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.

Thesis (Ph.D.) -- University of Glasgow, 2002. / Ph.D. thesis submitted to the Division of Infection and Immunity, Faculty of Biomedical and Life Sciences, University of Glasgow, 2002. Includes bibliographical references (p. 163-202). Print version also available.

Thesis (doctoral)--Københavns Universitet. / Bibliography: p. 207-231.

No description available.

Autism spectrum disorder (ASD) refers to a highly prevalent neuropsychiatric disorder, currently affecting one in every 68 children. ASD is understood as a heterogeneous disorder, and individuals with this condition vary considerably in terms of symptom presentation. This heterogeneity contributes to the difficulty faced by researchers and clinicians in trying to determine the precise underlying mechanisms and treatment for this condition. Furthermore, it remains unknown whether the variations in symptom manifestation are attributed to differences in underlying etiologies of the disorder or other factors as yet to be identified. Currently, it is believed that ASD is likely due to the interaction between different genetic and environmental factors. The maternal immune system is one example of where the environment may act upon genetic predispositions and lead to altered fetal brain development. Considering the importance of the immune environment during fetal development, maternal antibodies (Abs) directed against fetal proteins have been considered as potentially playing a critical function in the pathology of ASD. This thesis examines the literature focused on the role of maternal Abs in fetal development and their impact on the neuropathology of ASD. Studies have collected samples from mothers of children diagnosed with ASD and examined the reactivity patterns of the maternal Abs against fetal proteins. Through review and inspection of methodologies and results, this thesis highlights the important insights obtained as well as proposes possible reasons for the disparity in findings. Lastly, this thesis proposes future directions and therapeutic implications of identifying the maternal Abs that could be involved in at least a subset of ASD cases.

Guillain Barré Syndrome in humans is characterised by ascending paralysis. It is often associated with preceding infections two to four weeks prior to nadir and is fatal in five percent of cases. Antibodies specific to several nerve components are frequently associated with clinical symptoms in GBS. These antibodies were found to be specific to various gangliosides and ganglioside complexes. It was also found that antibody reactivity to gangliosides is affected by membrane components. The most prevalent (20-30%) immunoglobulin in GBS is anti-GM1 (20-30%), which also binds to the LPS of the PEN O:19 Campylobacter jejuni serotype. This is the most common infectious agent associated with GBS and emphasizes the importance of infection and anti-ganglioside antibodies in disease development. Intravenous infusion of pooled immunoglobulin from healthy donors, also called intravenous immunoglobulin (IVIg), halves the severity of disease manifestation. The action mechanism of IVIg in curing GBS is not clear, but intravenous immunoglobulin was shown to neutralize anti-ganglioside binding activity and its pathogenic effects. It was further found that anti-idiotypic antibodies in IVIg inhibit anti-ganglioside antibody activity. Treatment with IVIg is not equally effective in all GBS cases, which might be due to the inability of IVIg to neutralize anti-ganglioside antibodies in all patients adequately. Therefore, the treatment of GBS with IVIg needs to be better understood in order to improve its use as a cure for GBS. This study confirmed previous findings that the interaction of patient serum anti-GM1 antibodies and ganglioside auto-antigens is greatly impaired by components in healthy serum. Bound anti-GM1 antibodies could be displaced by (presumably) anti-idiotypic antibodies from healthy donor serum. This study found that the displacement potential between donor sera differs. Anti-GM1 antibody displacement was found to be dependent on the character of both anti-GM1 and anti-idiotypic antibody. This demonstrated the feasibility of improving the efficiency of treatment by IVIg by sourcing it from only those sera that test best for displacing auto-antibodies from their ganglioside antigens in ELISA. IVIg selection may therefore greatly benefit from the use of recombinant phage display antibodies to distinguish between the various types of GBS for treatment. To develop a method to characterize anti-ganglioside antibodies sensitively, an evanescent field biosensor was employed in which gangliosides were presented in a liposome environment. This provided a more physiological way of antibody antigen recognition. The optimized method determined the ganglioside binding specificity of purified IgG from a GBS patient, and mouse monoclonal anti-GM1 and anti-GD1a antibodies accurately. The results compared well with those from ELISA. The results obtained with purified IgG were far better than that obtained with whole serum analysis. This could be due to non-specific binding or the presence of inhibiting anti-idiotypic antibodies in patient sera. The biosensor method for antibody detection in GBS may allow the detection of anti-idiotypic antibodies in patients in future, because it requires no prior labelling of antibodies. Anti-idiotypic interaction may be detected by displacement of Ab1 from antigen, or by capturing Ab2 on Ab1 immobilized on the biosensor surface. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2008. / Biochemistry / MSc / unrestricted

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.