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Measurement of circulating protein biomarkers associated with disease can facilitate early detection, help guide treatment strategies and improve patient outcomes beyond current standards of care. The combination of inexpensive 3D-printed flow cells and electrochemical biosensors has recently emerged as a viable platform for low-cost, reliable biomarker measurements. Here, we report an electrochemical immunoassay system based on simple graphite electrode arrays, 3D-printed flow cells, and signal-generating magnetic bead bioconjugates for simultaneous detection of three biomarker proteins (cancer antigen 125 (CA-125), midkine (MK) and osteopontin (OPN)) associated with pediatric soft tissue sarcomas (PSTS). Magnetic bead bioconjugates are functionalized with large amounts of antibody and enzyme labels, electrode arrays are modified with gold nanoparticles and antibodies for specific capture of bioconjugate-labeled biomarkers, and 3D-printed flow cells facilitate their amperometric detection. Using this system, detection limits for CA-125, OPN and MK are 100 times lower than those obtained with commercial enzyme-linked immunosorbent assay (ELISA).

Ensuring correct analytical responses is of the utmost importance in laboratory workand interferences with analyses can give incorrect results. Work to improve the precision of the analyses is an ongoing process. An interference in the analysis of Interleukin-6 (IL-6), a cytokine that reflects inflammatory processes in the body, is biotin. Biotin interferes with EnzymeLinkedImmunoSorbentAssay (ELISA) analysis by binding in to streptavidin thereby causing falsely low analytical responses. An attempt to reduce that interference is to raise the threshold for biotin by introducing a new reagent. The aim of this project was to introduce a new reagent with elevated biotin interference threshold and carry out a precision measurement as well as a patient comparison. In addition, a sustainability study was carried out to investigate possible changes in IL-6 concentration when samples are stored in room temperature with open or closed vessels. Control material was analysed during three consecutive days. The patient comparison was performed by analysing 20 patient samples with both the older reagent and the new one. The sustainability study was preformed last and here were 7 patient samples used to perform this part of the study. The control material was analysed with good results, a total coefficient of variation (CV%) was calculated at 1.6% for the lower control and 3.0% for the higher one. The comparison showed good correlation with only a minor negative bias for the new reagent. The sustainability study showed, in line with what the supplier has indicated, a sample shelf life of 6 h at room temperature without sealing. Interesting to mention is that samples over 50 pg/mL showed a shelf life more than 24 h at room temperature without sealing. The decision was made by the medically responsible doctor that the reagent was approved for use in the laboratory.

Recombinant adeno-associated virus vectors are one of the most promising gene delivery tools for applications within gene- and cell therapy. The high level of wild-type adeno-associated virus infections in humans is a limitation due to the pre-existing immunity against the vector or its transgene product. An important tool to develop effective and safe therapies is the ability to measure the pre-existing immune responses against the virus capsids in humans. This master thesis at Gyros Protein Technologies aimed to investigate if the Gyrolab immunoassay system can be used to screen for pre-existing anti-capsid immunity in human sera by optimizing and evaluate three different assay formats: an indirect assay, a generic anti-AAV adsorption assay and a bridging assay. The evaluation focused on immunity against adeno-associated virus serotype 2. All immunoassay formats performed well and depending on application, the different formats offers different advantages. The generic anti-AAV adsorption assay offers the ability to easily screen for several viral serotypes without having to label the capsid, and the bridging assay provides high sensitivity. When screening 31 individual human sera, 58% were positive using the indirect assay and the generic anti-AAV adsorption assay and 65% using the bridging assay format. Provided, is automated and high throughout immunoassays where 16 individuals can be screened in one-two hours. It is shown that all three immunoassay formats can be used to screen for anti-adeno-associated virus antibodies, even though further optimization, cut off development and a larger data set is needed to obtain a fully sophisticated screening tool.

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Der Nachweis monoklonaler Proteine ist integraler Bestandteil in der Diagnostik von Monoklonalen Gammopathien. Derzeit wird die kombinierte Anwendung von elektrophoretischen Messverfahren und einem Immunoassay zur Bestimmung freier κ- und λ-Leichtketten im Serum angewandt, um monoklonale Proteine sensitiv zu detektieren und präzise zu quantifizieren. Der seit 2009 verfügbare Hevylite® Immunoassay ermöglicht die leichtkettenspezifische Bestimmung und Quantifizierung von intakten Immunglobulinen der Klassen IgA, IgG und IgM im Serum und bietet damit erstmals die Möglichkeit zur Beurteilung der Klonalität durch Berechnung des Verhältnisses von Igκ zu Igλ innerhalb eines Immunglobulin-Paares. Das zentrale Ziel dieser Studie war die methodische Validierung des Testverfahrens und der Vergleich des Hevylite® Immunoassays mit aktuell etablierten Messmethoden im Nachweis von monoklonalen Proteinen im Serum von Patienten mit Monoklonaler Gammopathie. Darüber hinaus untersuchten wir das klinische Potential des Assays im Vergleich mit derzeit validierten Markern zur Beurteilung der Tumorlast im Krankheitsverlauf von Patienten mit Multiplen Myelom. Für die Bewertung der analytischen Qualität des Hevylite® Immunoassays untersuchten wir Intra- und Interassay-Präzision, Linearität, Genauigkeit, Sensitivität und Spezifität für die Immunglobulin-Subklassen IgA, IgG und IgM. Für den Sensitivitätsvergleich wurden in einem zweiten Schritt Patientenseren mit gesicherter Monoklonaler Gammopathie durch eindeutigen Nachweis einer monoklonalen Bande in der Immunfixationselektrophorese mit dem Hevylite® Immunoassay untersucht. Für die Einschätzung des Hevylite® Immunoassays als Parameter zur Beurteilung der Tumoraktivität im Krankheitsverlauf wurden nachfolgende Seren von Patienten mit Multiplen Myelom nach Stammzelltransplantation über einen Zeitraum von 1 ½ Jahren gesammelt und die Ergebnisse des Assays mit konventionellen Markern verglichen. Zusammenfassend belegen unsere Daten analytische Leistungsmerkmale für alle aktuell verfügbaren HLC-Assays, die eine Anwendung in der Routinediagnostik erlauben. In der Detektion von monoklonalen Proteinen zeigt der Immunoassay vergleichbare Ergebnisse gegenüber etablierten Messverfahren. Jedoch entsteht durch die Limitation des Hevylite®-IgG-Assays eine diagnostische Lücke, so dass die Immunfixationselektrophorese als Goldstandart zum Nachweis von monoklonalen Proteinen nicht ersetzt werden kann. Wir konnten das Potential des Hevylite® Immunoassays in der Krankheitsüberwachung als alleinige Messmethode durch auserwählte Krankheitsverläufe von Patienten mit Multiplen Myelom zeigen. Unsere Befunde unterstützen die Ergebnisse verschiedener Arbeitsgruppen und belegen ebenfalls den Nutzen insbesondere des Hevylite®-IgA-Assays in der Krankheitsüberwachung von Patienten mit Multiplen Myelom Typ IgA als Alternative zu aufwendigen elektrophoretischen Messverfahren.:Abkürzungsverzeichnis 2 1 Einführung 3 1.1 Monoklonale Gammopathien 3 1.1.1 Definition und Epidemiologie Monoklonaler Gammopathien 3 1.1.2 WHO-Einteilung und diagnostische Kriterien Monoklonaler Gammopathien laut International Myeloma Working Group (IMWG) 4 1.1.3 Pathophysiologie und Klinik Monoklonaler Gammopathien 7 1.2 Labordiagnostische Möglichkeiten zum Nachweis Monoklonaler Gammopathien 8 1.2.1 Strategien und Kriterien der Diagnosefindung 8 1.2.2 Elektrophoretische Nachweisverfahren im Serum 9 1.2.3 Quantitative Bestimmung freier κ- und λ-Leichtketten mittels Immunoassay im Serum 10 1.2.4 Quantifizierung monoklonaler Proteine als entscheidender Parameter zur Überwachung der Tumoraktivität im Krankheitsverlauf 11 1.3 Leichtkettenspezifische Immunglobulinbestimmung zum Nachweis monoklonaler Proteine 12 1.3.1 Testprinzip und Interpretation des Hevylite® Immunoassays 12 1.3.2 Validierung und Vergleich des Immunoassays zur Bestimmung von leichtkettenspezifischen Immunglobulinen mit etablierten Messverfahren in der Diagnostik von Monoklonalen Gammopathien als wesentliches Ziel der vorliegenden Promotionsarbeit 13 2 Publikation 14 3 Aktuelle Anwendung und klinische Bedeutung des Hevylite® Immunoassays in der Erstdiagnostik und Beurteilung im Krankheitsverlauf Monoklonaler Gammopathien 15 4 Zusammenfassung der Arbeit 18 Literaturverzeichnis 21 Anlagen 26 Erklärung über die eigenständige Abfassung der Arbeit 27 Lebenslauf 28 Danksagung 30

The aim of this project was to make a comparison of the effects of antibody labeling chemistries on assays developed for the Gyrolab immunoassay platform. One of the labeling techniques was a heterogenous labeling technique targeting amino groups on the antibody. The other labeling technique was a site-specific labeling technique targeting the conserved Fc-glycan at the aspargine 297 residue on the IgG molecule. The site-specific labeling was performed using a kit from Genovis called GlyCLICK. The two labeling techniques were compared on four different assays developed for the Gyrolab platform. The assays tested in this project were two anti-drug antibody assays, a pharmacokinetics assay, a polyclonal antibody assay, and a monoclonal antibody assay. The drug tolerance was tested for the anti-drug antibody assays, resulting in better drug tolerance for reagents labeled with amino conjugation for the Humira assay with incubation overnight. A confirmatory analysis, testing the inhibition of negative control with addition of unlabeled drug in the Master Mix, was performed. This resulted in small differences in the inhibition between the different reagents, except for Keytruda on Gyrolab Bioaffy 200, for which the GlyCLICK labeled reagents led to a lower inhibition of the negative control. For all the assays the effects on signal to background ratio and limit of detection was investigated. The greatest advantages of GlyCLICK on the signal to background was observed for anti-drug antibody Keytruda assay and polyclonal antibody assay. For the polyclonal antibody assay, the results indicated potentially reduced need for the polishing step and for two wash solutions after addition of the detect reagent.

Biomarkers are measurable indicators of health status or disease state that can be used for diagnosis and may help guide patient treatment strategies. Enzyme-linked immunosorbent assays (ELISA) and other many clinical techniques currently used for measuring biomarker proteins lack sensitivity, demand high analysis cost, are often not well-suited for measuring multiple biomarkers in a single sample, and require long analysis times. Here, we demonstrate simple, low-cost 3D-printed flow-through devices with integrated electrodes modified with gold nanoparticles (AuNPs) for electrochemical immunoassays of S100B, a biomarker protein related to conditions like skin cancer and brain injuries. Flow-through devices are fabricated from photocurable-resin using a desktop digital light processing (DLP) projector-based 3D printer to produce 500-800 µm square cross-sectional fluidic channels. Threaded ports at the ends and center of the channel are included in the device design for connecting commercially available fittings for fluid delivery and integrating low-cost graphite electrodes for electrochemical biosensing.

Iron is an essential nutrient for energy and DNA replication. Its homeostasis is commonly perturbed by chronic inflammatory mechanisms. Chronic inflammation upregulates a cytokine, hepcidin, that degrades the iron export protein ferroportin. Without a way to export iron into the bloodstream iron availability in blood becomes depleted. Iron depletion in the blood stream hinders erythropoiesis and is termed anemia. Herein I investigate and inhibit the mechanism of hepcidin activation. Inhibition of hepcidin activation has released iron from tissues and alleviated anemic conditions in a cancer model. I have laid the foundation to investigate this pathway in a 3D spheroid model. The results show that hepcidin-25 inhibition is a promising treatment for anemia of cancer. More work needs to be done to confirm efficacy in an in vivo model. In addition to anemia of cancer I have also worked with diabetic rats and investigated their anemic state using common anemia diagnostic methods. I found that in this high fat high sugar diet Wistar rat model anemia was not induced. In addition to my studies on anemia I have investigated the use of portable x-ray fluorescence (pXRF) as an accessible and affordable elemental analysis technique for lateral flow immunoassays and biological samples such as cell lysates and animal tissue. While pXRF shows promising results more work needs to be done to increase its sensitivity and pixel size.

No description available.