Ultraviolet irradiation of DNA induces the formation of a number of mutagenic lesions. The most prolific of these is the cis-syn thymine dimer (formed maximally at 260 nm) and this has been implicated in the reaction pathways that lead to ultraviolet-induced carcinogenesis. In order that the molecular events underlying these neoplastic events be understood, it is imperative that the thymine dimers formed in ultraviolet-irradiated thymine containing systems be quantitated. In this laboratory, dimer quantitation is performed using reverse phase high performance liquid chromatography (HPLC) with ultraviolet (DV) detection and the data obtained has allowed a kinetic mechanism for lesion formation to be proposed. Such studies have used in vitro thymine containing substrates (aqueous thymine, thymidine, thymidylyl-3',5'thymidine, calf thymus DNA and pUC19 plasmid DNA) to generate the thymine dimer using DV irradiation. with the planned extension of this research to in vivo cellular systems (where DNA and hence thymine concentrations are intrinsically less than those of in vitro systems), a more sensitive technique for thymine dimer quantitation is required. An immunological approach to providing this technique was chosen. Here, DV-irradiated DNA was injected into rabbits whose immune system mounted a ' response (i.e. antibody production) to the DV-DNA antigen. Blood was drawn from the rabbits at regular intervals to obtain the antibodies. The technique of immunoblotting was chosen and developed to allow detection of the thymine dimer antigen. This involved the reaction between the UV-DNA antigen, the primary antibody (generated by the rabbit) and a secondary antibody conjugated to an enzyme, all of which were immobilized on a commercially available membrane system. Detection and quantitation of the immune complex immobilized on the membrane was performed using the technique of enhanced chemiluminescence. Upon addition of a chemiluminescent substrate (luminol) to the immune complex, the horseradish peroxidase enzyme catalysed the reaction of luminol, with one of the products being light of 425 nm to 430 nm. This light impinged on a luminescence film which was developed and printed using standard photographic techniques. The use of dilutions of the primary antibody in the immunoblotting protocol with enhanced chemiluminescent detection, allowed correlations of antibody dilutions with UV-DNA antigen to be made. This immunoblotting technique with enhanced chemiluminescent detection has been used successfully in detecting thymine dimer lesion formation at levels currently above the detection limit of the HPLC. It has also been used successfully in detecting and quantitating thymine dimers at levels undetectable by the HPLC. To this end it has proved to be 4000 to 8000 times more sensitive than the chromatographic technique. Any immunological technique requires that the antibody of interest be purified and characterized. Here, purification of the crude serum was performed using the classical technique of ammonium sulphate precipitation of proteins. As an alternative technique, affinity chromatography was performed on the crude serum using a Memsep 1000 affinity chromatography cartridge attached to a preparative HPLC system. Chromatographic data illustrating this purification are given. Characterization of the DV-DNA antigen was performed by considering the specificity of the antibody response in the laboratory animal. Support for the kinetic mechanisms previously proposed for pyrimidine dimer formation in DNA is also given in this work. Calf thymus DNA was irradiated and dimer yields obtained by immunoblotting. These were used in the computer programme CAKE together with the previously determined rate constants to determine simulated dimer yields. A good agreement between experimental and simulated data indicated the validity of the mechanism at a DNA concentration of 0.025 mg/ml. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Three economically important swine diseases: Porcine Reproductive and Respiratory Syndrome (PRRS), Porcine Circovirus Associated Disease (PCVAD) and Swine influenza cost the US swine industry more than a billion dollars each year. This study developed a fluorescent microsphere immunoassay (FMIA) to simultaneously detect antibodies to the causative pathogens: PRRSV, porcine circovirus (PCV2) and swine influenza virus (SIV). The results showed that the multiplex assay possessed the predicted specificities. In the case of PRRSV NA, the assay displayed higher sensitivity when compared to a commercially available ELISA. The assay was employed to measure both IgG and IgM responses. The FMIA was found to possess several advantages over standard ELISA which include reduced sample volume, time and cost and provides a new tool for veterinary diagnostics. The FMIA was applied for swine disease surveillance in Hawaiian and Texan feral swine populations. The antibodies against PCV2 showed the highest prevalence among these three pathogens in both Hawaii and Texas. Hence we consider PCV2 as the most prevalent pathogen in Hawaiian and Texan feral pigs and this pathogen poses the greatest threat to commercial pigs. SIV seroprevelance increased from 2007 to 2010 in Hawaii State, suggesting an increasing risk for commercial pigs. Moreover, yearly surveillance in Texas State shows growth in seropositive response to all pathogens, particularly PCV2. The development of FMIA for detection of antibodies to multiple swine pathogens in serum samples offers an important alternative for swine disease surveillance in commercial and feral herds.

In dieser Studie wurden zwei Immunoassays zur Bestimmung von TG- und TPO-Antikörpern hinsichtlich diagnostischer Trennschärfe sowie klinischer Relevanz in der Diagnostik der chronischen lymphozytären Thyreoiditis-Hashimoto (CLT) untersucht. Im Rahmen der vorliegenden Studie wurden zwei Patientengruppen erfasst: ein Kollektiv mit der Diagnose CLT (n=203) und das sogenannte Normalkollektiv, das 205 Probanden umfasste. Die diagnostischen Kriterien zur Diagnosestellung CLT ergaben sich aus dem Zusammenspiel von klinischem Befund, Ultraschalluntersuchung und Antikörpertiter. Verglichen wurden der an der Klinik und Poliklinik für Nuklearmedizin der Universität Würzburg für die Routinediagnostik eingesetzte manuelle VarELISA TG und TPO Antibodies Assay, PHARMACIA Diagnostics mit dem automatisierten BRAHMS anti-TGn bzw. anti-TPOn KRYPTOR Assay. Die Bestimmung der Ergebnisse bei TPO-AK zeigte, dass die von KRYPTOR gemessenen Werte im Mittel um 2670,51 U/ml höher lagen als bei VarELISA. Bei TG-AK wurden die Konzentrationen auf der Plattform KRYPTOR allerdings um 325,07 U/ml niedriger gemessen als bei VarELISA; es zeigte sich bei TG-AK somit ein umgekehrtes Verhältnis. Des Weiteren wurde eine relativ gute Übereinstimmung zwischen beiden Assays (Kappa-Koeffizient nach Cohen = 0,63) bei der Bestimmung der TPO-Antikörper festgestellt; 86,8% der Seren wurden als konkordant bewertet. Demgegenüber stehen 65,4% in ihrem subjektiven Urteil übereinstimmende Ergebnisse bei der TG-Antikörper Bestimmung, was für eine schwache Übereinstimmung der TG-AK-Werte spricht (Kappa-Koeffizient nach Cohen = 0,31). Zudem ist die diagnostische Trennschärfe bei TPO-AK höher (Area under Curve = 0,929) als bei TG-AK (Area under Curve = 0,805); somit unterscheidet KRYPTOR bei der Bestimmung der TPO-AK besser zwischen „gesunden“ und „erkrankten“ Patienten als VarELISA. Bei der Messung der TPO-AK auf der Plattform KRYPTOR zeigte sich bei dem dem Cut-Off (vom Hersteller auf 60 U/ml festgelegt) am nächsten liegenden Wert (59,9 U/ml) sowohl eine hohe Sensitivität (81,4%) als auch Spezifität (97,6%). Bei der TG-AK Messung lag bei dem Cut-Off Wert von 59,8 U/ml bei hoher Spezifität (99,5%) die Sensitivität sehr niedrig (43,6%), d.h. viele Patienten wurden als falsch negativ eingestuft. Aus der Auswertung geht ein optimaler Schwellenwert von 67,2 U/ml für TPO-AK und 40,7 U/ml für TG-AK hervor, wobei der vom Hersteller angegebene Cut-Off Wert für beide AK 60 U/ml beträgt. Mittels neu ermitteltem Cut-Off Wert (67,2 U/ml) konnte bei TPO-AK eine Steigerung der Spezifität auf 99,5% bei unveränderter Sensitivität erreicht werden. Dementsprechend erbrachte der Cut-Off Wert von 40,7 U/ml eine Steigerung der Sensitivät auf 50% bei gleich bleibender Spezifität bei TG-AK. Die Bestimmung des Antikörperprofils in den beiden Testsystemen zeigte somit, insbesondere bei TG-AK, häufig diskrepante Ergebnisse. Dies belegt erneut die bekannte Problematik bei der Labordiagnostik der CLT. Ursächlich sind Affinitätsunterschiede und unterschiedliche Kalibrierungen der verwendeten Tests sowie das Fehlen einer Standardisierung zu diskutieren. Darüber hinaus bestätigen die Ergebnisse die Notwendigkeit einer Definition eines institutionellen Cut-Offs. / A comparison of two different immunoassays in the clinical investigation of autoimmune thyroid disease was carried out. The manual Immunoassay VarELISA was compared to the fully automated KRYPTOR system regarding clinical relevance and diagnostic accuracy. On both assays thyroid peroxidase antibodiese (TPO-Ab) and thyroglobulin antibodies (Tg-Ab) were measured. The study was held on two groups: a normal population (n=205) and a group of patients (n=205) who had been diagnosed Hashimoto's thyroiditis. We found out "optimal" cut-offs (despite the manufacturer's data of 60 U/ml) for both antibodies regarding better sensitivity and specificity: these were 67,2 U/ml for TPO-ab and 40,7 U/ml for TG-Ab. Overall there was a benefit in the assessment of TPO-Ab on the fully automated Immunoassay KRYPTOR whereas we often found discrepant results in the measurement of TG-Ab.

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative (‘digital’) counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

This thesis was on the development of a high-throughput electrokinetically-controlled heterogeneous immunoassay (EK-IA) microfluidic chip for clinical application. Through a series of experimental studies, a high-throughput EK-IA was developed. This EK-IA was capable of automatically screening multiple analytes from up to 10 samples in parallel, in only 26 min. Flow control in an integrated microfluidic network was realized by numerical simulation of the transport processes. This EK-IA was successfully applied to detect E. coli O157:H7 antibody and H. pylori antibody from human sera with satisfactory accuracy. Simultaneous screening of both antibodies from human sera was also achieved, demonstrating the potential of this EK-IA for efficiently detecting multiple pathogenic infections in clinical settings. Preliminary work on the application of EK-IA to detect biomarkers of embryo development in embryo culture media also yielded good results. In addition to the experimental studies, the reaction kinetics of this microfluidic EK-IA has also been investigated, using both numerical simulation and a modified Damköhler number. Targeted towards a more sensitive assay, the influences of several important parameters on the reaction kinetics were studied. This EK-IA holds great promise for automated and high-throughput immunoassay in clinical environments.

No description available.

A mathematical model of Limiting Dilution Analysis for two limiting parameters is presented and investigated. Limiting Dilution Analysis is a microbiological cell assay developed for immunological application. In the given case we deal with the interaction between B lymphocytes, macrophage derived factor and T-independent antigens. The state of the art is that quantitative statements are only possible if one cell type (in general the B cells) is limiting and all others are in excess present. The basis for this thesis is a set of experiments in which B cells and macrophage derived factor are limiting and all other involved cells and factors are in saturating amounts present. It is shown that so far presented suggestions on modeling Limiting Dilution Analysis for two limiting cell-types are not suitable for this problem. Further, a mathematical model based on data is presented and interpreted in immunological terms with the help of a set of partial differential equations. The basis for the interpretation of the model are changes in affinity and saturation effects, both not incorporated in the so far presented models of the assay. In particular the relevance of mathematical interpretation of this process for the identification of new concepts as the saturation effects is stressed. The model of partial differential equations is highly non-linear but offers the possibility of interpreting the highly interrelated processes apart from each other. / Graduation date: 1994

This thesis was on the development of a high-throughput electrokinetically-controlled heterogeneous immunoassay (EK-IA) microfluidic chip for clinical application. Through a series of experimental studies, a high-throughput EK-IA was developed. This EK-IA was capable of automatically screening multiple analytes from up to 10 samples in parallel, in only 26 min. Flow control in an integrated microfluidic network was realized by numerical simulation of the transport processes. This EK-IA was successfully applied to detect E. coli O157:H7 antibody and H. pylori antibody from human sera with satisfactory accuracy. Simultaneous screening of both antibodies from human sera was also achieved, demonstrating the potential of this EK-IA for efficiently detecting multiple pathogenic infections in clinical settings. Preliminary work on the application of EK-IA to detect biomarkers of embryo development in embryo culture media also yielded good results. In addition to the experimental studies, the reaction kinetics of this microfluidic EK-IA has also been investigated, using both numerical simulation and a modified Damköhler number. Targeted towards a more sensitive assay, the influences of several important parameters on the reaction kinetics were studied. This EK-IA holds great promise for automated and high-throughput immunoassay in clinical environments.

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Thesis (Ph.D.)--University of Wyoming, 2008. / Title from PDF title page (viewed on August 5, 2009). Includes bibliographical references (p. 123-138).