Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas
| Autor: | Richard B. Meagher, Christine Milcarek, Linda Matsuuchi, Paul W. Price, Muthugapatti K. Kandasamy, Loren E. Sasser, Youliang Wang, Deborah G. Culver, Roger B. Deal, Elizabeth C. McKinney |
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| Rok vydání: | 2008 |
| Předmět: |
Receptor complex
medicine.drug_class Immunology Receptors Antigen B-Cell Monoclonal antibody Article Cell Fusion Mice Antigen Cell Line Tumor medicine Immunology and Allergy Animals Cell fusion Hybridomas biology Antibodies Monoclonal Membrane Proteins Cell sorting Flow Cytometry Molecular biology Cell culture biology.protein Immunoglobulin heavy chain Antibody Immunoglobulin Heavy Chains |
| Zdroj: | Journal of immunological methods. 343(1) |
| ISSN: | 1872-7905 |
| Popis: | Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igalpha and Igbeta of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igalpha expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS). |
| Databáze: | OpenAIRE |
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