Open AccessA High-Throughput Hybridoma Selection Method Using Fluorometric Microvolume Assay Technology
Author(s) -
Rozanne Lee,
Mylinh Tran,
Mark Nocerini,
Meina Liang
Publication year - 2008
Publication title -
slas discovery
Language(s) - Uncategorized
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057108314148
Subject(s) - antigen , monoclonal antibody , antibody , microbiology and biotechnology , high throughput screening , cell , ligand binding assay , chemistry , primary and secondary antibodies , biology , biochemistry , immunology , receptor
Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection.