HybriChip: An antigen microarray based screening tool, designed for a high-throughput production platform of mouse derived monoclonal antibodies

De Masi, Federico (2004) HybriChip: An antigen microarray based screening tool, designed for a high-throughput production platform of mouse derived monoclonal antibodies. PhD thesis, University of Glasgow.

Abstract

The incapacity to generate a sufficient number of mAbs to conduct large-scale analyses remains one of the most serious limitations in the field of functional genomics. Current methods of mAb production are very labour intensive and disappointingly low throughput, with a per capita production level of about 20 antigens per year. Here we present a modified hybridoma production method incorporating a novel screening assay which can be scaled up to generate antibodies in sufficient quantities for proteomics-scale analysis. Our method circumvents previous obstacles to increasing the throughput level of mAb production, in two ways. First, by immunizing a single mouse with multiple target antigens, we dramatically reduce the number of tissue culture operations normally necessary for performing multiple fusions simultaneously using only one antigen per animal. This minimises tissue culture operations and results in hybridomas that secrete antibodies specifically recognizing each of the target antigens. Second, we developed a novel antigen microarray assay (HybriChip) to screen supernatants generated by large-scale production. In this assay, an antigen chip is generated by coating an aminosilane treated slide with a single target antigen. Hybridoma culture supernatants from a fusion are consolidated and spotted as a microarray onto the antigen chip. After probing with a suitable fluorescently labelled secondary antibody, positive hybridomas are identified in a microarray scanner. The isotype of the bound antibody can be concomitantly determined by probing the antigen chip with mixtures of isotype-specific secondary antibodies, such as Cy5 -conjugated anti-mouse IgM and Cy3-conjugated antimouse pan-IgG (recognizing all mouse IgG isotypes). Different antigen chips can simultaneously be spotted in parallel with the same hybridoma culture supernatants, allowing rapid automated assay of multiple antibodies against many target antigens.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Bioinformatics, genetics.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Rosenthal, Prof. Nadia
• Date of Award: 2004
• Depositing User: Enlighten Team
• Unique ID: glathesis:2004-70987
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 09 May 2019 14:28
• Last Modified: 14 Jun 2021 15:11
• URI: https://theses.gla.ac.uk/id/eprint/70987