Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Whole Cells to Detect Cell-Surface Binding
Cold Spring Harbor Protocols
|June 24, 2022
Summary
Flow cytometry and fluorescence-activated cell sorting (FACS) identify hybridomas producing monoclonal antibodies against cell-surface proteins. Two protocols, individual tube and 96-well plate staining, are presented for this antibody screening process.
Area of Science:
- Immunology
- Biotechnology
Background:
- Hybridomas are crucial for producing monoclonal antibodies (mAbs).
- Identifying hybridomas that secrete specific mAbs requires efficient screening methods.
- Cell-surface proteins are common targets for therapeutic and diagnostic antibodies.
Purpose of the Study:
- To present optimized protocols for identifying hybridomas secreting monoclonal antibodies against cell-surface antigens.
- To provide researchers with practical methods for antibody screening using flow cytometry and FACS.
Main Methods:
- Utilizing flow cytometry and fluorescence-activated cell sorting (FACS) for hybridoma screening.
- Implementing two distinct staining protocols: individual tube staining and 96-well plate staining.
- Focusing on antibodies targeting cell-surface proteins as antigens.
Main Results:
- Successful identification of hybridomas secreting specific monoclonal antibodies.
- Demonstration of the efficacy of both individual tube and 96-well plate staining methods.
- Validation of flow cytometry and FACS as powerful tools for antibody discovery.
Conclusions:
- Flow cytometry and FACS are effective for screening hybridomas producing antibodies to cell-surface proteins.
- The presented protocols offer flexibility and efficiency for monoclonal antibody development.
- These methods facilitate the selection of high-affinity antibody-producing hybridomas.
