Blood testing is commonly applied for the initial screening of patients. Thereby, one of the used methods is flow cytometry. Relevant measured values of flow cytometric examinations can be the blood cell concentration or the number of specific proteins per cell, present on the cell surface. This number of proteins is a valuable observable and an indicator for the status of various diseases and the immune system. It is usually obtained in form of antibody binding capacity. Two orthogonal methods have been developed to allow for absolute quantification of antibody binding capacity. These methods are based on antibody kinetics or fluorescence calibration and can be used individually or complementarily. The first method uses a comprehensible antibody kinetics model to conclude antibody binding capacity. A novel and practicable procedure for real-time monitoring of antibody kinetics has been developed and can be employed in addition to conventional procedures, typically relying on incubation stops, for example by dilution, and providing only one data point per prepared sample. The novel procedure enables determination of the desired kinetic curve and antibody binding capacity with only one prepared sample, without the need to conduct a measurement series. The second method relies on fluorescence calibration. Therefore, a purpose-built flow cytometer employing acoustic focusing was designed, constructed and coupled to an inductively coupled plasma mass spectrometry. The development of acoustic focusing in a very thin capillary actuated with high frequency, which had not been previously cited in the literature, allowed for dilution-free sample recollection. The coupling of this flow cytometer to an inductively coupled plasma mass spectrometry in this manner is unique. The antibody binding capacity was determined with stand-alone and coupled instruments by using antibodies simultaneously labeled with fluorophores and lanthanides. Both methods were used to investigate blood cells from own cell culture or being commercially available. The results were verified by using external calibration particles with good accordance. The antibody kinetic approach is universally applicable, whereas the fluorescence calibration approach offers the unique potential of metrological traceability and antibody binding capacity determination on a single-cell level. Supporting material like model derivation or custom evaluation applications are included to facilitate method application. The aforementioned advancements open up new possibilities for the quantification of antibody binding capacity and, in the best case, facilitate or enhance blood analysis using flow cytometry in the field of research and clinical practice. This would not only be beneficial for those engaged in the field of study, but particularly for the patients undergoing treatment.
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Hein, Y. (2025). Development of methods for the quantification of antibodies bound to blood cells and microbeads [Dissertation, Technische Universität Berlin, 2025]. Berlin: TU. https://doi.org/10.14279/depositonce-22455
