occurred significantly more commonly in normal primigravid and multigravid pregnancies when compared with pregnancies of similar gestation which aborted. These antibodies were shown to be directed to MHC encoded antigens by family studies, but were not removed by platelet absorption, strongly suggesting that they were not class I MHC antigens. Studies in inbred rats demonstrated that these paternal antigens were encoded by the RT1A region of the rat MHC alone. Maternal alloantibody responses to RT1A antigens appeared to be suppressive because studies using the rat kidney allograft model showed that multiparous rats with EAI antibodies to paternal strain cells enjoyed prolonged graft survival. It was also found that pregnancies in which the paternal strain differed only by RT1A antigens induced a suppressive immune response in the mother. These results suggest that immune responses to MHC encoded antigens, possibly unique, may prevent fetal rejection in some instances.

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.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

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.