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Localization and quantification of clinically relevant binding epitopes on ultra flat red blood cell ghosts / submitted by Sarah Stainer, BSc
AuthorStainer, Sarah
CensorEbner, Andreas
PublishedLinz, 2018
Descriptionv, 56 Blätter : Illustrationen
Institutional NoteUniversität Linz, Masterarbeit, 2018
Document typeMaster Thesis
Keywords (DE)AFM / Rasterkraftmikroskop / TREC / Erythrozyt / rote Blutkörperchen / Rhesusfaktor
Keywords (EN)AFM / atomic force microscope / TREC / Erythrocyte / red blood cell / rhesus factor
URNurn:nbn:at:at-ubl:1-23285 Persistent Identifier (URN)
 The work is publicly available
Localization and quantification of clinically relevant binding epitopes on ultra flat red blood cell ghosts [11.72 mb]
Abstract (English)

With atomic force microscopy (AFM) a sharp tip is scanned over a surface. By using an optical deection read out system, images with nanometer resolution can be obtained. Due to its ability to measure in liquid environment and by minimizing the applied force on the substrate, biological samples can be analyzed. However, the ability of AFM goes further than its imaging capability. If a ligand is attached to the AFM tip, receptor-ligand interactions can be detected and analyzed. Furthermore, an application has been developed that combines the imaging mode and the recognition detection ability of the device. This technique is called ‘Si- multaneous Topography and Recognition Imaging‘ (TREC), where not only a topographical image is recorded, but simultaneously an image that unravels the location of receptor-ligand interaction is created. Within this thesis TREC has been applied to localize specic receptors on at red blood cell ghosts. ^Erythrocyte ghosts have been known since the last century and have been used e.g. as a model to study cell membrane properties as their cell content main- ly consist of hemoglobin and they can easily be depleted of their cytosolic content. Here, a special preparation protocol was developed and optimized to produce ‘ultra-at‘ erythrocyte ghosts immobilized on glass slides, creating a perfect substrate for TREC studies. Whole ‘ultra-at‘ erythrocyte ghosts were imaged to deduce the RhD antigen quantity and distribu- tion patterns over the cell. The rst approach included scanning the erythrocyte membrane in overlapping 2x2 m windows and merging them into an complete ‘ultra-at‘ erythrocyte ghost image. Due to measurement instabilities caused by the long measuring times and oset inconsistencies caused by the high number of image location changes, another measurement approach was tested. While holding the scan speed constant the image size was increased and a whole ‘ultra-at‘ erythrocyte ghost was scanned in one TREC image (10x10 m). With the second approach we were able to gain a stable TREC signal during the whole imaging procedure. We analyzed the produced data regarding dierences in epitope densities between the rim region and the dimple region of erythrocytes. An eective and stable approach was developed to elucidate erythrocyte antigen locations and quantities. With this work an im- pression on RhD antigen distributions on erythrocytes is given. A path for further mapping of erythrocyte antigen distributions of other clinically relevant binding epitopes like weak-D and partial-D RhD antigens was being paved with this work.

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