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- Monoclonal antibodies (3) (entfernen)
Organisationseinheit der BAM
Fraunhofer Life Science Day
(2015)
Their high affinity, selectivity and specificity make monoclonal antibodies (mAbs) very important tools in research, diagnostics and therapy. The production of mAbs is routinely performed by hybridoma technique. Hybridomas are generated by fusing antibody-producing B cells with indefinitely proliferating myeloma cells. Both, cell fusion and the indispensable identification and isolation of the desired antibody-producing hybridoma clone is still far from being trivial.
We describe a novel method that should help to optimize the cell fusion, and the screening for antibody-secreting hapten-specific hybridoma cells by using fluorescence activated cell sorting and single-cell fusion. Hybridoma cells specific for a hapten were incubated with a hapten-peroxidase conjugate (hapten-HRP), which was subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor® 488). To characterize the expression of membrane-bound immunoglobulin G (IgG) a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor® 647) was used. Successful staining was verified by confocal laser scanning microscopy (CLSM). We show that it is possible to specifically label hapten-specific hybridoma cells. It should also be possible to use this labelling approach for the isolation of hapten-specific B cells from the spleen and use these cells for single-cell fusion. Parallelized cell fusion chips were developed to increase fusion efficiencies.
Antibodies are the most used biomolecules in analytical research. Nevertheless, the sequence and structure information of antibodies is often limited, since manufacturers keep them secret or suppliers sell them under different names. This can make it difficult to reproduce even basic experiments performed in publications as the antibodies used might not be identifiable. To overcome these problems, we developed a simple and cheap method for antibody identification by MALDI-TOF-MS fingerprinting. This technique was used to generate a library of antibody fingerprints, which enables the identification and comparison of antibodies in short time.
Diclofenac is a widely used drug against fever, inflammation, pain, and rheumatic diseases. An average of 70 % of the ingested diclofenac is excreted in the urine. Thus, 63 tons are introduced into the water cycle in Germany. Due to insufficient removal of diclofenac in wastewater treatment plants, residues of diclofenac can be found in surface water and sometimes in drinking water It exist a polyclonal against diclofenac and monoclonal antibodies against diclofenac and it`s metabolites. There all based on direct couling of diclofenac on proteins via carboxylic functions. However it was imagined advantageous to introduce a heterobifunctional spacer between the analyte and the carrier protein.
Aceclofeac is commercial available. All other spacers can only be introduced with solid phase synthesis.