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Microarrays for the screening of monoclonal antibodies from supernatants

  • In this thesis a protocol based on a direct competitive and non-competitive immunoassay using the microarray format was developed to screen emulated hybridoma supernatants against the drug carbamazepine (CBZ). As support, epoxy slides were manufactured by coating microscope glass slides with epoxy silanes. The manufactured epoxy slides showed similar capacity for protein immobilization as commercially available NEXTERION slides, but showed a higher autofluorescence background. On the epoxy slides, protein A, Cys-A, G and Cys-G were immobilized as antibody capturing coating. It could be shown that the Cys-tag considerably increased the immobilization of all compared proteins, especially of protein G. Protein Cys-G immobilized at pH 8-9 was considered the most suitable protein for antibody immobilization due to good spot uniformity and a high binding capacity for IgG. Additionally, in immobilization experiments it could be shown, that 10-500 fold excess of bovine IgG, introduced by twoIn this thesis a protocol based on a direct competitive and non-competitive immunoassay using the microarray format was developed to screen emulated hybridoma supernatants against the drug carbamazepine (CBZ). As support, epoxy slides were manufactured by coating microscope glass slides with epoxy silanes. The manufactured epoxy slides showed similar capacity for protein immobilization as commercially available NEXTERION slides, but showed a higher autofluorescence background. On the epoxy slides, protein A, Cys-A, G and Cys-G were immobilized as antibody capturing coating. It could be shown that the Cys-tag considerably increased the immobilization of all compared proteins, especially of protein G. Protein Cys-G immobilized at pH 8-9 was considered the most suitable protein for antibody immobilization due to good spot uniformity and a high binding capacity for IgG. Additionally, in immobilization experiments it could be shown, that 10-500 fold excess of bovine IgG, introduced by two different fetal bovine serum (FBS) products, showed no significant competition on the immobilization of mice or goat IgG on protein Cys-G. To examine the captured antibodies for affinity to CBZ, the CBZ-TOTA-DY654 tracer was custom synthesized. It could be demonstrated, that the tracer antibody interaction was competitive with the tracer precursor, CBZ-TOTA-NH2 and CBZ. Furthermore, the synthesized tracer was also successfully used by externals to evaluate CBZ affine hybriodoma cells in flow cytometry. In the simulated screening, five antibodies with known affinity to CBZ were diluted in a cell culture medium. The screening method allowed to differentiate between affine antibodies (IC50 < 20 µg/ml) and unaffine antibodies (IC50 > 20 µg/ml), when concentrations of 0.1 µg/ml IgG or greater have been used. Of five applied antibodies, all highly affine clones (CE2 and B3212M) were reported as positives while no false-positive samples were observed. The screening included competitive, with around 89500 fold excess of CBZ, and non-competitive tracer incubation on different segments of the same chip, which resulted in total signal suppression on the competitive segment. The screening was performed in 20 hours, only a few nanoliters, of each simulated supernatant, were consumed in the screening process, furthermore in the method standard 96 well MTPs were used and no cleanroom facilities were required.zeige mehrzeige weniger

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Metadaten
Autor*innen:Martin Paul
Dokumenttyp:Sonstiges
Veröffentlichungsform:Graue Literatur
Sprache:Englisch
Titel des übergeordneten Werkes (Deutsch):Masterarbeit
Jahr der Erstveröffentlichung:2017
Gutachter*innen:Michael G. WellerORCiD, M. Linscheid
Verlag:Humboldt-Universität zu Berlin
Verlagsort:Berlin
Erste Seite:1
Letzte Seite:96
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Antibody immobilization; Carbamazepine; Competition; Epoxy silanes; Microscopic slides; Protein A cys; Supernatant
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:13.04.2022
Referierte Publikation:Nein
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