Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Permeabilized Cells to Detect Intracellular Binding

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Hybridomas are crucial for producing monoclonal antibodies (mAbs).
• Identifying hybridomas secreting antibodies to intracellular targets presents unique challenges.
• Flow cytometry and FACS are powerful tools for cell analysis and sorting.

Purpose of the Study:

• To describe a method for identifying hybridomas that secrete monoclonal antibodies targeting internal cellular proteins using flow cytometry/FACS.
• To highlight the critical step of cell permeabilization in this workflow.
• To outline essential control strategies for validating the assay.

Main Methods:

• Utilizing flow cytometry or fluorescence-activated cell sorting (FACS) for hybridoma screening.
• Implementing a cell permeabilization step to allow antibody access to intracellular antigens.
• Applying hybridoma supernatants to permeabilized cells for antibody detection.

Main Results:

• Successful identification of hybridomas secreting monoclonal antibodies against internal cellular proteins.
• Demonstration of the necessity of cell permeabilization for detecting intracellular antibody targets.
• Validation of the technique through the use of appropriate positive and negative controls.

Conclusions:

• Flow cytometry/FACS, with cell permeabilization, is an effective method for isolating hybridomas producing mAbs to intracellular proteins.
• Proper controls, including cell lines with primary/secondary antibodies and secondary antibody alone, are vital for assay accuracy.
• This technique enhances the ability to generate specific monoclonal antibodies for research and therapeutic applications.