Entwicklung von Antikörper-basierten Tests zum quantitativen Nachweis von Antibiotika in Serum und Plasma

Severe sepsis and septic shock are among the most urgent problems of modern medicine, accounting for 20-80% of illnesses and a mortality rate of 22-76% in ICU patients. The joint project "DiabKON" (Diagnostic Antibiotic Therapy Control), from which this dissertation emerged, attempts to improve the treatment of intensive care patients through controlled antibiotic therapy. The focus is on therapy monitoring, in which a renewed administration of antibiotics should depend on the timely determination of the antibiotic concentration in the patient's blood. The project was funded by the German Federal Ministry of Education and Research under the funding measure "KMU-innovativ: Medizintechnik" (FKZ 13GW0354 A-F). The aim of this work was to establish immunoassays that allow the rapid, cost-effective and quantitative determination of the antibiotics linezolid and ceftazidime from human blood samples. The results of the two immunoassays should be comparable with the reference method LC-MS/MS. To this end, monoclonal antibodies were first generated by immunizing mice with the antibiotics bound to carrier proteins. Spleen cells from mice with the highest antibody titer were then fused with myeloma cells to form hybridoma cells. Selection of the best positive clones was then performed by direct competitive ELISA over multiple rounds of screening. Assay development for linezolid and ceftazidime included optimization of the assay procedure with testing of multiple assay formats and establishment of a standard curve for quantitative detection. Optimization of the reagents used also significantly improved the stability of the assay and thus the reliability of the measurement. Following development, verification and validation of the two tests enabled all the required performance parameters of the assays to be determined. It was found that both assays met all regulatory requirements in terms of accuracy, analytical sensitivity, stability and reproducibility. The method comparisons showed very good comparability to the reference method LC-MS/MS. Both tests can be performed fully automatically on the DRG:HYBRiD-XL instrument from DRG Instruments GmbH. They are used for rapid quantitative detection of antibiotic concentrations of linezolid and ceftazidime in the blood and thus allow close and timely control of antibiotic therapy. The findings of this work contribute to the development of further tests against commonly used antibiotics, which can then be used for therapy monitoring of antibiotic treatment.