Screening Hybridomas for Cell Surface Antigens by High-Throughput Homogeneous Assay and Flow Cytometry

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. ( Journal of Biomolecular Screening 2008:210-217)

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Signal amplification in flow cytometry for cell surface antigen analysis

The Journal of Biochemistry ◽

10.1093/jb/mvz052 ◽

2019 ◽

Vol 166 (3) ◽

pp. 205-212 ◽

Cited By ~ 2

Author(s):

Takeshi Mori ◽

Yoshiki Katayama

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Surface Antigen ◽

Signal Amplification ◽

Surface Antigens ◽

Antibody Activity ◽

Cell Surface Antigens ◽

Promising Strategy ◽

Fluorescent Polymer ◽

Enzymatic Signal Amplification

AbstractSignal enhancing systems have been introduced to enable detection of cell surface antigens by flow cytometry. Cell surface antigens are important targets that describe the function and lineage of cells. Although flow cytometry is an effective tool for analysing cell surface antigens, this technique has poor sensitivity, which prohibits the detection of many important antigens on cell membranes. Thus, signal amplification is essential for developing practical tools for evaluating cell surface antigens by flow cytometry. Using a bright fluorophore or fluorescent polymer incorporated into antibodies is a straightforward strategy to improve flow cytometry sensitivity but may affect the functional characteristics of the labelled antibody. In contrast, enzymatic signal amplification is a more practical and efficient strategy to improve sensitivity that should not affect antibody activity. Although enzymatic signal amplification still has a number of drawbacks, this approach is a promising strategy to analyse cell surface antigens.

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