Method for the simultaneous visualization of alkaline Phosphatase and Peroxidase with the aid of Energy-Filtered-Transmission-Electron-Microscopy

Für das detaillierte molekulare Verständnis neuronaler Erregungskreise ist es notwendig ankommende Axone, die Targetneurone und weitere beteiligte Moleküle der Signaltransduktion auf ultrastruktureller Ebene zu untersuchen. Dies erfordert sehr anspruchsvolle Mehrfachmarkierungstechniken auf elektronenmikroskopischem Niveau. Eine zusätzliche von üblichen Doppelmarkierungsmethoden -z.B. Kombination von Immunogold- und Peroxidase-Techniken- unabhängigen Markierungsprozedur würde einen erheblichen Nutzen für solche Untersuchungen in sich bergen. In der vorliegenden Arbeit sollte hierfür eine immunenzymatische Markierungstechnik für alkalische Phosphatase auf elektronenmikroskopischer Ebene über einen Cer-Phosphat-Niederschlag entwickelt werden. Erstaunlicherweise waren etablierte Techniken für endogene alkalische Phosphatasen in verschiedensten Geweben nicht für eine Anwendung in der Immunzytochemie geeignet. Durch eine sorgfältige Charakterisierung und Optimierung der einzelnen Reaktionsbedingungen konnte eine entscheidende Steigerung der Sensitivität der Cer-Phosphat-H2O2-Methode erreicht werden. Das durch die neue Technik niedergeschlagene Cer-Präziptat erlaubt nun eine Detektion des immunenzymatischen Reaktionsproduktes im Elektronenmikroskop. Am Modellsystem Bulbus olfactorius der Ratte wurde anschließend gezeigt, dass die Kombination der neuen Technik mit der üblichen Peroxidase/Diaminobenzidin-Methode eine simultane Doppelmarkierung auf ultrastruktureller Ebene erlaubt. Das Cer-Präzipitat der alkalischen Phosphatase wurde mit Hilfe der Energiefilter-Transmissons-Elektronenmikroskopie identifiziert und konnte somit vom Cer-freien Niederschlag der Peroxidase eindeutig unterschieden werden. Auf diese Weise konnte festgestellt werden, dass ankommende serotininerge Axone aus den Raphe nuklei des Mittelhirns keine synaptischen Kontakte mit dopaminergen periglomerulären Neuronen im Bulbus olfaktorius der Ratte etablieren.To understand in detail the functional morphology of neuronal circuits it is important to identify at the ultrastructural level the incoming axon, its target neuron, and members of the signaling cascades involved. This, however, represents a formidable task, requiring highly sophisticated electron microscopic multiple labeling techniques. To extend available double labeling procedures such as combinations of immunogold and peroxidase methods, an additional, gold- and peroxidase-independent procedure would represent a considerable advantage. The present investigation therefore aimed to use alkaline phosphatase as immunoenzymatic label at the electron microscopic level via cerium phosphate precipitates. To my surprise I found that available techniques, which are well established for the visualization of endogenous enzymes in sections from various tissues, are not suitable for application to immunocytochemistry. Careful characterization of the individual reaction conditions, however, resulted in an optimized procedure with largely increased sensitivity. The novel technique yields cerium-containing precipitates, which are massive enough to allow the detection of the immunoenzymatic reaction product in the electron microscope. Using the rat olfactory bulb as model system I showed further that the new technique allows the combination with the peroxidase/diaminobenzidine system for ultrastructural double labeling. For this purpose, the alkaline phosphatase product is identified by its cerium content via energy filtered transmission electron microscopy and thereby differentiated from cerium-free peroxidase-derived precipitates. Doing so, I found that ascending serotoninergic fibers do not establish synapses with dopaminergic periglomerular cells in the rat olfactory bulb

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited